Bio-Plex Software 6.1 User Guide ™

Bio-Plex Software 6.1 User Guide ™
Bio-Plex Manager™ Software 6.1
User Guide
BioPlex_6.book Page i Friday, September 23, 2011 11:55 AM
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
BioPlex_6.book Page ii Friday, September 23, 2011 11:55 AM
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:
[email protected] (U.S.)
[email protected] (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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For More Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bio-Rad Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2
3
4
4
Chapter 2. Bio-Plex Manager™ Software Overview . . . . . . . . . 5
Software Editions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Software Licenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Compatibility with Luminex Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
General Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Quick Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Types of Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Key Software Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
6
6
7
8
9
9
Chapter 3. Software Installation . . . . . . . . . . . . . . . . . . . . . . . . 11
System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Required Screen Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Installing the Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Microsoft.NET. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bioplexdata.mdb File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Luminex LXR Directory and Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hardware Protection Key (HASP Key). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Uninstalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11
12
13
13
13
14
14
14
Chapter 4. Starting the System . . . . . . . . . . . . . . . . . . . . . . . . . 15
Starting Bio-Plex Manager . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Connecting with the Array Reader and Microplate Platform . . . . . . . .
Communication between the Array Reader and Microplate Platform .
Disconnecting and Reconnecting . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15
16
17
18
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Disconnecting with Sleep Mode. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Menu and Toolbars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Status Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Quick Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sample Needle Adjustment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19
19
19
20
21
Chapter 5. Controlling the System . . . . . . . . . . . . . . . . . . . . . . 25
Bio-Plex MCV Plate IV. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Start Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Optics Warm Up and Shut Down . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Opening the Calibration Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . .
Selecting/Entering Calibration Control Numbers . . . . . . . . . . . . . . . . .
Setting Up the Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Performing the Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Logging the Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Start Up and Calibrate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Wash Between Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Remove Air Bubbles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Unclog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Validation Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Validation Kit Control Number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Setting Up a Validation Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Control Number Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Validation Type Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Performing a Validation Run. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Validation Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Validation Log. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Platform Heater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Instrument Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Eject/Retract Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Additional Instrument Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cancel Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Shut Down . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Instrument Operations Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
iv
26
27
28
29
30
30
33
34
34
37
38
39
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41
41
41
42
42
43
44
44
45
48
49
49
49
51
51
51
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Chapter 6. Preparing Protocols . . . . . . . . . . . . . . . . . . . . . . . . . 53
Protocol Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Creating/Opening Protocol Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Saving Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reducing the File Size of Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . .
Multi-Assay Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Uses for Multi-Assay Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Creating a Multi-Assay Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Changing a Protocol from Single to Multi-Assay . . . . . . . . . . . . . . . . .
Protocol Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Step 1. Describe Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Step 2. Select Analytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Step 3. Format Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Plate Groupings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Step 4. Enter Standards Info . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Standard Lots. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Step 5. Enter Controls Info (Optional) . . . . . . . . . . . . . . . . . . . . . . . . .
Step 6. Enter Sample Info (Optional) . . . . . . . . . . . . . . . . . . . . . . . . . .
53
53
54
54
56
56
57
57
58
59
59
68
75
78
79
96
98
Chapter 7. Running Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Run Protocol Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bead Count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sample Timeout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reservoir Functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Skip Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Standard Curve Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Advanced Run Settings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bead Map Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sample Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Save Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sampling Errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Doublet Discriminator Gates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Plate Loading Guidelines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Running the Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Status Bar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Manually Stopping a Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Generating Results from a Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Histogram and Bead Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Histogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bead Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
100
101
101
102
103
104
104
104
105
105
105
107
107
108
110
110
110
111
112
115
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Bead Map Display Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Raw Data Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bead Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sampling Error Codes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Raw Data Display Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Set Number Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Other Table Formatting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Copying the Raw Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Rerun/Recovery Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
117
119
120
120
121
122
122
122
123
Chapter 8. Using the Security Edition . . . . . . . . . . . . . . . . . . . 125
Bio-Plex Manager™ Security Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Background on 21 CFR Part 11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Standard Mode vs. Secure Mode . . . . . . . . . . . . . . . . . . . . . . . . . . .
System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Installing and Starting Bio-Plex Manager Security Edition . . . . . . . . . . . . . . .
Users, Passwords, and User Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
User Level Restrictions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
User Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Enabling and Disabling Secure Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
User Authentication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Electronic Records . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
File Security and Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Calibration, Validation, and Instrument Operations Logs . . . . . . . . . . . . . . . .
Secure Protocol and Results Files . . . . . . . . . . . . . . . . . . . . . . . . . . .
Unsigned Protocol and Results Files . . . . . . . . . . . . . . . . . . . . . . . . .
Signed Protocol and Results Files . . . . . . . . . . . . . . . . . . . . . . . . . . .
Document ID Number and Signature . . . . . . . . . . . . . . . . . . . . . . . . .
Adjusting the Number of Unknown Samples in a Protocol . . . . . . . .
Generating a Results File from a Protocol File . . . . . . . . . . . . . . . . . .
Audit Trail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Viewing the Audit Trail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Protected Directories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Locking Bio-Plex Manager . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Logging Off. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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126
126
127
127
127
128
128
129
129
130
130
131
131
131
132
134
135
136
137
139
140
141
141
Chapter 9. Analyzing the Results . . . . . . . . . . . . . . . . . . . . . . 143
Results Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Opening a Results File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Saving a Results File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
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Reducing the Size of a Results File . . . . . . . . . . . . . . . . . . . . . . . . . .
Results Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Custom Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Viewing/Changing the Protocol Settings . . . . . . . . . . . . . . . . . . . . . .
Changing the Doublet Discriminator Gate Range . . . . . . . . . . . . . . .
Raw Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Setting the Plate ID. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Exporting the Raw Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Raw Data Table Error Codes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Printing the Raw Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Report Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Display Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Report Table Column Descriptions . . . . . . . . . . . . . . . . . . . . . . . . . .
Obs/Exp * 100 Column. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concentration in Range Column . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Showing/Hiding Outliers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Resizing the Columns. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Context Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sorting Report Table Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Copying the Report Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Printing the Report Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Exporting the Report Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Export Format. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Export a Subset of the Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Export Preferences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Use Multiple Worksheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Exclude Standard Curves. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Exclude Column Headers/Footers . . . . . . . . . . . . . . . . . . . . . . . . . . .
Exclude Table Error Codes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Regression Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Linear vs. Logistic Regression Methods . . . . . . . . . . . . . . . . . . . . . .
Copying the Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Exporting the Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Printing the Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Graphing Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Choosing Graph Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Viewing Information within Graphs. . . . . . . . . . . . . . . . . . . . . . . . . . .
Adding New Graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Editing an Existing Graph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
144
146
147
148
149
150
150
151
152
152
153
153
154
158
160
160
161
162
162
163
163
163
163
164
165
166
167
167
167
167
168
170
173
173
173
174
175
177
180
182
183
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Deleting a Graph. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Exporting Graphs to Other Applications . . . . . . . . . . . . . . . . . . . . . .
Adjusting Your Graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Printing Graphs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Document Export Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bio-Plex XML Export . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Output CSV File Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Using Stylesheets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Command Line Export . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Exporting the Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Importing Luminex xPONENT Output Files. . . . . . . . . . . . . . . . . . . . . . . . . . .
Exporting Results Data to Bio-Plex Data Pro™ . . . . . . . . . . . . . . . . . . . . . . .
184
184
185
185
186
187
189
191
192
193
194
195
Chapter 10. Standard Curve Optimizer . . . . . . . . . . . . . . . . . . 199
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Standard Curve Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concentration in Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Determining Outliers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Recommended Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Standard Curve Optimizer Report . . . . . . . . . . . . . . . . . . . . . . . . . . .
Residual Variance and Fit Probability. . . . . . . . . . . . . . . . . . . . . . . . .
Logistic Weighting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
200
200
201
201
201
202
204
204
Chapter 11. Data Normalization . . . . . . . . . . . . . . . . . . . . . . . 207
Normalization Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Selecting Internal Controls/Housekeeping Genes . . . . . . . . . . . . . . .
Assigning a Control Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Report Table Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Predefined Normalization Report Schemes . . . . . . . . . . . . . . . . . . . .
Normalization Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Calculations Based Upon Your Settings . . . . . . . . . . . . . . . . . . . . . .
Normalization Factor Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . .
207
208
209
210
211
212
212
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’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Norm Ratio’’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Normalized Fluorescence Values (Norm FI) . . . . . . . . . . . . . . . . . . . .
Ratio (Simple) and the Grouping Function . . . . . . . . . . . . . . . . . . . . .
Basic Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reporter Fluorochromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Excitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Microsphere Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Microsphere Dispersion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Probe Sonicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bath Sonicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Enumeration of Microsphere Suspensions . . . . . . . . . . . . . . . . . . . .
Microsphere Separation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . .
Microsphere Agitation During Assay . . . . . . . . . . . . . . . . . . . . . . . . .
Microsphere Stability and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . .
Software Warranty. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reporting Problems to Bio-Rad . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Report Table Error Codes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
224
224
225
226
227
227
227
228
228
229
229
229
229
230
230
231
231
232
233
234
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
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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
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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™ 6.1 software. New angiogenesis, acute
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.
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)
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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”
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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:[email protected] (U.S.)
[email protected] (International)
Web:
4
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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
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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.
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General Workflow
ANALYZING LUMINEX DATA IN BIO-PLEX MANAGER
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.
.
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
Figure 2. Bio-Plex Manager software workflow
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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
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Types of Files
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)
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Bio-Plex Manager Software 6.1 User Guide | Key Software Features
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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 Windows XP Professional
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 × 1024
1280 x 1024 (Windows 7)
Screen colors
256 colors
24-bit True Color
Ports for connecting
1 RS232 serial port and
1 RS232 serial port and 1 USB 2.0
instrument (required for 1 USB port
port
Instrument Control
license only)
Port for connecting
1 USB port
1 USB 2.0 port
the HASP key
Other software
Internet Explorer 6.0 or later
Internet Explorer 8.0
Microsoft Excel 2003 or later
Microsoft Excel 2007
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.
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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.
Figure 4. Changing the screen resolution settings
12
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Installing the Software
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.
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.
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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.
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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.
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Bio-Plex Manager Software 6.1 User Guide | Starting Bio-Plex Manager
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.
HTF
Serial
port
Computer
USB1
USB2
Serial port
Power
Power
Array Reader
P1
P2
Power
Microplate Platform
Power
Figure 5. System cable connections
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Serial port
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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
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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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Menu and Toolbars
Disconnecting with Sleep Mode
When your computer goes into sleep mode, the instrument is disconnected
from Bio-Plex Manager. When you awaken your computer from sleep mode,
you will not be able to reestablish the connection to the instrument using the
Reconnect option on the Instrument menu, and you will need to restart BioPlex Manager.
Bio-Plex Manager overrides any sleep mode settings on your computer.
Therefore, as long as it is running, your computer will not go into sleep mode.
However, Bio-Plex Manager does not prevent you from actively putting your
computer into sleep mode. It is recommended that you do not do this as long
as Bio-Plex Manager is running.
Menu and Toolbars
Bio-Plex Manager includes a menu bar and main toolbar at the top of the
application window.
The pulldown menus contain all the major functions of the software. Note that
these menus change depending on whether you are displaying a Protocol
window, a Results window, or neither.
You can move quickly between any number of open Bio-Plex Manager
windows. Hold down the Ctrl key, then press Tab.
The main toolbar includes the major instrument controls, including Start up &
Calibrate, Shut Down, Wash, Unclog, etc. The various Protocol and Results
windows also contain their own toolbars, with commands specific to those
windows. These are described in greater detail in the following chapters.
Status Bar
The Instrument Status Bar shows the current state of the instrument
(Calibration, Warm Up, Ready, Pressurizing, etc.). It includes a Cancel button
for canceling the current operation (see page 51).
Figure 8. Instrument status bar
When the array reader performs an operation, the time remaining for the
operation shows in the instrument status bar.
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Bio-Plex Manager Software 6.1 User Guide | Quick Guide
The Bio-Plex Manager software status bar is below the instrument status bar,
and provides information about the command under the cursor. This
information is continuously updated as you move your cursor over the
software window.
The software status bar also shows the current user and, if you are using the
Security Edition of the software, whether you are in Secure Mode (shown as a
"locked" symbol
) or Standard Mode (shown as "unlocked"
). See
page 5 of this manual, and the Bio-Plex Manager 6.1 Software Upgrade and
Configuration Guide for more information about the Security Edition.
The software status bar also shows the Caps Lock, Scroll Lock, and Num
Lock keyboard status.
Quick Guide
When Bio-Plex Manager first opens, a Quick Guide toolbar appears in the
upper right corner of the screen. You can use the Quick Guide to guide you
through the typical workflow, from start up and calibration through shut down.
Figure 9. Bio-Plex Quick Guide
By default, the Quick Guide opens automatically when you start Bio-Plex
Manager. To disable, deselect the Show at startup checkbox at the bottom of
the guide.
To open the Quick Guide after closing, select Quick Guide from the View
menu.
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Sample Needle Adjustment
Sample Needle Adjustment
See the Bio-Plex 200 System Hardware Instruction Manual or the Luminex
manual for instructions on installing the sample needle.
WARNING: To protect hands and fingers, keep them out of the microplate
platform when the needle is not in the down position.
The height of the sample needle must be adjusted when the microplate type
has changed, and/or when the sample needle is replaced. The MCV plate
included with your system provides a method for adjusting sample needle
height for standard flat-bottom plates (catalog #171-025001), filter plates
(Millipore catalog #MSBVS1210), or PCR plates.
Follow these steps to adjust the sample needle height.
1. Turn on the array reader and microplate platform.
2. Launch the Bio-Plex Manager software.
3. Click Instrument in the menu bar and choose Adjust Needle. The
following dialog box appears.
Figure 10. Adjust Needle dialog
4. Click Eject/Retract to eject the plate holder.
5. Place the MCV plate on the microplate platform with the black arrow
facing toward the array reader.
6. Click on the Eject/Retract button to retract the plate.
7. Tape the access door of the microplate platform open. It will be
necessary to be able to see inside the access door.
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Bio-Plex Manager Software 6.1 User Guide | Sample Needle Adjustment
8. Select the plate type to adjust the needle height appropriately. Choose
Standard Plate if you are using a Bio-Plex Pro™ flat-bottom microplate
for magnetic beads or the Millipore filter plate. Your other choice is PCR
Plate.
9. In the Adjust Needle window, click on the Up/Down button. The needle
will move to the down position.
10. With the needle in the down position, loosen the needle height
adjustment thumbscrew at the top of the needle so that the needle
housing can move up and down freely.
NOTE: All adjustments to the needle height must be made when the needle is
in the down position.
Needle height
adjustment thumbscrew
Figure 11. Sample needle assembly
11. By holding onto the needle height adjustment thumbscrew on the
needle arm, manually move the needle so that it just touches the
bottom of the needle adjustment well of the MCV plate. Move the
needle up and down gently a couple of times to verify that the needle is
barely touching the bottom of the well.
12. Tighten the needle height adjustment thumbscrew so that it is no longer
possible to manually move the needle up and down. Take care to
ensure that the needle does not move while you are tightening the
screw. Do not overtighten.
13. In the Adjust Needle window, click on the Up/Down button to move the
needle up and down. Look inside the microplate platform at the MCV
plate. The needle should just touch the MCV plate at the bottom of the
cutout (use flashlight for better viewing). Readjust the needle height if
necessary.
14. Save these settings. This allows Bio-Plex Manager to warn you which
type of plate the system will use, before it starts a run.
15. When the needle is adjusted properly, click the Eject button.
16. Remove the MCV plate from the microplate platform.
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Sample Needle Adjustment
17. Perform a Wash Between Plates step to remove any air introduced into
the lines.
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Bio-Plex Manager Software 6.1 User Guide | Sample Needle Adjustment
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Bio-Plex Manager Software 6.1 User Guide
5
Controlling the System
This chapter covers the Bio-Plex Manager™ 6.1 software commands that
control basic system functions, such as instrument start up, warm up,
calibration, validation, and shut down.
NOTE: The startup, warm up, and calibration functions must be performed
prior to running an assay with Bio-Plex Manager, all of which together take
about 45 minutes. The Start up & Calibrate function allows you to complete
these steps with one command.
WARNINGS:
• The sheath fluid container and the waste fluid container should be
closely monitored
• The sheath fluid bottle must be placed at the same level as the
array reader, unless you are using the Bio-Plex High-Throughput
Fluidics (HTF) or Luminex Sheath Delivery System (SDS). The fluid
level should be below the air inlet connection and above the sheath
outlet connection. Always check the sheath fluid level before
starting a run or procedure
• If you are using the Bio-Plex HTF or Luminex SDS, it should sit on
the counter next to the array reader, while the sheath fluid cube
should be placed 3-4 feet below the array reader (for example, on
the floor)
• The waste fluid container receives waste from the system. Do not
allow the waste container to overflow. Empty the waste bottle each
time the sheath fluid bottle is filled. The waste container should be
placed on the bench next to the instrument. Never place this
container on top of the instrument. All waste containers should
have vented caps
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Bio-Plex Manager Software 6.1 User Guide | Controlling the System
Bio-Plex MCV Plate IV
Bio-Plex Manager 5.0 and later requires the use of the Bio-Plex MCV
(Maintenance, Calibration and Validation) Plate IV. This plate contains wells
marked for the different types of fluids used in validation, washing, calibration,
and other functions. The Bio-Plex MCV Plate IV has been modified from the
previous MCV plate to work with Bio-Plex Validation Kit 4.0 and its Reporter
and Classify bead sets. It also includes two open needle wells for adjusting
needle heights for microplates or PCR plates.
NOTE: Bio-Plex Manager 5.0 and later require the use of the Bio-Plex MCV
Plate IV, the Bio-Plex Validation Kit 4.0, and the Bio-Plex Calibration Kit.
Figure 12. Bio-Plex MCV Plate IV
When a particular procedure such as calibration requires you to add solutions
to the Bio-Plex MCV Plate IV, the dialog box describing the procedure
includes a diagram of the MCV Plate IV with the wells to be loaded highlighted
in blinking yellow.
Wells highlighted
in yellow
Figure 13. Highlighted wells in diagram indicate wells to be filled
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Start Up
Start Up
Startup is a series of fluidic functions that prepares the array reader to acquire
data. This process requires the Bio-Plex MCV Plate IV, distilled water, and
70% isopropanol. Startup takes approximately 10 minutes.
NOTE: The startup procedure can be performed while the optics are warming
up, but no validations, calibrations or runs should be performed before the
optics are warmed up. This takes an estimated time of 30 minutes.
Click the Start Up button
on the main toolbar or select the command
from the Instrument menu. Follow the step-by-step directions in the dialog
box for preparing the MCV Plate IV.
Figure 14. Start Up dialog
Insert the prepared plate into the microplate platform and click OK to begin
the startup process.
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Bio-Plex Manager Software 6.1 User Guide | Controlling the System
Optics Warm Up
and Shut Down
To ensure accurate and reproducible results, the optics (that is, the lasers) in
the array reader must warm up for at least 30 minutes prior to calibration,
validation, and reading assays. Optics warm up begins when you first turn on
the array reader.
You can proceed with the array reader startup procedure described in the
previous section while the optics are still warming up. However, if you try to
perform calibration, validation, or an assay reading, you will receive a warning
message.
You can cancel the warm up procedure using the Cancel Operation command
on the Instrument menu or the Cancel button in the status bar; however, this is
not recommended. Results for identical readings may vary if the optics have
not reached optimal operating temperature.
NOTE: If you attempt to start a reading during warm up, you can eject the
plate carrier using the Eject/Retract Plate command
on the main toolbar
and remove the plate for storage until warm up is complete, without canceling
the warm up procedure.
If the array reader is idle for more than four hours, the optics automatically
power down (though the array reader itself remains on). Depending on the
length of the shutdown, a full 30-minute warm up period may be required
before more readings can be taken.
Click the Warm Up button
on the main toolbar or from the Instrument
menu to begin warm up after automatic power down.
If you attempt to perform a reading after automatic power down, you receive a
warning message and the optics begin warming up.
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Calibration
Calibration
Calibration of the array reader is essential for optimal performance and dayto-day reproducibility of results. Calibration is required:
• Each day after the startup procedure is complete and the optics
have warmed up
• If the array reader temperature changes by more than 2ºC during
the course of the day. (If the temperature changes by more than
2ºC, a message box prompts you to recalibrate)
• Before data acquisition, if you switch between Bio-Plex Manager
and Luminex software installed on the same computer (see page 6)
NOTE: Before calibrating, make sure that optics warm up is complete.
The Bio-Plex Calibration Kit contains calibration microspheres (CAL1 and
CAL2 beads) with stable fluorescent intensities in the RP1, CL1, and CL2
wavelength ranges. The calibration process uses these microspheres to
adjust voltage settings for optimal and consistent microsphere classification
and reporter readings over time and across different instruments. Current
calibrated settings are automatically applied to any new session.
Calibration using Bio-Plex Manager requires the Bio-Plex MCV Plate IV, CAL1
beads and CAL2 beads from the Bio-Plex Calibration Kit, and distilled or
deionized water. The CAL1 beads calibrate the array reader's doublet
discriminator and classification channels, while the CAL2 beads calibrate the
array reader's reporter channel for reporter fluorescence detection.
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Bio-Plex Manager Software 6.1 User Guide | Controlling the System
Opening the Calibration Dialog Box
To begin calibration, click the Calibrate button
on the main toolbar or
select Calibrate from the Instrument menu. The Calibrate dialog box opens.
Figure 15. Calibrate dialog
At the top of the dialog box, enter your name in the field. If you are using the
Security Edition of the software in Secure Mode, your user name will be listed
and grayed out.
The time and date of the last calibration using Bio-Plex Manager are listed, as
is the temperature at the time of that calibration. If the temperature has not
changed by more than 2ºC in a single day, it is not necessary to recalibrate the
instrument.
Next, select the calibration type by clicking on the CAL1 & CAL2, CAL1 Only,
or CAL2 Only button. You should perform both CAL1 and CAL2 calibration
daily.
Selecting/Entering Calibration Control
Numbers
Under Select Control Numbers in the Calibrate dialog box, you can either
select existing control numbers for your CAL1 and CAL2 microspheres, or
enter new control numbers.
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Calibration
Select existing control numbers from the pulldown list, and the target values
for the control numbers appear in the appropriate fields.
When you receive a new Bio-Plex Calibration Kit, you must add the new CAL1
and CAL2 control numbers and target values to the Calibrate dialog. These
numbers are printed on the bottles containing the beads.
NOTE: It is critical that you enter the correct target values for your CAL1 and
CAL2 calibrators. Entering incorrect values results in an incorrectly calibrated
array reader, which adversely impacts assay results.
To add a new CAL1 control number, click the Add button under CAL1 Control
Number in the Calibrate dialog. The Add New CAL1 Control Number dialog
box opens.
Figure 16. Adding a new CAL1 control number
In the dialog, enter the control number from the CAL1 bottle in the Enter
Control Number field. If the expiration date is printed on the bottle, select the
option button next to the date field under Expiration Date and click the
pulldown button next to the field to open the calendar selection box.
Figure 17. Calendar selection box
Scroll through the calendar using the scroll buttons at the top. Click to select a
particular date. Use the up/down arrow keys to highlight and change
individual date components.
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Bio-Plex Manager Software 6.1 User Guide | Controlling the System
Next, enter the DD, CL1, and CL2 target values for the control number, as
printed on the bottle, in the appropriate fields.
NOTE: Use only the DD target value on the Bio-Plex CAL1 bottle. Using
Bio-Rad’s calibrators is highly recommended, because other manufacturers’
DD target values have not been validated.
Click Add to close the dialog box and save your changes.
To add a new CAL2 control number, click the Add button under CAL2 Control
Number in the Calibrate dialog box. The Add New CAL2 Control Number
dialog box opens.
Figure 18. Adding a new CAL2 control number
In the dialog, enter the control number from the CAL2 bottle in the field. Select
the expiration date, if printed on the bottle, as described above. Next, enter
the Low RP1 target value for the control number, as printed on the bottle, in
the RP1 field.
NOTE: The CAL2 calibration bottle label lists two RP1 (also known as PMT, for
photomultiplier tube) target values: Low RP1 and High RP1. The High RP1
target value cannot be used in Bio-Plex Manager 5.0 or higher. All calibration
is done at the Low RP1 target value, which must fall between 3000 and 4000.
The High RP1 target value remains on the bottle label because it can be used
with earlier versions of Bio-Plex Manager. Assays that require calibration using
the High RP1 target value can be run by selecting the checkbox labeled “Run
at High RP1 Target” in the Run Protocol window.
When you are done, click Add to close the dialog box and save your changes.
Your new control numbers are added to the selection lists in the main
Calibrate dialog.
To delete a particular control number, first select it in the Calibrate dialog box,
and then click the Delete button.
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Calibration
Setting Up the Calibration
When you have specified the control numbers of your calibration
microspheres and specified the type of calibration you want to perform (CAL1
& CAL2, CAL1 Only, or CAL2 Only), click OK in the Calibrate dialog.
Another dialog box lists step-by-step instructions for preparing the Bio-Plex
MCV Plate IV.
Figure 19. Preparing the Bio-Plex MCV Plate IV for CAL1 & CAL2 calibration
NOTES: When preparing the MCV Plate IV, do the following:
• Important: Before vortexing, remove the calibration beads from 2 to
8°C (36 to 46°F) storage, and allow them to warm to room
temperature. Vortex each bottle for 30 seconds. Proper suspension
of the microspheres is essential for efficient calibration
• Never dilute the calibration beads, and be careful to limit their
exposure to light. Store at 2 to 8°C immediately following
calibration
• Load 6 drops of beads (approximately 200 μl) per reservoir (CAL1
or CAL2)
• The DI H2O well of the MCV Plate IV holds about 3 ml of distilled or
deionized water
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Bio-Plex Manager Software 6.1 User Guide | Controlling the System
Performing the Calibration
Click the Eject/Retract Plate button to eject the microplate platform plate
carrier. Place the MCV Plate IV in the carrier with the arrow facing toward the
platform, and then retract the plate. Click OK to start the calibration process.
As calibration proceeds, the status bar at the bottom of the Bio-Plex Manager
window monitors the progress of calibration. The number of beads per
second should be 100 or higher. Fewer beads per second may indicate a
problem with the fluidics system.
Number of beads per second should be 100 or higher
Figure 20. Status bar during calibration
Because calibration beads are highly concentrated, calibration is followed by
three wash cycles using distilled water.
An alert box notifies you whether calibration has succeeded or failed. You also
receive an alert which informs you how many days have elapsed since you
last ran a validation procedure.
If the calibration process failed, check the MCV Plate IV to verify that the
correct beads were added to the wells. Then perform a fluidics wash and
repeat the above steps. For more information, consult the Troubleshooting
chapter.
Logging the Calibration
The Calibration Log provides a list of past calibration dates and times, results,
instrument settings, and other data obtained by Bio-Plex Manager. These data
are stored in a log file called bioplexdata.mdb. By default, the file is saved in
the main Bio-Plex Manager application folder on your computer.
NOTE: This database is not compatible with versions of Bio-Plex Manager earlier
than 4.0. If you have an earlier version of Bio-Plex Manager, installing version 4.0
or later copies data from your existing database (bioplex.mdb) into the new
database. A copy of the old database remains in the application folder.
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Calibration
To open the Calibration Log, go to the View menu and select Calibration Log.
The Calibration Log viewer opens.
Figure 21. Calibration Log viewer
Each calibration is listed by date and time. If you are using Bio-Plex Manager
Security Edition in Secure Mode, the User and Access Level columns list
information about the user who was logged into the application when each
calibration was performed. The Result column notes the result of each
calibration, including whether it passed or failed.
NOTE: If you see a drastic change in a detector's voltage from one calibration
to the next, it could indicate a problem with the instrument. A steadily
increasing detector voltage may indicate that the laser is decreasing in
intensity.
On the toolbar, click Calibration Detail
to display additional information
for each calibration, including bead count, system temperature and pressure,
and the reader software and firmware versions. Click Calibration Log
return to the default view.
to
For CAL1-only calibrations, the RP1 column displays an entry of NA (not
applicable). For CAL2-only calibrations, the DD, CL1, and CL2 columns
display entries of NA.
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Bio-Plex Manager Software 6.1 User Guide | Controlling the System
To print the calibration report, select Print from the File menu. Print Preview
displays the report as it will appear in a printout. Print Setup allows you to
select some standard print settings.
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Start Up and Calibrate
Start Up and Calibrate
Each time you use Bio-Plex Manager, the array reader must go through the
start up, warm up, and calibration procedures. The Start up & Calibrate
command allows you to perform these steps with a single command. Click the
Start up & Calibrate button on the Quick Guide or main toolbar, or select the
command from the Instrument menu.
Refer to the following for more information on the individual functions:
• Start Up on page 27
• Optics Warm Up and Shut Down on page 28
• Calibration on page 29
If you prefer, you may perform each function separately. The individual
functions are available on the main toolbar or from the Instrument menu.
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Bio-Plex Manager Software 6.1 User Guide | Controlling the System
Wash Between Plates
You should wash the fluidics lines between each plate reading to prevent
traces of sample or other debris from building up inside the system.
Click the Wash Between Plates button
on the Quick Guide or main
toolbar, or select the command from the Instrument menu. A dialog box
guides you through the steps for preparing the MCV Plate IV for a wash
procedure.
Figure 22. Steps for washing between plate readings
Add 70% isopropanol and distilled water to the appropriate reservoirs, insert
the plate in the microplate platform, and click OK. This procedure performs a
series of fluidics operations and takes several minutes. After initial system
pressurization (5 to 20 seconds), the time remaining in the operation displays
in the Bio-Plex Manager status bar.
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Remove Air Bubbles
Remove Air Bubbles
Microscopic air bubbles in the cuvette may cause a sudden shift in the bead
regions during an assay reading. If the array reader detects such a shift, the
reading stops and you are prompted to perform an alcohol wash to force air
bubbles out of the system.
Click the Remove Bubbles button
on the main toolbar or select the
command from the Instrument menu. A dialog box guides you through the
steps for preparing the MCV Plate IV for the procedure.
Figure 23. Steps for removing air bubbles from the fluidics system
Add 70% isopropanol and distilled water to the appropriate reservoirs, insert
the plate in the microplate platform, and click OK. This procedure performs a
series of fluidics operations and takes several minutes. After initial system
pressurization (5 to 20 seconds), the time remaining in the operation displays
in the Bio-Plex Manager status bar.
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Bio-Plex Manager Software 6.1 User Guide | Controlling the System
Unclog
If the array reader detects an unusually low bead count during a reading, you
are prompted to perform an Unclog operation to remove possible
obstructions from the fluidics lines.
1. Click the Unclog button
on the main toolbar or select the command
from the Instrument menu. An instruction box guides you through
preparation of the MCV Plate IV.
Figure 24. Steps for unclogging the fluidics system
2. Add 70% isopropanol solution and distilled water to the appropriate
reservoirs.
3. Add 5 drops of CAL1 beads to the CAL1 reservoir.
4. Insert the plate in the microplate platform, and click OK.
This procedure performs a series of fluidics operations and reads a sample of
CAL1 beads to verify that the fluidics are operating properly. It takes several
minutes. After initial system pressurization (5 to 20 seconds), the time
remaining in the operation displays in the Bio-Plex Manager status bar.
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Validation
Validation
Validation of the array reader is a formal process for documenting that the
instrument is fit for its intended use and that it is kept in a state of
maintenance and calibration.
NOTE: Validation is performed after startup and calibration have been
performed. Validation is dependent on successful calibration for accuracy.
You should perform a validation reading:
• Once a month
• Each time you move the array reader, or
• To diagnose any problems with the array reader that cannot be
solved by other procedures (calibrating, washing, unclogging, etc.)
Validation Kit
Bio-Plex Manager requires the use of Bio-Plex Validation Kit 4.0 to perform
validation. It must be used in conjunction with the Bio-Plex MCV Plate IV.
Each Bio-Plex Validation Kit consists of reagents and procedures to evaluate:
• Optical alignment
• Reporter channel performance
• Efficiency of multiplexing
• Integrity of fluidics
The Bio-Plex Validation Kit validates the operation of all of the primary
components of the array system, and can also be used to discriminate
between assay and instrumentation problems.
Validation Kit Control Number
Each Bio-Plex Validation Kit has a control number linked to the specifications
for the kit. You must use the mini-CD included in the kit to install the control
number on your computer. After installation, you can select the control
number in Bio-Plex Manager, as described in the following section.
See the Bio-Plex Validation Kit 4.0 Instruction Manual for more information.
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Setting Up a Validation Run
To set up a validation reading, click the Validation button
on the main
toolbar or select the command from the Instrument menu. The Validation
dialog box opens.
Figure 25. Validation dialog
Enter your user name in the top field. If you are using the Security Edition of
the software in Secure Mode, your user name is listed and grayed out.
Control Number Selection
In the Validation dialog box, select the Control Number for your specific BioPlex Validation Kit from the Control Number pulldown list. The expiration date
for the control number is listed below the field.
If the control number is not available in the list, or the list is too long and you
want to limit it to the control numbers you are using, click the Add/Remove
button.
Figure 26. Add/Remove Control Numbers dialog
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Validation
The Add/Remove Control Numbers dialog lists all the available validation
control numbers. To make control numbers available in the Validate dialog
pulldown list, add them to the Selected List. To select all the numbers in the
Available List, click the Add All>> button. To add control numbers individually,
double-click them, or select multiple numbers using Shift + Click and Ctrl +
Click key combinations, and click the Add>> button. Use the <<Remove and
<<Remove All buttons to unselect them. At least one control number must
remain in the Selected List.
When you click OK, the control numbers in the Selected List are added to the
pulldown list in the Validate dialog.
To view the specifications for a control number, select the number from the
pulldown list and click the Show Specifications button. The Control Number
Specifications dialog box lists the validation specifications.
Figure 27. Control Number Specifications dialog
Validation Type Selection
In the Validate dialog, select the type of validation you want to perform using
the Validation Type option buttons: All, Optics
and Classify
, Fluidics
, Reporter
,
.
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Performing a Validation Run
When you have made your selections in the Validation dialog, click OK. The
dialog box for the selected type of validation opens.
Figure 28. Dialog box for performing All Validation procedures
See the Bio-Plex Validation Kit 4.0 manual for information about the bottles in
the Validation Kit, and instructions on loading the Bio-Plex MCV Plate IV for
validation. When you have prepared the plate according to the instructions,
click Eject/Retract Plate in the dialog box, place the plate on the plate carrier,
and click OK.
Validation Results
After a run, the Validation Results dialog box automatically opens. You can
also view this dialog by going to the View menu and selecting Validation
Results.
Figure 29. Validation Results dialog
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Validation
The Validation Results dialog box includes tabs for each type of validation. If
you selected and performed All Validations, all the tabs will be available.
Otherwise, only the tab for the validation you performed (Optics, Fluidics,
Reporter, or Classify) will be available and displayed.
Click Create Report to generate and display a Microsoft Excel spreadsheet
showing the validation results. Note that Microsoft Excel 2000 or higher must
be installed on your computer for this function to work. (Excel comes
preinstalled on all computers supplied with the Bio-Plex suspension array
system.)
See the Bio-Plex Validation Kit 4.0 Instruction Manual for interpreting the
results of a validation run.
Validation Log
Each validation run is recorded in the Validation Log. This log is stored in a
secure database file called bioplexdata.mdb. By default, this file is saved in
the main Bio-Plex Manager application folder on your computer.
NOTE: If you have an earlier version of Bio-Plex Manager, installing version
4.0 or later will copy the data from your existing database (bioplex.mdb) into
the new database. A copy of your old database will remain in the application
folder.
To view this log, go to the View menu and select Validation Log.
Figure 30. Validation Log Viewer
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The validation results are listed by date and time in the upper window, and the
specifications associated with each Control Number are listed in the lower
window. If you are using Bio-Plex Manager Security Edition in Secure Mode,
the User and Access Level columns list information about the user who was
logged into the application when each validation was performed.
Use the buttons in the upper left corner of the window to view the different
validation types (Optics
, Fluidics
, Reporter
, and Classify
To print the Results window, click the Print Report Results button
).
.
To print the Specifications window, click the Print Specifications button
.
To show or hide the specifications data in the Validation Log viewer, click the
Show/Hide Specifications Window button
.
To create a spreadsheet report for a particular validation run, click the row for
the run in the Results window. It will appear selected, as will the specifications
associated with the Control Number for that run in the lower window.
Then click Create Report
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Validation
A Microsoft Excel spreadsheet is automatically generated and displayed
showing all the validation data for that run. Note that Microsoft Excel 2000 or
higher must be installed on your computer for this function to work.
Figure 31. Validation results spreadsheet (Microsoft Excel)
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Platform Heater
The microplate platform has a heater for warming samples. The heater has an
operating range of 35ºC to 60ºC (95ºF to 140ºF), and can be controlled
through Bio-Plex Manager.
To access the heater controls, select Platform Heater from the Instrument
menu, or click the button on the main toolbar
.
Figure 32. Platform Heater dialog
In the Platform Heater dialog, click the Turn Heater on checkbox to turn on the
heater, and set the target temperature within the range 35 to 60ºC using the
scroll buttons.
To automatically turn the heater off at the end of a run, select the Turn Heater
off after run checkbox.
The dialog box and the Bio-Plex Manager status bar indicate the current
platform temperature. The status bar also indicates whether the heater is
turned on.
Figure 33. Status bar indicating the heater is on
If the heater is in the process of warming up or cooling down, the temperature
is shown in red. If the heater is on and has reached the specified temperature,
the temperature is shown in green. If the heater is off and is at ambient
temperature, the temperature is displayed in black.
NOTE: If you initiate a run before the heater has warmed up or cooled down
(that is, the temperature is shown in red), you have the option of waiting until
the specified temperature is reached or proceeding with the run immediately.
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Instrument Information
Instrument Information
The array reader continuously monitors its internal temperature, voltage,
pressure, and other systems for diagnostic purposes. You can access this
information by clicking the Information button
selecting Information from the Instrument menu.
on the main toolbar or
Figure 34. Instrument Information dialog
Click the tabs in the dialog to access information about the instrument. See
the hardware manual for detailed instrument specifications.
Eject/Retract Plate
The Eject/Retract Plate command
on the main toolbar and Instrument
menu ejects and retracts the plate carrier in the microplate platform.
Note that every function that requires insertion of a plate into the platform (for
example, startup, wash, calibrate, etc.) includes an Eject/Retract button in its
dialog box.
Additional Instrument Functions
The Additional Functions submenu under the Instrument menu contains
secondary instrument fluidics functions. Selecting one of these commands
opens a dialog box to guide you through the procedure. Many of these
functions require the use of the Bio-Plex MCV Plate IV.
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NOTE: Major fluidics functions, such as Wash Between Plates, Remove
Bubbles, and Unclog, consist of combinations of the following functions.
To perform each of the following functions, select it from the Additional
Functions submenu of the Instrument menu and follow the steps in the dialog
box that appears.
WASH
The Wash command performs a single fluidics system wash using distilled or
deionized water. It can be used to remove traces of sample in the fluidics
pathway. Note that this procedure is less comprehensive than Wash Between
Plates (page 38).
DRAIN
This command completely drains the fluidics system of the array reader—for
example, if you need to move the instrument. Make sure you have enough
room in your waste fluid container to prevent overflow.
PRIME
This command primes the fluidics system with sheath fluid.
SANITIZE
This command performs a comprehensive cleansing of the fluidics system
using 10% bleach.
ALCOHOL WASH
This command performs a single fluidics system wash using 70%
isopropanol.
ALCOHOL FLUSH
This command performs a comprehensive cleaning of the fluidics system
using 70% isopropanol.
BACK FLUSH
This command flushes sheath fluid through the fluidics system in the opposite
direction of sample flow, to remove particle obstructions from the cuvette.
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Shut Down
Cancel Operation
The Cancel button, located in the status bar at the bottom of the Bio-Plex
Manager window, can be used to cancel many of the instrument functions,
including calibration, validation, or any of the fluidics functions. Click the
Cancel button to cancel the current operation. An alert box prompts you to
confirm the cancellation.
Figure 35. Cancel button in the status bar
Shut Down
The shutdown procedure consists of a series of functions designed to clean
the fluidics lines and prevent a buildup of debris within the system.
To initiate this process, select Shut Down
from the main toolbar or
Instrument menu, and follow the instructions in the dialog box.
Figure 36. Shut Down dialog
Instrument Operations Log
Each instrument operation you perform with Bio-Plex Manager is recorded in
the Instrument Operations Log. This log is stored in a secure database file
called bioplexdata.mdb. By default, this log is saved in the main Bio-Plex
Manager application folder on your computer.
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NOTES: If you install Bio-Plex Manager 4.0 or later, the data from your
existing database (bioplex.mdb) copies into the new database
(bioplexdata.mdb). A copy of your old database will remain in the application
folder.
This Instrument Operations Log did not exist in Bio-Plex Manager versions
before 4.0, so if you are installing Bio-Plex Manager 6.1 over versions earlier
than 4.0, no Instrument Operations data will be copied.
To view this log, go to the View menu and select Instrument Operations Log.
The log viewer opens.
Figure 37. Instrument Operations Log Viewer
Each instrument operation is listed by date and time. If you are using Bio-Plex
Manager Security Edition in Secure Mode, the User and Access Level
columns list information about the user who was logged into the application
when each operation was performed.
To print the log, click the Print button
.
To copy the information in the log to the Windows clipboard and paste it into
another document, drag your cursor over the rows and columns in the table to
select them, and use the Ctrl + C key command to copy the data.
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Bio-Plex Manager Software 6.1 User Guide
6
Preparing Protocols
Protocol Files
A protocol file contains the parameters of a Bio-Plex Manager™ 6.1 software
reading. It specifies the analytes used in the reading, the plate wells to be
read, sample information, the values of standards and controls, and
instrument settings.
When the reading is complete, the parameters in the protocol file are copied
into the Results file, along with the data from the reading.
You can save protocol files and reuse or modify them for other readings. Raw
data from the most recent reading is stored in the protocol file, and you can
generate a new Results file from this data.
Creating/Opening Protocol Files
To create a new protocol file, click the New Protocol button
on the main
toolbar or Quick Guide, or select New Protocol from the File menu. A new
Protocol window opens.
To open an existing protocol file, click the Open Protocol button
on the
Quick Guide or main toolbar, or select Open Protocol from the File menu.
Locate and select the file using the standard Windows Open dialog.
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Standard protocol files have the file extension *.pbx. Secure protocol files,
created using Bio-Plex Manager Security Edition, have the file extension
*.spbx.
Multi-assay Standard Edition Protocols (see page 56 for more information)
have the file extension *.mpbx, and those saved with the Security Edition have
the file extension *.spbx.
NOTES:
• Secure protocol files can be opened only as read-only files using
the Standard (non-Security) Edition version of Bio-Plex Manager
• Protocol files created or modified by the current Bio-Plex Manager
cannot be opened by earlier versions of the software
You can generate a new protocol file from the settings stored in a Results file
(see page 143 for information about Results files). The new protocol may
include the data from the Results file or not, depending on the option you
select. From the File menu, select New Protocol from Results - Exclude Data,
or select New Protocol from Results - Include Data.
Saving Protocols
To save the settings in a protocol, click the Save button
toolbar or select Save from the File menu.
on the main
After you have performed a reading (see Running the Protocol on page 108),
the data from that reading are also saved with the protocol file. These data are
overwritten in the protocol file whenever you rerun the protocol. To preserve
the data from multiple readings, you must save separate Results files (see
page 144).
Use Save As on the File menu to save a protocol under a different name. You
can create a new protocol by opening an existing protocol, modifying the
settings, selecting Save As, and specifying a new name for the modified
protocol.
Reducing the File Size of Protocols
Protocol files that include all the data from a reading can be very large
(>5 MB). Much of this file size is due to the raw bead event data from the
reading, which is used to generate the bead map and histogram displays. If
you require a smaller file size (for instance, to send the protocol file via email),
you can save a copy of the file without this raw bead event data, but with the
other numerical data from the reading intact.
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Protocol Files
NOTE: We strongly recommend keeping a copy of the original protocol file
with the raw bead event data intact. These data are necessary for changing
the DD gate range in the Results file, and can also be exported in XML file
format from the Results file for further analysis.
In the Save As dialog box, select the Compressed mode checkbox to save the
protocol file without the bead map and histogram data. All other data are
preserved.
Figure 38. Save As dialog for protocols
NOTE: This compressed file option is not available for secure protocol files
generated using the Security Edition of the software.
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Multi-Assay Protocols
Bio-Plex Manager 6.1 has the ability to run multiple assays at one time. You
describe each assay, using Describe Protocol, in the same way as describing
single assays.
If you have chosen New Multi-Assay Protocol from the File menu, the
information you enter for each assay becomes its own tab, as outlined in red
in the figure below.
Figure 39. New Multi-Assay Protocol tabs
When an assay tab is clicked, the window displays all information connected
with the corresponding independent assay.
The assay number of the assay being displayed also appears in the title bar.
Uses for Multi-Assay Protocols
This functionality allows you to test the same, or different, samples with two or
more separate assays simultaneously. You can also run the same assay under
different conditions; for example, when you are in the optimization phase of
assay development.
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Multi-Assay Protocols
Creating a Multi-Assay Protocol
1. Choose New Multi-Assay Protocol from the File menu. This action
creates two tabs on the left pane, called Assay 1 and Assay 2.
2. Enter the protocol settings for Assay 1, then save.
3. Click on the Assay 2 tab, currently located at the bottom of the left
pane. It will jump to a position under the tab for Assay 1.
4. Enter the protocol settings for the second assay into the right hand
pane, then Save.
NOTE: Results of multi-assay protocols are always stored in separate results
files. Each assay results file is stored under its own descriptive name, such as
Bio-Plex Pro Human Group II.rbx.
NOTE: Choosing the New Results from Protocol command from the File menu
will generate results only for the assay that is currently open.
Changing a Protocol from Single to
Multi-Assay
A Multi-Assay Protocol can also be created from a single assay protocol.
1. Open the single assay protocol and right click on the Protocol Settings
tab in the left pane of the Protocol window.
2. Select Add Assay. A message will open telling you that a new MultiAssay Protocol must be created.
3. Select Yes, then complete the protocol settings for each assay.
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Protocol Window
The Protocol window steps you through a series of windows..
Figure 40. Protocol window
The buttons are numbered to guide you through defining your protocol:
1. Describe Protocol (optional)
2. Select Analytes
3. Format Plate
(see page 59).
(see page 68).
4. Enter Standards Info
5. Enter Controls Info
(see page 59).
(see page 78).
(see page 96).
6. Enter Samples Info (optional)
7. Run Protocol
58
(see page 108).
(see page 98).
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Protocol Window
Click each setting button and enter the necessary information, then select the
next setting. After all the settings have been specified, you are ready to run
the protocol.
Step 1. Describe Protocol
The Protocol description window displays when you first open a protocol.
(Otherwise, click the Describe Protocol button to display this window.)
Entering a description of your protocol is optional.
If you are using the Standard Edition of the software, the Author field contains
your computer login name. You can enter a new name in the field, if desired,
and a description of the assay reading. If you are using the Security Edition,
the field contains your user name, which cannot be changed. Enter a
description of the assay reading in the Description field.
The Assay Lot field can be used to maintain a record of the lot number of the
assay. If you are using the Security Edition, Supervisor-level users are the only
users who can edit the Assay Lot field in the Describe Protocol window. If you
want to control the lot number associated with a protocol, enter it on the
Describe Protocol window. Then, whenever the protocol is run, the assigned
lot number will be associated with the results. If, however, the protocol will be
used with different assays, then leave the field blank on the Describe Protocol
window. Any users who run the protocol will be able to enter the assay lot
number for that particular experiment in the Run Protocol window, and this
information gets stored with the results.
Supervisor-level users can change the Assay Lot value in both the Describe
Protocol and Run Protocol windows. It is assumed that when a value is
entered in the Describe Protocol window, the purpose is to associate a
particular lot number with both the protocol and the results, and the Assay Lot
field is synchronized in both places. If the Supervisor then changes the value
in the Run Protocol window, the value in the Describe Protocol window is
updated with this new value.
If you are using the Standard Edition of the software, there is no restriction on
who may edit the Assay Lot field when setting up or running the protocol.
Step 2. Select Analytes
In this step, you select the analytes that you want reported in the reading.
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NOTE: During a reading, the array reader always detects all the analytes in the
sample, including any you have not selected. However, analytes that were
detected but not selected will, by default, not appear in the final reports and
tables. After a reading, you can go back and correct your selections in the
Protocol window and/or the Results file, and the detected analytes will appear
in the tables.
Click the Select Analytes button to view the analyte selection window.
Figure 41. Select Analytes window
Bio-Plex Manager groups analytes by panels. Preconfigured panels of
analytes are built into the software; these panels correspond to off-the-shelf
Bio-Plex assays, and include human, mouse, and rat cytokines and
phosphoproteins, plus the newer human angiogenesis, diabetes, isotyping
and acute phase assays. You can add to these existing panels of analytes, or
create new panels that contain only the analytes you use in your experiments.
Bio-Plex cytokine, phosphoprotein, and total target assays are available as
custom-mixed x-Plex™ assays. For repeat sample testing, these multiplex
assays are offered in one or ten 96-well plate formats. Select from the current
list of available assays, and Bio-Rad will mix and quality-test the coupled
beads and detection antibodies to order. You can obtain product information
and a catalog number for your custom-designed x-Plex assay by going to
www.bio-rad.com/bio-plex/x-plex.
First select the panel of analytes that you want reported, then select the
specific analytes.
SELECTING ANALYTES
Go to the Panel pulldown menu and select from the list of preconfigured
panels. The analytes in the selected panel display in the Available list.
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Protocol Window
Each analyte is listed by name and region number. The region number refers
to the region of a fluorescent color map used to identify the analyte's bead
set. Each bead set is embedded with specific quantities of two fluorescent
dyes; the combination of these fluorochromes, as detected by the array
reader, places the bead set within a unique region on the color map, thereby
identifying the set and its associated analyte.
To move an individual analyte into the Selected list, double-click it. Doubleclick an analyte in the Selected list to move it back.
Hold down the Shift or Ctrl key and click multiple analytes in the Available list
to select them as a group. Then click the Add >> button to move them over to
the Selected list. Use the << Remove button to return multiple selected
analytes back to the Available list.
Use Add All>> to move all the available analytes into the Selected list, and
<<Remove All to move them back.
Figure 42. Selecting the analytes to be reported
NOTE: Some analytes share the same region. Only one analyte per region can
be added to the Selected list.
CUSTOMIZING ANALYTES AND PANELS
Bio-Plex Manager comes preconfigured with a selection of panels and
analytes that Bio-Rad provides as Bio-Plex assays. You can edit these and/or
create new ones.
NOTE: If you have assay kits for analytes that are not included in Bio-Plex
Manager, you must add these analytes using the commands described in this
section.
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CREATING A NEW PANEL OF ANALYTES
Click the Add Panel button
panel of analytes.
in the Select Analytes toolbar to create a new
Figure 43. Creating a new panel of analytes
Enter a name for the new panel in the top
field. Select the matching Assay type
from the Assay drop-down menu, which
is used to ensure the correct defaults.
Then click Add to add the analytes. The
Add Analyte dialog box opens.
Figure 44. Adding a new analyte
Enter the bead region number of the first analyte in the Region field, and the
analyte name in the Name field.
NOTE: The bead region number must be correct for proper detection of
analytes. Confirm this number is correct before proceeding.
Click Add Continue to add the analyte to the panel and continue adding more
analytes. When you have entered your last analyte, click the Add button to
add it to the list and close the Add Analyte dialog.
After you add several analytes to the panel you are creating, you can use the
Sort buttons at the bottom of the Add Panel dialog to sort them.
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Protocol Window
Click an analyte in the list to
select it, then click the
appropriate arrow to move it up
or down in the list.
Figure 45. Sorting analytes
You can also remove an analyte
from the list by selecting it and
clicking the Remove button, or
you can remove all analytes, by
clicking the Remove All button.
To edit an analyte in the list, select it, then click the Edit button. A dialog box
opens in which you can change the analyte region and name.
When you are finished creating the panel, click OK to save your changes and
return to the Protocol window.
IMPORTING A PANEL OF ANALYTES
Follow these steps to import a new panel.
1. Open Excel and create a Regions and Analytes list as shown. You may
choose to include the Panel name, as shown, or omit it.
2. Save this as a CSV file by choosing Save As from the File menu.
Choose CSV (MS-DOS) from the Excel Save as type drop-down list.
Figure 46. Saving a CSV File in Excel
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3. Import this CSV file into Bio-Plex Manager by choosing the import
button in the Add Panel dialog.
Figure 47. Add Panel Dialog Box
4. Navigate through your file system to where you have saved the CSV file.
5. Click on the file to highlight, and press the Open button.
Figure 48. Importing the CSV File
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Protocol Window
6. The new panel will be added to the list of panels in Bio-Plex Manager.
Figure 49. Imported Panel
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EDITING A PANEL OF ANALYTES
To edit an existing panel of analytes, first select it from the Panel pulldown
menu in the Select Analytes window. Then click the Edit Panel button
The Edit Panel dialog box opens.
.
Figure 50. Editing a panel
Use the New, Edit, Remove, Remove All, and Sort buttons as described on the
preceding pages to change the analytes in the panel.
If you change the Panel Name, the changes are saved under the new name,
and the old panel will not be overwritten.
NOTE: The preconfigured Bio-Plex panels (Human Cytokines, Mouse
Cytokines, etc.) can be edited but not overwritten; therefore, you must change
the panel name to preserve your changes. When you are satisfied with your
changes, click OK.
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Protocol Window
DELETING AND RENAMING CUSTOM PANELS
To delete a custom panel of analytes:
1. Select it from the Panel pulldown menu in the Select Analytes window.
2. Click the Remove Panel button
want to delete the panel.
. You are asked if you are sure you
Figure 51. Delete Panel Alert
To rename a custom panel of analytes:
1. Select it from the Panel name pulldown menu.
2. Click the Rename Panel button
.
3. Enter a new name in the pop-up dialog box, and click OK.
Figure 52. Rename Panel Dialog
COMBINING PANELS OF ANALYTES
To combine entire panels of analytes (for example, Human Cytokines I and
Human Cytokines II) without going to the trouble of creating custom panels:
1. Select the panels individually from the pulldown list.
2. Use the Add All>> command to move the analytes from each panel into
the Selected list.
3. Save the entire protocol as you would normally.
Your analyte selections are saved when you save the protocol.
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Step 3. Format Plate
After you have selected analytes, you are ready to specify the format of your
96-well microtiter plate using the plate template in the Protocol window. The
formatting in the plate template tells the array reader which wells to read, and
tells Bio-Plex Manager how to analyze the different sample types in each well.
NOTE: Only formatted wells will be read by the array reader. Be careful to
format all the wells that you want to read before running a protocol.
Click the Format Plate button in the Protocol window to display the plate
template and the formatting controls.
Figure 53. The plate template and formatting toolbar (Plate Formatting view)
The Format Plate window has two views: the Plate Formatting view and the
Plate Groupings view. You can toggle between the views using the tabs above
the plate diagram. As you toggle, the plate template remains the same, but the
commands toggle between formatting controls and grouping controls.
• Plate formatting tools are used to define the types of wells in a
plate (sample, control, standard, blank, etc.)
• Plate groupings tools are used to organize the well types into
groups, with one member of each group defined as the Reference
(or Primary) member, as in a Western blot kinase assay. The ratio of
each member's fluorescent intensity to the fluorescent intensity of
the Reference can then be calculated
NOTE: Plate formatting is required to perform a reading, because the array
reader will read only formatted wells, whereas plate groupings are optional
and can be defined later.
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Protocol Window
PLATE FORMATTING
To format the well types on the plate, select the Plate Formatting tab. Then
use the buttons on the toolbar above the plate template to define the wells on
the microtiter plate. These formatting commands are also located on the
Format Options menu.
Figure 54. Plate formatting tools (Plate Formatting view).
The different well types you can select are:
Unknown sample
Standard
Control
Blank
Undefined*
*Remember that all undefined wells are not read by the array reader.
DEFINING UNKNOWN SAMPLE WELLS
To define wells containing unknown samples, first click the Unknown Sample
button
, then click or drag the wells in the template. Use the Autofill
buttons as described on the following page for defining multiple unknown
wells and/or replicate groups of unknowns.
Figure 55. Defining unknown sample wells
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Note that the cursor changes to an Unknown Sample cursor as you move it
over the template. As you select each well, it is marked with a square and a
number at the center. The square identifies the well as containing an unknown
sample, and the number identifies the specific sample.
The Set Number of Unknown Samples command is available only in the
Security Edition of the software. For more information about the Security
Edition, see the Appendix on page 219.
WELL NUMBERING AND REPLICATE GROUPS
For unknowns, standards and controls, wells with different numbers contain
different samples. Wells with the same number contain the same sample, and
are defined as a replicate group. For each replicate group, a mean value is
calculated in the Report table, along with the standard deviation and
coefficient of variation.
AUTOFILLING WELL NUMBERS
To define multiple single wells as different samples, you can click them
individually, or select Autofill Across
or Autofill Down
cursor over the wells to number them sequentially.
and drag the
Figure 56. Defining multiple sample wells with Autofill
Note that Autofill Across numbers the wells sequentially left to right, then top
to bottom, while Autofill Down numbers them sequentially top to bottom, then
left to right.
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DEFINING A REPLICATE GROUP
To define multiple wells as a replicate group, select Turn Off Autofill
from
the formatting toolbar or Format Options menu, then drag your cursor over
several wells that contain the same sample. The wells are labeled with the
same number.
Figure 57. Defining a replicate group of unknowns
For each replicate group, a mean value is calculated, along with the standard
deviation, standard error, and coefficient of variation.
AUTOFILLING REPLICATE GROUPS
If you have multiple replicate groups of wells in rows or columns, you can
autofill these by selecting Autofill Across or Autofill Down as before, and then
entering the number of wells in the replicate group (up to 96) in the number
field next to the autofill buttons. (You can also select well numbers up to 10
from the pulldown menu.)
Figure 58. Entering the number of replicate wells in an Autofill sequence
Now when you drag your cursor, the sequential sets of replicate wells are
defined.
Figure 59. Autofilling a sequence of replicate groups
For each replicate group, a mean value is calculated, along with the standard
deviation, standard error, and coefficient of variation.
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CHANGING A REPLICATE GROUP
To change the replicate group of a well or group of wells:
1. Click the Select tool
, and click or drag the wells you want to change.
The wells appear highlighted.
2. Right-click the selection, and select Set Replicate Group from the
context menu.
3. In the pop-up box, select the desired replicate group number from the
pulldown list.
4. Click OK. The selection is changed to the specified group number.
Figure 60. Selecting a different replicate group
RESEQUENCING WELL NUMBERS
If your well numbering is out of sequence and you want to correct it, select
Resequence Well Names from the Format Options menu. All the wells in the
plate are renumbered sequentially, based on whether you have selected
Autofill Across or Autofill Down. This is a cosmetic change, and does not
affect the analysis in any way.
DEFINING STANDARD WELLS
Standard wells contain analytes of known concentration, which are used to
generate a standard curve of fluorescence intensity versus analyte
concentration. The regression equation for the curve is then used to calculate
the concentrations of your unknowns.
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To define the wells containing standards in your microtiter plate, click the
Standard button
, then click or drag the wells in the template. Use the
Autofill buttons as described on page 70 to define multiple standard wells
and/or replicate groups of standards.
Figure 61. Defining a row of standard wells
NOTES:
• The cursor changes to a Standard cursor as you move it over the
template. Standard wells are marked in the template by a circle
with a number inside it.
• After you have defined the standard wells, you are ready to enter
the concentrations of the standards as described on page 91.
DEFINING CONTROL WELLS
Control wells contain samples of known concentration; the expected vs.
observed concentrations of the controls can be calculated at the end of the
reading.
To define the wells containing controls in your microtiter plate, click the
Control button
, then click or drag the wells in the template. Use the
Autofill buttons as described on page 70 to define multiple control wells and/
or replicate sets of controls.
Figure 62. Defining a row of control wells
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Note that the cursor changes to a Control cursor as you move it over the
template. Control wells are marked in the template by an octagon with a
number inside it.
NOTE: After you have defined the control wells, you are ready to enter the
concentrations of the controls as described on page 96.
DEFINING BLANK WELLS
In certain types of assays such as the Bio-Plex phosphoprotein assay, it may
be useful to subtract the assay background from the readings of standards,
controls, and unknown samples. To do this, you can prepare "blank" wells
containing all the assay components except sample. Wells prepared as blanks
are read along with the rest of the assay, and then Bio-Plex Manager subtracts
the mean background reading of these wells from the fluorescence intensity
values of the wells containing standards, controls, and unknowns.
To format blank wells in the plate template, click the Blank button
, then
click or drag the blank well(s) in the template. Note that the cursor changes to
a Blank cursor as you move it over the template.
Figure 63. Defining a row of blank wells
Blank wells are marked in the template with a diamond shape containing the
letter B.
Multiple blank wells in a template are treated as a single replicate group. A
mean value, standard deviation, standard error, and coefficient of variation are
calculated for the group, and the mean is used as the background value.
DELETING OR CHANGING WELL FORMATTING
To delete the formatting from a well or set of wells, select Undefined
from
the toolbar or Format Options menu, then click or drag the well(s) you want to
clear. To clear the formatting from all wells in the template, select Clear Plate
from the Format Options menu. To change the formatting of a well or group of
wells, simply select the appropriate tool and overwrite the old formatting, as if
you were defining the wells for the first time.
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Plate Groupings
After you have formatted the wells (see previous section), you can organize
them into groups and identify one member of each group as the Reference, or
Primary member. You can then calculate the ratio of each member's
fluorescence intensity to the fluorescence intensity of its Reference. This type
of analysis is similar to that performed in Western blotting.
Click the Plate Groupings tab to display the plate grouping tools.
Figure 64. Plate Groupings view
The buttons on the toolbar are used to group the wells on the microtiter plate
diagram and identify the Reference well(s). These formatting commands are
also located on the Format Options menu.
Figure 65. Tools in the Plate Groupings view
First, select the ratio you want to calculate for each group — Reference/
Member or Member/Reference — using the Ratio field pulldown list.
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DEFINING A GROUP
To define an assay group, click the Group button
across the wells you want to group.
and drag the cursor
NOTES:
• Wells must be defined as Unknown samples, Standards, Controls,
or Blanks before they can be grouped.
• You can group across well types (for example, a single group can
include Standards, Unknown samples, Controls, and/or Blanks).
• Replicate wells are automatically placed in the same group (even if
you drag over only one well in the replicate set).
Figure 66. Defining a group of wells
The grouped wells change color to indicate their grouping, and the first
member of the group is highlighted to indicate that it is defined as the
Reference well.
Each group you create will be designated with a different color.
To remove individual members of a group or the entire group, click the
Ungroup Samples button
and click or drag over the members/group.
Note that you can also use the Select tool
to first select a group of wells,
and then use the Set Sample Group command on the Format Options menu
or context menu to select the group.
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CONTEXT MENU
Right-click and drag over a number of formatted cells to bring up a context
menu, that allows you to change several settings, including dilution factor,
replicate group, and sample description.
Figure 67. Right-click context menu
DEFINING THE REFERENCE
To change the Reference member of the group, click the Reference
button and click the desired Reference member in the group. For replicates,
all replicate wells of the same number within the group are automatically
designated as the same Reference.
You can also use the Select tool
to first select the Reference well within a
group and then use the Set Sample as Reference Member command on the
right-click context menu to set the Reference.
PRINTING THE PLATE FORMAT
To print the plate format for your records, select Print from the File menu, or
click the Print button
on the main toolbar. For a preview of the printed
format, select Print Preview from the File menu.
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Step 4. Enter Standards Info
This section describes how to enter information about your standards. To
enter information for standard wells defined on the current plate, make sure
the Standards Info tab is selected.
Standards are analytes of known concentration. They are used to generate a
standard curve of values using one of the several regression methods built
into Bio-Plex Manager. This curve is used to calculate the concentrations of
your unknowns.
The standards you select depend on the performance characteristics of your
assay, and the type of curve-fitting model you want to use. Due to variations in
assay preparation, we recommend using a minimum of eight standard
concentrations to generate the standard curve. See the appropriate Bio-Plex
Assay Instruction Manual for recommendations of standard concentrations.
To see what manuals are available, visit www.bio-rad.com/bio-plex.
The minimum number of non-zero standards required for each type of
regression method available in the software is shown in the following table.
Method
Minimum number of standards
Logistic-5PL
6
Logistic-4PL
5
Cubic spine
4
Linear (linear and semi-log)
2
Point to point (linear and semi-log)
2
Before entering the concentration values of your standards, you must select
the analytes that you will be analyzing in the reading as described on page 59.
If your current plate contains your standards, you must also identify the
standard wells as described on page 72.
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Click the Enter Standards Info button in the Protocol Settings pane to enter
information about your standards.
Figure 68. Entering information about standards
The Enter Standards Info window allows you to define standards on your
current microtiter plate, and/or import external standards data from another
plate. Use the tabs above the data fields to select between standards on the
current plate and external standards.
NOTE: You can define standards on the current plate and import external
standards in the same protocol. This is useful if you want to run standards for
some analytes on the current plate, but include external standards for other
analytes in your concentration calculations. Note that if external standards
and current standards are defined for the same analyte, the external
standards will always be used to calculate the concentrations of that analyte.
Standard Lots
You can create, reuse, and import standard lots to ensure repeatable results
between experiments and/or researchers in a workgroup.
This function eliminates the need for you to enter or re-enter this data
manually.
Click the Enter Standards Info button in the Protocol Settings pane. Choose
the Standards Info tab.
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The Standard Concentration Lot window is at the top of the screen.
Figure 69. Standard Concentration Lot window
LOADING A STANDARD LOT INTO YOUR PROTOCOL
If you have previously saved a standard lot or it is otherwise present in your
current version of Bio-Plex Manager, you can quickly load it into your protocol
by using the load function.
Figure 70. Load Standard Lot button
This will result in the following window which will display all the lots loaded
into your version of Bio-Plex Manager. Choose the lot you want to use and
press OK. This will load the concentration and dilution information associated
with this lot.
Figure 71. Load Standard Lot dialog
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MANAGING STANDARD LOTS
You can import new lots, export lots to collaborators, or delete lots on your
Bio-Plex Manager by using the Manage Standard Lots function.
Figure 72. Manage Standard Lots
Pressing the Manage Standard Lots button brings up a dialog that allows you
to import, export and delete standard lots.
Figure 73. Manage Standard Lots screen
Importing Standard Lots—Standard lots will be added continually to the
Bio-Rad web site, which can be accessed in the help menu of Bio-Plex
Manager. Download the lot file (which is in XML format) from the web site to
your PC and then choose Manage Lots (above) and press the Import button
from the resulting window. You can import all or a subset of the lots present in
this file.
Exporting Standard Lots—You can export all or a selected number of lots to
collaborators by pressing the Export button. When you export the lots they
are saved in an XML format which can be sent to other users of Bio-Plex
Manager for import.
Deleting Standard Lots—Unneeded standard lots are deleted by pressing
the Delete button.
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ENTERING STANDARD LOTS MANUALLY
If your lot is not preloaded in the software or you cannot find it on the Bio-Rad
web site, you can enter the information manually from the product insert. You
can save any manually entered standard information as a new standard lot, as
described above.
Kits fall into two categories; those which have various starting concentrations
and those which have the same starting concentration for each analyte.
When starting concentrations vary across all analytes—There are a
number of ways to edit values and apply dilutions. The following outlines the
most efficient approach.
1. Enter the starting concentration of each analyte directly in the
spreadsheet within the interface. Enter each analyte before proceeding
to the next step. Make sure the “Apply same concentration to all
analytes” box is unchecked (default)
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2. Once each analyte’s starting concentration is entered, apply the dilution
factor which is appropriate for your experiment.
Figure 74. Applying the Dilution Factor
3. The software will calculate all the concentrations for each analyte at
each dilution, as seen below.
Figure 75. Bio-Plex Manager calculates concentrations
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When starting concentrations are the same across all analytes—follow
the following steps.
1. Check the box next to Apply same concentration to all analytes. This is
not the default setting.
2. Enter the starting concentration in the box indicated in the image below.
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3. Enter the dilution factor and press calculate.
4. The software calculates all the concentrations for each analyte and at
each dilution, as seen below.
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DELETING STANDARDS
To delete your Standards entries, click in the spreadsheet title called “Std” to
highlight all entries as shown below. Press the Delete button on your keyboard.
Figure 76. Deleting Standards Entries
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SELECTING EXTERNAL STANDARDS
This section describes how to select a Results file from which to import
external standards data into a protocol.
Click the Select External Standards tab. In the window, click the Fill Available
List button, navigate to the directory containing the Results file with your
standards of interest, and select the file. (Note that the Open dialog displays
only Results files.)
Figure 77. Selecting the Results file that contains external standards
The Available External Standards list will be populated with the standards
from the selected file. To add additional standards, repeat the procedure,
selecting a different file.
To delete all standards from the Available list, click the Clear Available List
button.
To make the available external standards accessible in the current protocol,
you must add them to the Selected External Standards list (right-hand pane).
To add all of the standards in the Available column, click the Add All>> button.
To add available standards individually, double-click them, or select multiple
analytes using Shift + click and Ctrl + click key combinations and click the
Add>> button.
(Use the <<Remove and <<Remove All buttons to deselect them.)
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NOTE: Each external standard you select must match the region of a selected
analyte in the protocol. For example, if you selected analytes in regions 19, 32,
52, and 73 in the Select Analytes window (page 59), you can select only
external standards in those same regions. (Note that the analyte name does
not have to match, only the region.) Also, each analyte must come from a
different region.
When you are finished, the Selected External Standards list should include all
the analytes that you want to use as external standards, and their source
file(s).
Figure 78. Selected external standards
Click the External Standards Info tab to enter additional information about
your external standards.
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NOTE: The Standards Info and External Standards Info tabs contain the same
fields, but you cannot alter the concentrations under the External Standards
Info tab (they appear grayed out), as shown in Figure 79.
Figure 79. Standards Info window (top) and External Standards Info window
(bottom)
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The analytes that you selected in the Select Analytes window appear in the
Assign Standards Information table. The most efficient method for entering
standard concentrations is to enter them directly into the table. Enter the
highest concentration into the first row for each analyte.
Figure 80. Selecting a standard analyte
NOTE: For the settings described on the following pages, you can select
different settings for each analyte, or select the same settings for all analytes.
Use the controls shown in the red rectangle to access your standard lots.
To save a Standard Lot for reuse, name it and enter an expiration date, if
required. The calendar under the Expiration Date pulldown menu enables you
to set a specific date.
Save places your new Standard Concentration Lot into the Load Standard Lot
window so it can be reused.
Figure 81. Load Standard Lot window
Load . . . brings up the Load Standard Lot window so that you can choose
between previously-saved standard lots.
Manage Standard Lots . . . brings up the Manage Standard Lots window that
allows you to import or export your standard lots. You can also select any
number of Standard Lots for deletion.
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ENTERING CONCENTRATIONS
(FOR CURRENT STANDARDS ONLY)
To calculate them automatically from a dilution series, select Enter
Automatically.
Enter the high standard concentration (S1) into the first row of the table, for
each analyte. Then select the Dilution Factor, check the Apply dilution to all
analytes box, and click Calculate. The table fills with the standard
concentrations.
If you are using external standards, the concentrations are automatically
entered, and you can skip to the next section on selecting concentration units
(page 93). To enter concentrations, make sure the Standards Info tab is
selected.
ENTERING CONCENTRATIONS MANUALLY
You can enter your concentrations manually. The table lists your standard
wells (S1, S2, S3, etc.) These are the wells that you defined in the Format
Plate window (page 68). For the selected analyte, enter the concentration for
each standard in the concentration column.
Figure 82. Entering standard concentrations manually
If the Apply same concentrations to all analytes checkbox is selected, the
concentration values you enter are applied to all the analyte standards.
Deselect the checkbox if you want to enter different concentrations for each
analyte standard.
You can also enter additional information about your standards in the
description column for each well.
NOTE: You can use the Cut, Copy, and Paste commands on the Edit menu to
copy your descriptions and concentrations among analytes. You can also
copy and paste from and to other applications, such as Microsoft Word or
Excel. Click a cell, row, or column in the table to copy and paste.
If you are entering different concentrations for each analyte standard, repeat
the procedure by selecting each analyte from the pulldown menu as described
above.
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CALCULATING CONCENTRATIONS AUTOMATICALLY
If a dilution series was created for the standards, the software can
automatically calculate the concentrations of each standard.
1. Select the appropriate Most Concentrated Standard well number. (The
most concentrated standard must be in either the first standard well or
the last.)
Figure 83. Entering standard concentrations automatically
2. Enter the concentration of the most concentrated standard in the
Concentration of S1 field and the dilution factor for the remaining
standards in the Dilution Factor field.
NOTE: Calculations are made by dividing by the dilution factor, so do not
enter fractional values for the dilution factor. If each standard is half the
concentration of the previous standard, enter 2 for the dilution factor. If each
standard concentration is one-fourth the previous, enter 4, and so on.
3. Click the Calculate button when all the information is entered. The
concentrations in the table are automatically updated.
If the Apply same concentrations to all analytes checkbox, below the
concentrations table, is selected, the concentration values are applied to all
the analyte standards. Deselect the checkbox if you want to enter different
concentrations for each analyte standard, and repeat the steps.
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CONCENTRATION UNITS
You can enter the units of concentration in the Concentration Units field (units
are not required and do not affect calculations). These units appear in your
reports. If all your analytes have the same concentration units, select the
Same Units for All Analytes checkbox.
REGRESSION METHODS
Using the Std Regression Curve pulldown list, choose from among seven
different regression methods to generate the standard curve (page 168):
• Logistic-5 PL
• Logistic-4 PL
• Linear
• Cubic spline
• Point to point
• Linear (semi-log)
• Point to point (semi-log)
Figure 84. Selecting a regression method
You can change your selection later in the Standard Curve window of the
Results file. See page 170 for a more detailed discussion of regression
methods.
You can also select the regression-curve axis scale from four different
combinations of linear and logarithmic scales. Select the axis scale from the
Axis Transformation pulldown list.
Figure 85. Selecting a regression-curve axis transformation
Note that the axis transformation only changes how the curve is displayed,
not the calculated values.
NOTE: The regression method and axis transformation you select in the
Protocol window is used to calculate your initial results. However, these
settings do not affect the reading, and you can change these settings and
recalculate your results after a reading is complete.
Finally, if you want to use different regression methods for different analytes,
deselect the Same Regression Type for All Analytes checkbox. Then, when
you select a different analyte from the pulldown list, you can select a new
regression method as well.
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LOGISTIC WEIGHTING
If you select a logistic curve fitting method (Logistic-5PL or Logistic-4PL), the
software automatically weights the points in the curve. Click the Logistic
Weighting button to access the weighting algorithm controls.
Figure 86. Logistic Weighting dialog
There are eight models for calculating the variance in the logistic weighting
algorithm:
• Linear Variance
• Logarithmic Variance
• Exponential Variance
• Power Law Variance (default)
• Linear CV
• Logarithmic CV
• Exponential CV
• Power Law CV
COEFFICIENT A
Weighting coefficient a will be calculated automatically using the following
equation:
a = .002 * pow ((m_YMax + m_YMin) / 2 + blank,.2)
Alternatively, you can enter your own value by deselecting the Auto calculate
coefficient a checkbox and entering a value in the Coefficient a field.
COEFFICIENT B
Weighting coefficient b has a default value of 1.8. You can enter a different
value in the Coefficient b field.
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RECOVERY PERCENTAGE RANGE
Because the standard curve (page 168) is critical for calculating the
concentrations of your unknowns, Bio-Plex Manager includes a mechanism
for assessing the fit of the curve to the standards. This is the recovery
percentage. See Standard Curve Optimizer on page 199 for how Bio-Plex
Manager incorporates Recovery Range data automatically.
For each analyte standard, an observed concentration is back-calculated from
the standard curve and the fluorescence intensity. This is divided by the
expected concentration as entered in the Enter Standards Info window and
multiplied by 100 to give the recovery percentage:
Recovery percentage = Observed conc/Expected conc * 100
Recovery percentages are also used with controls (page 96) to determine the
overall accuracy of an assay.
In the Enter Standards Info window, use the Acceptable Recovery Percentage
Range pulldown menu to select an acceptable recovery range.
Figure 87. Selecting the recovery percentage range
For example, if you select a range of 70 to 130%, the observed concentration
should be within 70 to 130% of the expected concentration.
The actual recovery rate for each standard and control is shown in the Obs/
Exp * 100 column of the Report table (see page 160). Unknown
concentrations that fall in regions of the curve where standards fall outside the
selected range will be reported as OOR< or OOR> in the Conc in Range
column of the Report table. (See Report Table Error Codes on page 234.)
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Step 5. Enter Controls Info
(Optional)
Controls are samples of known concentration whose values are measured
during a reading in the same manner as the unknown samples. You can then
compare the calculated concentrations of the controls after a reading with the
expected concentrations to determine the overall accuracy of the assay. (This
is sometimes called the "spiked" recovery method, because you are "spiking"
your assay with known quantities of analytes.)
In this step, you enter the concentration values of your controls. First, you
must select the analytes you will be analyzing in the reading as described on
page 59, and identify the plate well(s) containing your controls as described
on page 73.
NOTE: Only formatted wells are read by the array reader. Be sure to define all
your control wells before running a protocol.
Click the Enter Controls Info
information about your controls.
button in the Protocol window to enter
Figure 88. Enter Controls Info dialog
The analytes you selected in the Select Analytes window are available under
the Analyte pulldown menu. Select the first analyte for which you want to
enter controls.
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The table lists your control wells (C1, C2, C3, etc.). These are the wells that
you defined in the Format Plate window (page 68). Enter the concentration of
the selected analyte in the Conc column for each well.
Figure 89. Entering concentrations of the controls
If the Same Conc. Values for All Analytes checkbox is selected, the
concentration values you enter are applied to all the available control analytes.
Deselect the checkbox if you want to enter different concentrations for each
control analyte. You can enter additional information about your controls in the
description column for each well.
If you are performing a dilution of your controls, you can enter a dilution factor
for each control in the Dilution column. Note that a control diluted 1 in 2 would
have a dilution factor of 2. This feature allows for parallelism studies, whereby
a control diluted by different dilution factors is analyzed to verify that the final
concentration is the same for all samples.
Figure 90. Entering Controls with different dilution factors
If you are entering different concentrations for each analyte standard, repeat the
procedure by selecting each analyte from the pulldown menu as described above.
To enter the same dilution factor for all controls, enter the value in the Dilution
Factor field, and click the Set All Dilution Factors button. The dilution factor
you enter for one analyte is automatically applied to all analytes.
NOTE: You can use the Cut, Copy and Paste commands on the Edit menu to
copy your descriptions, concentrations, and/or dilutions between analytes.
You can also copy and paste from and to other applications such as Microsoft
Word or Excel. Click a cell, row, or column in the table to copy and paste.
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Step 6. Enter Sample Info (Optional)
Before entering information about your unknown samples, select the analytes
you are analyzing in the reading, as described on page 59, and then define the
plate well(s) containing your unknown(s), as described on page 69.
NOTE: Only formatted wells are read by the array reader. Be sure to define all
your unknown sample wells before running a protocol.
Click the Enter Sample Info button in the Protocol window to enter information
about your unknown samples.
Figure 91. Enter Sample Info dialog
The information table lists all the unknown sample wells as defined in the plate
template for the reading (page 69).
You can enter a description and a dilution factor for each unknown in the
appropriate columns. Note that a sample diluted 1 in 2 would have a dilution
factor of 2. To enter the same dilution factor for all unknowns, enter the factor
in the Dilution Factor field and click the Set All Dilution Factors button.
NOTE: You can use the Cut, Copy, and Paste commands on the Edit menu to
copy your descriptions and/or dilutions from and to other applications such as
Microsoft Word or Excel. Click a cell, row, or column in the table to copy and
paste.
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7
Running Protocols
After you have prepared the Protocol as described in the previous chapter,
you are ready to run it.
Click the Run Protocol button
settings and run the Protocol.
in the Protocol window to select the final
WARNINGS:
• When performing the following operations, the sheath fluid
container and the waste fluid container should be closely
monitored
• The sheath fluid bottle must be placed at the same level as the
array reader, unless you are using the HTF (High-Throughput
Fluidics, (see the next Warning). The fluid level should be below the
air inlet connection and above the sheath outlet connection. For
more information, refer to the Bio-Plex 200 System Hardware
Instruction Manual. Always check the sheath fluid level before
starting a run or procedure
• If you are using the HTF, it should sit on the counter next to the
array reader, while the sheath fluid cube should be placed ~3–4
feet below the reader (for example, on the floor)
• The waste fluid container receives waste from the system. Do not
allow the waste container to overflow. Empty the waste bottle each
time the sheath fluid bottle is filled. Do not place it on the
instrument. All waste containers should have vented caps.
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Run Protocol Window
The Run Protocol window has controls for performing the run, and two views
for displaying the status of the run: the Raw Data table view and the
Histogram/Bead Map view.
Click the Show/Hide Histogram/Bead Map button
to display only the
Raw Data table, or the histogram/bead map display with the Raw Data table
below it (maximize the window to see both the table and the display).
Figure 92. Run Protocol window—Raw Data table view
Figure 93. Run Protocol window—Histogram/Bead Map view
The Histogram and Bead Map (see page 111) provide graphical data about a
run, and are continually updated throughout the run. The Raw Data table (see
page 119) displays the raw data of the reading after it is complete.
The controls for performing the run are described in the following sections.
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Bead Count
Bead Count
The Beads field in the Run Protocol window lists the number of beads to be
detected before a reading is complete. Note that this number can be beads
detected per region or total beads detected, depending on the pulldown
selection.
If you select per region as the counting method, you can enter a number
between 1 and 10,000 in the Beads field. For most assays, including the
Bio-Plex cytokine assay, we recommend a value of 50 beads per region. This
ensures that at least 50 beads in each region will be acquired before sample
acquisition is complete. If the region has less than 25% of the specified beads
per region or there are fewer than 20 beads (whichever number is greater), a
low bead warning is triggered. If you specify a bead count that is less than 20,
then a low bead warning is triggered any time the bead count is less than the
specified number.
If you select total as the counting method, you can enter a number between 1
and 100,000 in the Beads field. Note that if you choose to measure total
beads, the instrument will stop acquisition as soon as it has acquired the total
number of beads in any combination of bead sets. If you are detecting
multiple analytes, this means that some bead sets may be under represented
in the final tally.
NOTE: For the Bio-Plex™ phosphoprotein assay, we recommend specifying
25 beads per region to avoid triggering a low bead count warning.
Sample Timeout
The Sample Timeout field works in conjunction with the Beads field to ensure
that a well reading does not continue for an extended length of time. If the
specified bead count in the Beads field is not reached for a particular well (that
is, because the well does not contain all of the selected analytes), the array
reader will continue the well reading until the time limit specified in the Sample
Timeout field. If no Sample Timeout is specified, a default time based on the
sample volume is used.
You can enter a time limit in seconds from 0 to 200, or make a selection from
the pulldown list. Select <none> or enter 0 if you want no time limit. The
Sample Timeout value for each well will be noted in the Raw Data table.
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Bio-Plex Manager Software 6.1 User Guide | Running Protocols
Reservoir Functions
The Bio-Plex Manager™ 6.1 software reservoir functions allow you to
incorporate standard maintenance processes directly into the Run Protocol.
This enables you to perform the Run Protocol without needing to replace the
microplate with the MCV plate. The maintenance processes you can select
with the Reservoir Functions window are:
• Wash
• Wash between plates
• Prime
• Drain
• Back flush
• Sanitize
• Alcohol wash
• Start up
• Shut down
• Remove bubbles
NOTE: Two of the most common functions users want performed
after the plate is run are Shut Down and Wash Between Plates.
For users’ convenience, these functions can also be selected
directly from the Run Protocol window.
These functions are available to the Protocol before the run and after the run.
When running the Protocol, the status bar indicates which function is in
process.
Figure 94. Reservoir Functions dialog
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Reservoir Functions
Skip Functions
The Run Protocol - Reservoir Functions window contains a drop-down menu
that allows you to skip reservoir functions built into existing Protocols when
they are not necessary.
For instance, if you have paused to troubleshoot before a plate run, and know
that the Protocol calls for the instrument to perform a function you performed
manually, you may choose to skip that function in order to save time.
You can choose to skip the following functions:
• Functions that will happen before a plate run
• Functions that will happen after a plate run
• All instrument functions
NOTE: If you are running multi-assay Protocols, the reservoir functions can be
run between the assays as well.
NOTE: Other MCV functions and operations, such as Calibration, Validation,
Alcohol flush, and Unclog, require using the MCV plate.
The available volume in the reservoir limits the number of times each function
can be performed with a Protocol. The total allowable number of repetitions
for each function includes the total of functions selected Before Plate Run,
Between Assay Run, and After Plate Run tabs.
The following table shows the total allowable number of repetitions for those
individual functions that use reagent from the reservoir.
Reservoir Function
Max. # of Operations
Wash
20
Alcohol Wash
6
Sanitize
2
To access these functions, use the Reservoir Functions button from the Run
Protocol dialog.
In the Reservoir Functions dialog, in the Before Plate Run tab, select a
function in the Available column and move it to the Selected column by using
the right arrow button. Do the same in the After Plate Run and Between Assay
Run tabs. You can change the order of the functions by selecting one and
moving it up or down with the arrow buttons. Click OK.
To save these settings as the default for future Protocols, select the Save As
Default for New Protocol checkbox.
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Standard Curve Optimization
When Protocols are run, by default, the software will optimize the fit of the
standard curves. This can be turned off from the Run Protocol window, if
optimizing is not desired. In Security mode, only users with permissions are
allowed to change this setting. For more information on standard curve
optimization see Standard Curve Optimizer on page 199.
Advanced Run Settings
Click the Advanced Settings button to access additional reader settings and
controls.
Figure 95. Advanced Run Settings dialog
Bead Map Selection
Bio-Plex Manager software identifies each bead in an assay by its
classification region—that is, the region in the bead map where the bead falls,
based on its fluorescence as measured by the classification channels in the
array reader (see page 115). The default bead map is the 100-region map,
which is appropriate for assays that use xMAP microspheres (such as
Bio-Plex assays, most Bio-Plex Pro assays, and Bio-Plex COOH beads). A
25-region map is also available.
The 25-region map includes select regions from the default 100-region map.
The result is that the regions are larger and more spread out. The 25-region
map is not available when creating a new panel, as it applies only to a specific
subset of pre-installed Bio-Rad assays. It will be applied correctly to these
preinstalled assays.
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Advanced Run Settings
Sample Size
The Sample Size is the amount of sample that will be aspirated from the
microplate well by the reader. The default sample size of 50 μl is
recommended. You can specify a sample size as small as 10 μl or as large as
200 μl.
NOTE: To avoid air uptake, make sure the sample needle is properly aligned
(see page 21) and that each well to be read contains at least 125 μl of sample.
The maximum sample volume per well is approximately 140 μl.
(The array reader rinses an additional 160 μl into each well after a reading is
complete; the maximum well capacity is 300 μl.)
Save Options
You can automatically save the Results file generated by the reading. With the
Auto Save After Run checkbox selected, when you click Start to begin a
reading, you are prompted to enter a name for the Results file.
NOTE: In Bio-Plex Manager Security Edition, Secure Mode, this checkbox is
selected and cannot be deselected.
If the Auto Save After Run checkbox is selected, you can also select the Auto
Export After Run checkbox. With this checkbox selected, Bio-Plex Manager
automatically generates an XML file and saves it as specified in the XML
Export Properties dialog).
Sampling Errors
During a reading, the system continuously monitors the flow of beads, the
bead count, the bead regions, and the platform temperature. If the system
detects a problem in any of these areas, it can stop the reading and trigger a
warning. This feature is enabled if any or all of the checkboxes under
Sampling Errors are selected.
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Select the Pause Run If Error Occurs checkbox to select all the possible error
conditions, or select/deselect the individual error condition checkboxes. By
default, these are not selected (that is, the run will not pause for any error
condition).
Figure 96. Sampling Errors checkboxes in Advanced Settings window
NOTE: All errors are logged in the Raw Data and Report tables, whether or not
the checkboxes are selected (see page 120).
The reading errors and their possible causes are listed in the following table.
Error
Possible Cause*
1. Low bead number
Too few beads in the assay; buffer volume in well is too
low; plate was not shaken properly before analysis;
microbubbles in the cuvette.
2. Aggregated beads
Bead clumping in the assay; sheath fluid is empty; waste
reservoir is overfull.
3. Classify efficiency
Microbubbles in the cuvette; beads have become
photobleached; percentage of beads outside the
selected bead region is too high.
4. Region selection
Incorrect beads were selected in the Protocol or assay;
too few beads are present in the assay.
5. Platform temperature
Platform temperature has fluctuated ± 2ºC during the
reading.
*See Troubleshooting on page 215 for a detailed list of reading errors, causes,
and suggested solutions.
With the checkboxes selected, the reading stops and an alert box describes
the problem and suggests steps for correcting it. After you have performed
the suggested steps, you can rerun the reading.
NOTE: If you are rerunning wells in Rerun/Recovery Mode, you may want to
disable the Low bead number checkbox, because wells that have already
been read once contain fewer beads.
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Plate Loading Guidelines
Doublet Discriminator Gates
During a reading, a doublet discriminator (DD) channel measures the amount
of light scatter from particles that flow past the red laser. The light scatter is
directly proportional to particle size, and an internal DD “gate” is designed to
identify particles smaller or larger than a single microsphere, including
microspheres that are clumped or aggregated.
In the Advanced Run Settings dialog, the default DD Gates range for the
100-region map is dependent on the type of bead used. For the 5.5 micron,
non-magnetic, polystyrene beads the gates are 4335–10000, whereas the
gates for the 6.5 micron, magnetic beads are 5000–25000. Some Bio-Rad
assays are built on a larger 8.1 micron, magnetic bead which uses DD gates of
5000–32000.
If you are using different-sized beads or want to change the default settings,
you can change the size of the gate by selecting the Override Gates checkbox
and entering new values in the Low and High fields.
You can also change the range by dragging the red lines on the Run Protocol
window histogram (see page 149).
NOTES:
• The values in the Low and High fields are channel numbers
corresponding to the amount of light scatter from a particle. If you
want to manually change the range, you need to know the
approximate channel numbers corresponding to the size of the
beads you are using
• You can change the DD gate range in the Protocol before a reading
or in the Results file after a reading (see page 113). If you change
the DD gate range in the Results file, a record of the range during
the reading is preserved
• You can set the gates on only the DD channel, not the RP1, CL1, or
CL2 channel
• For Bio-Plex assays or Luminex-type assays from other
manufacturers, the default DD gate settings should work well.
However, if you are developing your own assay, the position of the
DD peak may shift and thus the DD gates may need to be shifted
accordingly. Note that you can adjust the DD gate range after a
reading in the Results file and reanalyze the data
Plate Loading Guidelines
Before starting the run, note the following plate handling guidelines and
warnings:
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WARNINGS:
• Protect your assay microspheres from light. Once photobleached,
the beads are no longer usable. Proper care of microspheres must
be observed to maintain the benefits of your product warranty
• When using a filter plate, avoid touching the bottom of the wells
when wrapping the plate. Sample may wick or leak from the
bottom of the well, causing problems when reading the plate
• Make sure that you have added at least 125 l of sample to all the
wells specified in your plate template before starting a reading. If
the array reader attempts to draw sample from an empty well, air is
sucked into the sample loop and injected into the flow chamber. As
a result, bubbles form in the cuvette and interfere with the analysis.
If this happens, perform a Remove Air Bubbles procedure
(page 39) and rerun the Protocol
• Shake the microplate on a plate shaker for 30 seconds prior to
performing a reading
Running the Protocol
1. When you have selected all of your Protocol options and prepared your
microplate, click Start.
The microplate platform automatically ejects.
NOTE: If you are running the Security Edition, you are prompted
to enter your Electronic Signature after you click Start.
Figure 97. Run Protocol dialog
2. Place the microplate on the microplate platform.
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Running the Protocol
3. In the Save As dialog, enter the name of the Results file, and click Save.
If you selected reservoir functions, the Run Protocol Reservoir Function
window opens.
If you did not select any reservoir functions, skip to Step 5.
4. In the Run Protocol Reservoir Function window, follow the steps
described and choose any functions you want to skip from the Skip
Functions drop-down list. Click Next.
5. Check that the needle height is set for the type of plate you are using,
either Standard or PCR plate.
6. Enter your user name in the field. If you are using the Security Edition of
the software in Secure Mode, your user name will be entered and
cannot be changed.
7. Enter the Assay Lot number. If you are using the Security Edition of the
software and this field is filled in, only a Supervisor or Service user may
change it.
8. If your microplate has a barcode number or other type of ID number,
enter it in the Plate ID field.
9. Click OK to begin the reading.
The following conditions may delay the start of a reading:
• If the array reader optics have not warmed up, the reading is
delayed until warm up is complete
• If the pressure is too low in the fluidics system, you receive a
warning message prompting you to check the sheath fluid level,
hose connections, etc.
• If the platform heater is not at the specified temperature, a warning
message will appear and you will be prompted to wait for the
temperature to reach the specified temperature
When the reading begins, the sample needle lowers, draws sample from a
plate well, and retracts to its original position. The sample is drawn from the
needle into the sample loop and the reading is taken.
After initial system pressurization (5 to 20 seconds), a typical well reading may
take up to 2 minutes; in most cases, individual wells are read in a matter of
seconds.
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Status Bar
As the sample is analyzed, the status of the reading displays in the status bar
at the bottom of the Bio-Plex Manager window.
Figure 98. Status bar during a reading
The status bar provides a running count of both the Number of beads and the
Beads per second. Within these categories, the array reader keeps track of
the following information:
• The Region fields monitor only those beads that fall within one of
the regions you selected in the Protocol (page 60)
• The Gate fields monitor all beads that pass an internal
discriminator gate (DD gate range) designed to eliminate particles
smaller or larger than a microsphere (including microspheres that
are clumped or aggregated)
• The Total fields report all particles measured by the array reader,
including those that do not pass the internal discriminator gate
Manually Stopping a Reading
While the sample is being read, the Start button changes to a Stop button in
the Protocol window. You can click Stop to cancel the reading. An alert box
notifies you that you can generate a report with the incomplete results, or
rerun the Protocol as described on page 123.
Generating Results from a Protocol
The reading stops when you click Stop, when an error stops the reading, or
when all samples have been analyzed. At the end of the reading, the sample
needle lowers and the syringe pump purges the sample loop and needle using
sheath fluid. Then it sends a fraction of sheath fluid (approximately 160 l)
back into each well.
At the end of a complete reading, the Results are automatically calculated and
displayed.
Note that the Results file automatically is saved if you selected Auto Save
After Run (page 105) in the Protocol. Otherwise, you must save your Results
as a separate step (see Saving a Results File on page 144).
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Histogram and Bead Map
To generate a new Results file from a Protocol, select New Results from
Protocol
from the Run Protocol toolbar or the File menu.
Histogram and Bead Map
The histogram and bead map provide a graphical display of the raw data from
a reading. These graphs are continuously updated during the reading in the
Run Protocol window of a Protocol file. They also can be displayed after a
reading in the Raw Data window of a Results file.
The histogram and bead map share window space with the Raw Data table. If
the histogram and bead map are not visible, click the Show/Hide Histogram/
Bead Map button
to display them.
Figure 99. Histogram and bead map
You can see some rows of the Raw Data table below the histogram and bead
map display if you maximize the window.
During a run, the well number being read is displayed in blue in the upper left
corner of the histogram. After the run, select the wells to display by selecting
rows in the table below the graphs.
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When viewing the histogram and bead map, you can choose to display all
analytes, each analyte separately, or all beads that pass through the discriminator gate. Make your selection from the pulldown menu above the histogram.
Figure 100. Selecting the analyte(s) to display
To print the histogram and/or bead map, click the Print Histogram/Bead Map
button
. This opens a small dialog in which you can select the
histogram, bead map, or both to print.
Histogram
The histogram plots the number of events per channel number for the
selected well, analyte(s), and channel type. An event is generated when
particles such as a bead or aggregated beads pass through the path of the
lasers. Each event generates signals in different channels:
• The fluorochromes embedded in each bead generate signals in the
Classification 1 and Classification 2 channels
• The fluorescent molecules bound to the analytes generate a signal
in the Reporter 1 channel
• The light scatter of each bead generates a signal in the Doublet
Discriminator channel. The amount of light scatter is directly
proportional to bead size
The signals are reported as channel numbers. In the histogram, the y-axis
represents the events, and the x-axis represents the channel numbers from 1
to 32,766 for the selected channel type.
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Histogram and Bead Map
DOUBLET DISCRIMINATOR GATE RANGE
The Doublet Discriminator (DD) range specified under the
Run Protocol Advanced Settings (see page 107) is shown
on the histogram as two red lines perpendicular to the xaxis. In the Protocol window, you can change the DD gate
range by positioning your cursor over the red lines and
dragging them to the right or left. This also changes the
range as listed in the Advanced Settings dialog.
NOTE: The DD gate range cannot be changed while the
Protocol is running.
Figure 101. Changing the DD gate range
You can show or hide the gate lines by right-clicking on the
histogram and selecting Show or Hide from the Gate context menu.
To reset the gates to their default values, click the Restore Default Gates
button
above the histogram.
SELECTING THE CHANNEL TYPE
You can change the histogram to display different channel types. Click the
histogram with the right mouse button and select X-Axis from the context
menu.
Figure 102. Histogram right-click context menu
From the X-Axis submenu, select from the following channel types:
• The Doublet Discriminator channel measures the forward light
scatter from particles as they pass through the red laser. Light
scatter correlates to particle size; this channel allows you to
discriminate single beads (“singlets”) from aggregated beads in the
histogram. This is the default selection
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•
•
•
The Reporter 1 channel measures fluorescence from the reporter
molecules bound to the analytes on each bead as they pass
through the green laser. The amount of fluorescence per bead type
is directly proportional to the amount of analyte present in the
assay. This can be useful for viewing the distribution of the
fluorescence signal of a particular analyte
The Classification 1 channel measures fluorescence from the first
classification dye embedded in each bead as it passes through the
red laser
The Classification 2 channel measures fluorescence from the
second classification dye embedded in each bead as it passes
through the red laser
NOTE: Changing the channel type changes the channel numbers and the
histogram display.
SCALING THE HISTOGRAM DATA
If the histogram is too big or too small to fit comfortably within the display
range, click the Auto Scale button
on the histogram toolbar. The height of
the y axis changes to better fit the data. You can also select this command
from the right-click context menu.
Select Set Scale from the right-click histogram context menu to manually set
the range of the y-axis. In the pop-up box, enter the maximum number of
events to display on the y-axis between 20 and 100,000.
The default view of the data is log scale. From the toolbar, click the Log/Linear
button
to change the display from logarithmic to linear. Click again to
return to log scale.
MAGNIFYING AND RESIZING THE HISTOGRAM
Click the Zoom button
on the histogram toolbar to magnify the display.
With Zoom selected, the cursor changes to a hand symbol
when you
position it over the histogram. Drag the hand cursor to view different
magnified regions of the histogram.
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Histogram and Bead Map
Click the Maximize button
to maximize the histogram portion of the
window within the larger Protocol window. Click the Restore button
return the histogram to its default state.
to
Figure 103. Histogram maximized in the Protocol window
Bead Map
The bead color map is a density dot plot of the events in a reading for a
selected well. Different colored dots in the map represent different numbers of
events at those data points. The number of available regions in the bead map
(25 or 100) is listed above the map, and is based on the selection in the
Advanced Settings dialog (see page 104).
In the default bead map, the x-axis is the Classification 1 channel and the yaxis is the Classification 2 channel. These channels measure the embedded
fluorochromes in each bead, which are used to identify the bead set and
corresponding analyte. The resulting data point clusters in the map represent
the different bead sets in the assay and their associated analytes.
NOTE: Each bead set generates a cluster of data points, rather than a single
point, because of minute variations in fluorochrome levels and intensity
among the beads in a set.
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The white areas on the map indicate the expected regions of the selected
analytes. If you move your cursor over these areas, the name of the analyte
appears in a pop up window. The data point clusters should fall within these
regions.
Figure 104. Bead map, with pop-up window indicating the analyte region
under the cursor
If one or more clusters do not fall within the white areas, there may be a
problem with the beads such as photobleaching, or the analyte selections in
the Protocol may be incorrect. If all the data clusters have shifted out of the
white regions, there may be microbubbles in the cuvette. See the
Troubleshooting chapter on page 215 for troubleshooting suggestions.
In this way, the map can be used to confirm that the bead sets are correctly
selected, measured, and identified.
CHANGING THE X- AND Y-AXES OF THE MAP
You can change the channel types of the axes. Right-click the bead map and
select one channel from the x-axis submenu, and the other channel from the
y-axis submenu. The available channels are:
• Doublet Discriminator —This channel measures light scatter from
the beads as they pass through the red laser. Light scatter
correlates to particle size
• Reporter 1 —This channel measures the reporter molecules bound
to the analytes on each bead
• Classification 1—This channel measures the first classification dye
embedded in each bead
• Classification 2—This channel measures the second classification
dye embedded in each bead
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Histogram and Bead Map
If you are developing your own assay, you may want to plot the Reporter 1
channel against the Doublet Discriminator channel to evaluate the distribution
of the reporter signal. This yields information about the specificity of the
antibodies in the assay.
Figure 105. Changing the channel displayed on the x-axis
SCALING THE BEAD MAP DATA
The default view of the data is log scale. From the toolbar, click the Log/Linear
button
log scale.
to change the display from log to linear. Click again to return to
MAGNIFYING AND RESIZING THE BEAD MAP
Click the Zoom button
in the bead map toolbar to magnify the display.
With Zoom selected, the cursor changes to a hand symbol
when you
position it over the map. Drag the hand cursor to view different magnified
regions of the bead map.
Click the Maximize button
to maximize the bead map portion of the
window within the larger Protocol window. Click the Restore button
return the bead map to its default state.
to
Bead Map Display Options
To access additional Bead Map display settings, right-click the bead map and
select Options.
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DENSITY FILTERING
Each colored dot in the bead map represents a certain number of events
measured by the reader at that data point. Different colors are used to
distinguish among different numbers of events. You can adjust the display so
that data points with low numbers of events are not shown, or to set the
number of events at before which the data point color changes.
Figure 106. Bead map display options
NOTE: These settings only change the bead map display, and do not affect
the underlying data in any way.
First, enter a number in the Level Multiplier field. This number is used
exponentially to determine the number of events required before a data point
changes color. For the default value of 2, the color level changes at 2, 4, 8, 16,
etc. events.
If you want to filter lower numbers of events from the display, select the Filter
Levels checkbox and use the arrow keys next to the Display Above Level field
to select the exponent level below which no data points will be displayed. For
example, if the Level Multiplier is set to 2, and the Display Above Level field is
set to 3, data points are not displayed until 8 or more events are registered;
the first color level occurs at 8 events, the second color level occurs at 16
events, the third color level occurs at 32 events, etc.
The color levels in the display are, in ascending order, dark blue, purple, dark
green, light blue, blue, light green, orange, black, and brown.
LOGARITHMIC/LINEAR DISPLAY
Select whether you want to display the bead map data on a logarithmic or
linear scale using these option buttons.
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Raw Data Table
Raw Data Table
The Raw Data table displays the raw numerical data of a reading, as well as all
instrument settings and information related to the system hardware and
software used in the reading (version numbers, voltage, temperature, gain,
etc.).
NOTE: The instrument hardware and software information included in the Run
Info window of previous Bio-Plex Manager versions is now provided in the
Raw Data table.
This table is available in both the Run Protocol window and in the Raw Data
window of the Results file. (Note that you can only print and export the Raw
Data table from a Results file, not a Protocol window.
The first few lines of this table are visible below the bead map and histogram.
Alternatively, hide the histogram/bead map display by clicking on the Show/
Hide Histogram/Bead Map button
to maximize the table.
Figure 107. Raw data from a reading
During a reading, the table is filled in line by line as each well is read. After the
reading is complete, all the data are displayed.
In the table, the fluorescence intensities (FI) of the analytes you selected are
listed for each well number.
NOTE: During a reading, the array reader always detects all the analytes in the
sample, including any that you have not selected. However, analytes that were
not selected in the Protocol are not included in the table, even if they were
detected. After a reading, you can go back and change your selections under
Select Analytes, and the new analytes, if detected, will appear in the table.
For online Help information about the Raw Data table, click the Display Help
for Table Legend button
.
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Bead Statistics
To display statistical data for the individual beads as well as the entire well,
click the Show/Hide Bead Statistics Columns button
on the table
toolbar. The number of beads collected for each analyte displays in
parentheses after the analyte fluorescence intensity.
Figure 108. Analyte fluorescent intensity and bead count
With Show/Hide Bead Statistics Columns selected, the following columns are
also displayed:
• Total—total bead count in the well
• Region—number of beads that fell within defined regions
• Gate—number of beads that passed the DD gate (see page 107)
• % Agg Beads—percentage of aggregated beads in the well
calculated as (100 – [Gate/Total*100])
Figure 109. Well data
NOTE: The number in the Region column is flagged by an asterisk (*) if that
well did not acquire the number of beads per region specified for the reading.
Sampling Error Codes
During a reading, the flow of beads, bead count, bead regions, and platform
temperature are continuously monitored. The Sampling Errors column reports
errors in any of these areas for each analyte. These errors are reported in the
table even if you chose not to pause the run due to an error condition (see
page 105).
The sampling error codes in the column are listed in the following table.
120
Error Code
Indication
1
Low bead number detected in the well
2
Aggregated beads detected in the well
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Raw Data Table
Error Code
Indication
3
Bead classification efficiency problem detected in the well
4
Region selection problem detected in the well
5
Platform temperature problem detected
Raw Data Display Options
To select the columns to display in the Raw Data table, and other display
settings, click the Raw Data Display Options button
command from the Table Options menu.
or select the
Figure 110. Raw Data Display Options dialog
In the Raw Data Display Options dialog, select the columns to display using
the checkboxes. You can also choose between predefined report settings by
selecting Standard or Diagnostic in the Report Scheme field; these selections
display different sets of columns.
Select the Show Bead Region Counts and Show Histogram and Bead Map
checkboxes to set these display options as the default for the Raw Data table.
Select the Save Settings as Default for New Protocol checkbox to use the
selections in this dialog as the default for new Protocol files.
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Set Number Format
In the Raw Data Display Options dialog, click the Set Number Format button
to select the number format for each column in the Raw Data table.
Figure 111. Set Column Number Format dialog
In the dialog, select the column from the pulldown list, then specify the
number of decimal places for the value of that column in the Decimal Places
field. Select the Scientific Notation checkbox to display the number in
scientific notation. Select the Same Number Format for All Columns checkbox
to use the selected number format for all the columns.
NOTE: Changing the number format changes only how the numbers are
displayed and printed, and does not affect calculations in any way.
Click OK to make the changes, or Reset Defaults to return all columns to their
default number formats.
Other Table Formatting
To display the description of each sample in the table, click the Show/Hide
Description Column button
displays.
above the table. The Description column
To automatically size the columns to fit the width of the text, click the Autofit
button
on the toolbar.
Copying the Raw Data
You can copy the contents of the table to the Windows clipboard. Select rows,
columns, cells, or the entire table, then select Copy from the Edit menu. The
values you copy can be pasted into other Windows applications.
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Rerun/Recovery Mode
Rerun/Recovery Mode
After a reading has stopped—because of an error or user intervention, or
because the reading is complete—the Rerun/Recovery Mode checkbox in the
Run Protocol window will be selected. This allows you to rerun some or all of
the samples.
With Rerun/Recovery Mode selected, checkboxes appear next to each well
number in the Run Protocol table. Select the checkboxes next to the wells you
want to read again.
Figure 112. Selecting wells to rerun
NOTE: To rerun wells in a filter plate, you must first vacuum the wells using the
vacuum filter apparatus. The beads remain on top of each filter plug after
vacuuming. Resuspend the beads with 125 μl of resuspension buffer in each
well. See the Bio-Plex Cytokine Assay Manual for detailed instructions.
Each well may be read twice without adversely affecting the results.
Because there are fewer beads in a well that has already been run once, be
sure to select 100 beads per region or fewer in the Run Protocol settings (see
page 101). Also, click the Advanced Settings button and deselect the Low
bead number error condition checkbox (page 104). Otherwise, a low bead
error may stop the reading.
Click Start to begin the reading.
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Bio-Plex Manager Software 6.1 User Guide
8
Using the
Security Edition
Bio-Plex Manager™ Security Edition
Bio-Plex Manager™ 6.1 Security Edition software works in conjunction with
the built-in security features of the Windows XP Professional or Windows 7
operating systems to provide a secure environment for the maintenance,
verification, and tracking of all electronic records generated by the
application. These records include Protocol and Results files, and the
calibration, validation, and instrument operations logs. The tools provided in
the software include:
• Access controls and authority checks via the use of user
identification codes and passwords
• Electronic record security via the use of protected directories
• Time- and date-stamped audit trails
• Electronic signature of electronic records (user authentication)
When properly configured and administered, these tools ensure compliance
with the rules for secure handling of electronic records as outlined in Title 21,
Part 11, of the Code of Federal Regulations (CFR).
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Background on 21 CFR Part 11
Effective August 20, 1997, the United States Food and Drug Administration
(FDA) released Part 11 “Electronic Records; Electronic Signatures” of Title 21
of CFR. This rule states the conditions under which the FDA considers
electronic signatures and electronic records to be trustworthy, reliable, and
equivalent to traditional handwritten signatures.
Bio-Plex Manager Security Edition is designed to enable organizations and
institutions using the Bio-Plex suspension array system (or Luminex system)
to comply with the rules laid out under 21 CFR Part 11. It enables system
administrators to ensure that Bio-Plex Manager operates in compliance with
21 CFR Part 11 within a “closed system.” A closed system is defined as “an
environment in which system access is controlled by the persons who are
responsible for the content of electronic records that are on the system”
(Section 11.3 (b) (4).
NOTES:
•
•
The security controls built into Bio-Plex Manager Security Edition
must be properly configured and administered by the system
administrator(s) in your organization. Otherwise, the system will not
be secure and will not be in compliance with 21 CFR Part 11
Bio-Rad makes no claim that Bio-Plex Manager Security Edition is
CFR-compliant in and of itself, nor does it guarantee compliance
for the user. Your organization must establish policies and standard
operating procedures that work in conjunction with the tools
provided by Bio-Rad to ensure compliance with 21 CFR Part 11
Standard Mode vs. Secure Mode
Bio-Plex Manager Security Edition can run in Standard Mode, in which all the
security and audit trail features are disabled so that software functions like the
standard version of Bio-Plex Manager, or it can run in Secure Mode, with the
security functions enabled. This chapter assumes you are running the
software in Secure Mode, unless otherwise noted.
When you are in Standard Mode, the status bar displays an “unlocked”
symbol. When you are in Secure Mode, the status bar display a “locked”
symbol.
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Installing and Starting Bio-Plex Manager Security Edition
System Requirements
Bio-Plex Manager Security Edition has the same system requirements as the
Standard Edition, except that the security functions are not supported under
the Windows XP Home Edition operating system. The software must be
installed on a computer running the Windows XP Professional or Windows 7
operating system for full Secure Mode functionality.
Installing and Starting Bio-Plex Manager
Security Edition
Security Edition is installed and starts up just like Standard Edition. After
installation, the software will run in Standard Mode until an administrator-level
user (see Security Edition: User Access by Function on page 219 in the
Appendix) enables Secure Mode.
When you start Bio-Plex Manager in Secure Mode, you are prompted to log in
with your user name and password.
NOTE: User names and passwords must be set up on your computer or
computer network as described in the Bio-Plex Manager 6.1 Upgrade and
Configuration Guide before you can use the application in Secure Mode. This
guide is provided on your software CD.
Users, Passwords, and User Levels
In Secure Mode, Bio-Plex Manager Security Edition requires that users log in
with a user name and password to run the software. User names and
passwords are set up by your Windows system administrator as described in
the Bio-Plex Manager 6.1 Upgrade and Configuration Guide.
NOTES:
• Contact your Windows system administrator about issues
regarding your user name and password
• Each user is assigned to a particular user level. There are six user
levels in Bio-Plex Manager Security Edition, and each level gives
the user access to specific features and functions of the software
A table listing the specific features and functions accessible at each user level
is provided on page 219 in the Appendix. The following is a general summary
of each user level.
• Administrator — Users at this level can enable or disable Secure
Mode. Administrator-level users also can view log files. Access to
all other features and functions of the software is restricted
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Bio-Plex Manager Software 6.1 User Guide | Users, Passwords, and User Levels
•
•
•
•
•
Supervisor — Users at this level have full access to all features
and functions of the software, except that they cannot enable or
disable Secure Mode
Service — Users at this level have full access to all features and
functions of the software, except that they cannot enable or disable
Secure Mode
Clinician 2 — Users at this level can perform instrument
operations, run existing Protocols, and view Protocol files, results
files, and log files. They can also change the number of unknown
samples in the plate format (using the Set Number of Unknown
Samples command) and enter sample descriptions. All other
access is restricted
Clinician 1 — Users at this level can perform instrument
operations, run Protocols, and view Protocol files, Results files, and
log files, but cannot change any settings. All other access is
restricted
Reviewer — Users at this level can view and sign Protocol and
Results files, and can view log files. All other access is restricted
User Level Restrictions
If you attempt to select a command or perform an action not allowed by your
user level, you receive a pop-up warning. In addition, many fields and settings
that are restricted by your user level appear grayed out in the application.
User Information
To display information about the current user, including user name, domain,
full name, and access level, go to the Tools menu and select User Info. You
can also click the user name displayed in the application status bar.
Figure 113. User Information dialog
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Enabling and Disabling Secure Mode
Enabling and Disabling Secure Mode
Only users with Administrator-level access can enable or disable Secure
Mode. Enabling or disabling Secure Mode requires that you close all
documents and restart the application.
When Secure Mode is enabled, the status bar displays the locked symbol.
Figure 114. Secure Mode disabled (left) and enabled (right)
To enable Secure Mode, go to the Tools menu and select Preferences. In the
Preferences dialog, select the Enable Secure Mode checkbox to enable
Secure Mode, or deselect the checkbox to disable it.
If your user name and password are set up on a Windows network server,
enter the network domain where they reside in the Domain to Be Used for
Authentication field. Contact your Windows system administrator if you are
unsure of your domain.
NOTE: Local groups can be set up on the local machine and authenticated
using domain users. Click the box to use local user groups for establishing
user security levels.
When you click OK, you are prompted to sign in as an Administrator-level user
to authenticate the change. You then need to restart the software.
User Authentication
Bio-Plex Manager Security Edition requires user authentication (entering a
user name and password) at key points during operation. User authentication
creates an electronic audit trail of user activity that cannot be altered or
deleted.
Figure 115. Log In dialog
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Bio-Plex Manager Software 6.1 User Guide | Electronic Records
Users are required to enter their user name and password when they:
• Start the application
• Start a reading
• Unlock the application
• Sign a document, such as a Protocol or Results file
Different users may authenticate operations. For example, a Clinician
1-level user may be logged into Bio-Plex Manager, but a Supervisor-level user
may enter his user name and password to authenticate a particular reading.
Note that in this case, the Clinician 1-level user will remain logged onto the
application.
Your organization must establish its own guidelines for user authentication of
specific operations.
Electronic Records
Bio-Plex Manager Security Edition supports the creation and control of secure
“electronic records,” as defined by 21 CFR Part 11. In Bio-Plex Manager, the
following are electronic records:
• Protocol files
• Results files
• Calibration Log (included in the bioplexdata.mdb file)
• Validation Log (included in the bioplexdata.mdb file)
• Instrument Operations Log (included in the bioplexdata.mdb file)
These records are accessible only through Bio-Plex Manager and its
associated utilities.
File Security and Validation
The files used by Bio-Plex Manager Security Edition cannot be opened or
edited using other programs. Bio-Plex Manager checks the integrity and
validity of a file each time it opens. If a file becomes damaged or shows signs
of tampering with an outside application, it will not open in Bio-Plex Manager.
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Calibration, Validation, and Instrument Operations Logs
Calibration, Validation, and Instrument
Operations Logs
These logs provide a permanent chronological record of all calibration,
validation, and instrument operations events. You can view, copy, and print
the data from these logs. They are available from the View menu in Bio-Plex
Manager.
NOTE: If the application is in Secure Mode, each event in a log includes the
name and access level of the user who performed/authorized the action. If the
application is in Standard Mode, the event is flagged as having occurred
outside Secure Mode and the access level is labeled as “unrestricted.”
These logs are stored in a database file called bioplexdata.mdb, which by
default is saved in the main Bio-Plex Manager application folder on your
computer (you can specify a different location during installation).
NOTE: The bioplexdata.mdb database is not compatible with versions of
Bio-Plex Manager earlier than 4.0. If you have an earlier version of the
software, installing version 4.0 or later will copy the data from your existing
database (bioplex.mdb) into the new database. A copy of your old database
will remain in the application folder.
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.
Secure Protocol and Results Files
Bio-Plex Manager Security Edition supports the creation of Secure Protocol
and Results files. Secure files are documents that have been electronically
“signed” by a user. Once signed, these files are controlled documents that
cannot be overwritten by Bio-Plex Manager and that preserve a built-in audit
trail of all saved changes.
Unsigned Protocol and Results Files
When you create a new Protocol file in Bio-Plex Manager Security Edition, it is
initially unsigned and is not a secure file. Protocol or Results files created with
previous versions of the software or created in Standard Mode are also
unsigned, uncontrolled documents, and you are notified that these files are
not secure when you open them in Secure Mode. You can save and overwrite
these files without restrictions and no audit trail will be generated for any
changes.
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If you make changes to a signed Protocol or Results file and save them as a
new file, the new file will not be signed; however, the audit trail (see page 137)
will be preserved in the new file and the file will remain a secure file.
NOTE: You cannot generate a new unsigned, uncontrolled Results file in
Secure Mode; all Results files generated in Secure Mode must be signed.
Signed Protocol and Results Files
Protocol and Results files become secure files when they are electronically
signed using a user name and password. Once signed, these files become
read-only; you can make changes to the file, but they must be saved under a
different file name. An audit trail of changes is built into each Secure Protocol
and Results file.
Secure Edition Protocol files have the file extension *.spbx. Secure Edition
Results files have the file extension *.srbx.
To sign a Protocol or Results file, go to the File menu and select Sign
Document. The Electronic Signature dialog will open. This dialog opens
automatically when a Results file is generated from a Protocol file.
Figure 116. Electronic Signature dialog
NOTE: In the dialog, the signing party enters his or her user name and
password in the appropriate fields. (This may be someone other than the
current user.) The reason for signing the document must be entered in the text
field. Typical reasons may include review, approval, responsibility, or
authorship.
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Calibration, Validation, and Instrument Operations Logs
When you click OK, a Save As dialog opens.
Figure 117. Save As dialog for Secure Protocol files
Enter a name for the file or use the default name and click Save.
After a file has been signed, it is flagged as read-only and cannot be modified.
Any subsequent changes must be saved as a new file. Also, any changes
made to a file after it has been signed will generate an audit trail documenting
each change, as described starting on page 137.
A new file generated from a signed file can be signed again to verify and lock
in any changes made to it. As before, the new signed file is flagged as readonly and any subsequent changes must be saved under a new file name.
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Document ID Number and Signature
Each signed Protocol and Results file has a unique document identifier
(Document ID) number. To view this number, go to the File menu and select
Document Properties.
Figure 118. Document Properties dialog
The Document Properties dialog also provides information about the
signature.
The full name of the user who signed the document is displayed in the title bar
of the File window.
Figure 119. Signed and read-only status of file shown in title bar
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Calibration, Validation, and Instrument Operations Logs
All printouts and exports of a signed document also include the signature and
Document ID number.
Figure 120. Document ID and signature in an Excel export file
Adjusting the Number of Unknown
Samples in a Protocol
A Supervisor-level user can create a “master” Protocol file with a defined plate
format, including a set number of unknown sample wells. A Clinician 2-level
user can then open the Protocol and adjust the number of unknown samples
to be read in a particular reading. This enables Clinician 2-level users to use
the same Protocol file in multiple readings with different numbers of samples.
NOTES: Clinician 2-level users do not have access to the other plate
formatting tools in Protocols. See Defining Unknown Sample Wells on
page 69 for information on defining unknown sample wells and replicates in
Protocols.
With the Protocol file open and the Format Plate window displayed, click the
Set Number of Unknown Samples button
in the formatting toolbar or
select the command from the Format Options menu.
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In the Set Number of Samples dialog, the maximum number of unknown
samples defined in the plate template will be noted next to the field.
Figure 121. Set Number of Unknown Samples dialog
The maximum number of unknown samples is determined by the number of
formatted unknown sample wells and the number of wells per replicate group
defined in the template. For example, a template with 48 wells defined as
unknowns and four wells per replicate will have a maximum number of 12
samples.
Enter the number of samples to use in a particular reading in the field and click
OK. The plate format will change to indicate the new number and layout of the
sample wells. Only those wells will be read during a reading.
NOTE: You can specify only the number of unknown samples using this
command—not standards, controls, or blanks.
If you save the Protocol file after changing the number of unknown samples,
the number of samples you specified will become the new maximum number
of samples. To preserve the original number of unknown samples, save the
changed Protocol file under a different name.
Generating a Results File from a Protocol
File
The Results file must be signed before a reading can begin. In Secure Mode,
the Auto Save After Run checkbox in the Run Protocol window (see page 100)
is automatically selected and cannot be deselected. You are prompted to sign
and save the Results file at the start of each reading.
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Audit Trail
NOTE: In Secure Mode, each reading, including reruns of plate wells,
generates a new Results file that cannot be overwritten.
Figure 122. Electronic Signature required for reading Results file
You are also prompted to sign and save the Results file if you select the New
Results from Protocol command from the File menu (see page 143).
Audit Trail
Any changes made or actions performed after a Protocol or Results file has
been signed automatically generates an audit trail documenting each change/
action. If you make changes to a signed, read-only file and save it as a new
file, the audit trail is preserved in the new file (with the signature of the
previous file noted as part of the audit trail). Also, the audit trail for a Protocol
file is preserved in the Results file generated from that Protocol.
All major actions and changes are audited. Examples of auditable actions
include:
• Signing a file
• Changing Protocol settings (plate format, analyte selection, etc.)
• Changing the standard curve regression type, variance model, etc.
• Flagging outliers
• Performing readings (audited in the Results file)
Minor changes that affect only the display, such as selecting different columns
in the Report Table or changing the axis transformation in the Standard Curve,
are not audited.
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Bio-Plex Manager Software 6.1 User Guide | Audit Trail
Each change you make to a signed Protocol or Results file or a new file saved
from a signed file must be documented in the Reason for Change dialog. The
information in this dialog is used to generate the audit trail. The dialog opens
automatically whenever you make a change and either save the document or
switch among subwindows in the Protocol or Results file.
Figure 123. Reason for Change dialog, used to generate the audit trail
The dialog notes the date and time of each auditable action and describes the
change/edit made. You must enter a reason for each change in the bottom
field.
If you have made more than one change, use the Next and Back buttons to
scroll through the changes. When you have entered a reason for each change,
click the Finished button.
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Audit Trail
Viewing the Audit Trail
To view the audit trail for a Protocol or Results file, go to the View menu and
select Audit Trail. The audit trail is displayed in a subwindow of the Protocol or
Results file.
Figure 124. Audit trail for a Protocol file
Figure 125. Audit trail for a Results file
The audit trail notes the date and time of each action, the user, the access
level, the action, a description of the action (automatically generated by the
software), the reason for the action (entered by the user), and the version of
the software.
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Bio-Plex Manager Software 6.1 User Guide | Audit Trail
The audit trail also notes each time the file has been signed. In the example
below, note that each time the file was signed and made read-only, the audit
trail was continued in the new file.
Figure 126. Example of a Protocol audit trail with signatures
To display the complete contents of the Description and Reason cells in the
table, hold your cursor over a table cell and the information for that cell is
displayed in a context pop-up box. Or you can click the Expand Audit Entries
button
in the Audit Trail window toolbar to expand all the cells.
Figure 127. Example of a pop-up information box in the audit trail
To copy the contents of the audit trail to the Windows clipboard, click the
Copy to Clipboard button
click the Print button
in the window toolbar. To print the audit trail,
.
Protected Directories
Your Windows system administrator may set up protected directories on your
computer or computer network for the secure storage of Protocol and Results
files. These protected directories must be set up using Windows tools, and
are keyed to your Windows user name and password (which may be different
than your Bio-Plex Manager user name and password). Consult your system
administrator for information about protected directories.
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Locking Bio-Plex Manager
Locking Bio-Plex Manager
If you are stepping away from the computer and want to prevent anyone else
from using Bio-Plex Manager Security Edition, you can lock the application.
Once locked, the application can only be unlocked by the user who locked it
or a Supervisor-level user.
NOTE: Locking Bio-Plex Manager will not shut down the application or end
any instrument operations that are in progress.
To lock the application, go to the Tools menu and select Lock Application. You
will be notified that the application is locked, and the Log In dialog will be
displayed.
To unlock the application, enter your name and password and click OK.
Logging Off
When you close Bio-Plex Manager Security Edition, you will be automatically
logged off. You can also log off without closing the application.
Note that you cannot log off while the array reader is in operation. Logging off
will close all open documents. You will be prompted to save any changes
before logging off.
To log off, go to the Tools menu and select Log Off Application. You will be
notified that you have successfully logged off and the Log In dialog will be
displayed.
If a Supervisor-level user unlocks the application, that Supervisor-level user
will then be logged in as the current user.
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Bio-Plex Manager Software 6.1 User Guide
9
Analyzing the Results
Results Files
After a reading is complete, a Results file generates and opens automatically.
The Results file contains the data from a reading, the Protocol parameters
used to collect that data, and analysis tools for interpreting the data. It
includes all the information necessary to analyze raw data, including a
description of the assays, well types, concentrations, dilutions, and the
regression method used for calculating the concentrations of unknowns from
standards. It also includes information about the instrument and the run
conditions at each well at the time of data acquisition.
In the Results file, you can change many of the parameters copied from the
original Protocol file—including the plate format, analyte selection, standards
info, etc.—and recalculate the results. However, you can change only the
parameters of wells that have already been read, and you can calculate only
results based on the analytes that were present in the sample.
NOTE: If you selected Auto Save After Run in the Protocol window, the
Results file is automatically saved after a reading. Otherwise, you must save
your Results file as a separate step.
To generate a Results file from an incomplete reading or a previous reading,
open the Protocol file that contains the raw data for that reading and select
New Results from Protocol
menu.
from the Run Protocol toolbar or the File
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Opening a Results File
To open an existing Results file, click the Open Results button
on the
Quick Guide or main toolbar, or select Open Results from the File menu.
Locate and select the file using the usual Windows Open dialog.
Standard Edition Results files have the file extension *.rbx. Secure Edition
Results files have the extension. *.srbx. See Using the Security Edition on
page 125 for more Security Edition information.
NOTES:
• Secure Edition Results files can be opened only as read-only files
using the Standard Edition of Bio-Plex Manager™
• Results files created or modified by the current version of Bio-Plex
Manager cannot be opened by earlier versions of the software
• From Bio-Plex Manager, you can import a Luminex xPONENT file.
For more information, see Importing Luminex xPONENT Output
Files on page 194
Saving a Results File
To save a Results file, with the file open, click the Save button
toolbar or select Save or Save As from the File menu.
on the main
Reducing the Size of a Results File
Results files that include all the data from a reading can be very large
(>5 MB). Much of this file size is due to the raw bead event data from the
reading, which is used to generate the bead map and histogram displays. If
you need a smaller file size (for example, to send via email), you can save a
copy of the file without this raw bead event data, but with the other numerical
data from the reading intact.
NOTE: We strongly recommend keeping a copy of the original Results file with
the raw bead event data intact. These data are necessary for changing the DD
gate range in the Results file (see page 149), and for exporting the data in XML
file format.
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In the Save As dialog box, select the Compressed Mode checkbox to save the
Results file without the bead map and histogram data. All other data are
preserved.
Figure 128. Save As dialog for Results files
NOTE: This compressed file option is not available for Secure Protocol files
generated using the Security Edition of the software.
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Results Window
The Results window is divided into subwindows, each containing different
options for viewing, formatting, and calculating your data.
Figure 129. Results window
Navigate among the different subwindows using the buttons along the left
side of the Results window:
146
•
Raw Data
(see page 150)
•
Report Table
•
Standard Curve
•
Graphing Functions
(see page 153)
(see page 168)
(see page 175)
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Custom Reports
The custom report function is located in the view menu and is active when you
are viewing a results file.
Figure 130. Custom Reports
Choose from available report templates by using the pull down menu, or by
navigating to a folder location.
Figure 131. Custom Reports Dialog
Bio-Rad plans to release custom report templates for specialized applications
periodically via the web. Bio-Plex Manager 6.1 includes the Gene Manager, an
Excel-based analysis application designed for genotype (including SNP) and
Present vs. Absent analysis. It contains its own online help documentation
and is launched from the Custom Report dialog box.
Sample results files with genotype data are present in the Bio-Plex Manager
example file directory. The default directory is
C:\Program Files\Bio-Rad\Bio-Plex Manager\Example Files.
You can save different versions of Gene Manager with your own analysis
preferences, which show up in the list of available reports. This is particularly
useful if you are continually rerunning the same assay.
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Viewing/Changing the Protocol Settings
In the Results window, you can review and in some cases change the plate
formatting, analyte selections, sample concentrations, etc., from the original
Protocol file to display different data or recalculate data in the Results file
tables. These controls are located in the toolbar across the top of the Results
window.
NOTE: Any changes you make in the Results file will not change the original
Protocol file or the data from the reading. They will change only how these
data are displayed in the Results file tables.
Click the Describe Protocol button
in the Results toolbar to review or
change the description of the Protocol, as described on page 59.
Click the Select Analytes button
in the Results toolbar to review the
analyte selection or select different analytes, as described on page 59.
NOTE: During a reading, the array reader always detects all the analytes in the
sample, including any you have not selected. However, analytes that were
detected, but not selected, will not appear in the final reports and tables. After
a reading, you can go back and change your analyte selections, and any
detected analytes will appear in the tables.
Click the Format Plate button
in the Results toolbar to review or change
the formatting of the plate. Plate formatting is described on page 68.
NOTE: Only formatted wells are read by the array reader. You must define all
the wells you want to read before running a Protocol. However, you can
change the formatting of a defined well (for example, from unknown to
standard) after a reading, and the analysis of that well will change.
Click the Enter Controls Info button
in the Results toolbar to review or
change the concentrations of the controls, as described on page 96.
Click the Enter Sample Info button
in the Results toolbar to review or
change the sample information, as described on page 98.
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Changing the Doublet Discriminator Gate
Range
You can change the doublet discriminator (DD) gate range (see page 107) in
the Results file and recalculate the data based on the new range. The original
DD gate range set in the Protocol file and used during the reading is preserved
and can be displayed in the Raw Data table.
To reset the DD gate range, go to the Edit menu, open the Reanalyze Results
submenu, and select Change DD Gates. The Change DD Gates dialog opens,
displaying the histogram and bead map with controls for changing the DD
gate range. (See page 111 for a complete description of other histogram and
bead map controls.)
Figure 132. Change DD Gates dialog.
In the Change DD Gates dialog, first select the well you want to change or
select the Same DD Gates for All Formatted Wells checkbox. Also, you can
select a specific analyte, all gated analytes, or all analytes from the pulldown
menu above the histogram. Then either drag the red dashed lines in the
histogram display to adjust the DD gates, or enter new values in the DD Low
and/or DD High fields. Click the Update Gates button.
When you have made your changes, click OK. The data in the Raw Data table
(see next section) recalculate based on the new settings.
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Raw Data
The Raw Data window in the Results file includes the same histogram, bead
map, and numerical Raw Data table as the Run Protocol window. These
features are described in the previous chapter on the following pages:
• Histogram and bead map—see page 111
• Raw Data table—see page 119
Figure 133. Raw Data window in Results file
The Raw Data window in the Results file includes additional exporting and
printing functions not available in the Protocol window.
Setting the Plate ID
If you have an identification number for the microplate used in a reading, you
can specify the ID number in the Results file. Right-click anywhere in the Raw
Data table and you can select Set Plate ID from the context menu.
Figure 134. Set Plate ID dialog
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Raw Data
In the dialog, enter the plate ID number in the Plate ID field. If you want to
associate the same ID number with all the wells in the reading, select Apply
Plate ID to All Wells checkbox. If you want to associate different ID numbers
with different wells, select the well for this number from the Well pulldown list.
Click OK to save the changes.
The plate ID number is displayed for each well in the Raw Data table.
Exporting the Raw Data
To automatically export your raw data to Microsoft Excel, click the Export to
Excel button
in the toolbar or select the command from the File menu.
This command automatically launches Excel and displays a spreadsheet
containing your data.
Figure 135. Example of an Excel spreadsheet with exported data.
You can also export the raw data to a text file. With your raw data displayed,
go to the File menu and select Export Table to File. This command opens a
Save As dialog box in which you can specify a file name and location for the
text file.
NOTE: You can copy any or all values in the table to the Windows clipboard.
Select the entire table, or the desired rows, columns or entries, and select
Copy from the Edit menu. The values you copy can be pasted into other
Windows applications.
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Raw Data Table Error Codes
If data cannot be measured or a sampling error occurs in a well, it will be
flagged in the Raw Data table. The possible error conditions in the table are
listed below.
Column
Error Code
Cause
F1
***
No data present
Sampling
Errors
1
Low bead number detected in the well
2
Aggregated beads detected in the well
3
Classify efficiency problem detected in the well
4
Region selection problem detected in the well
5
Platform temperature problem detected in the well
Printing the Raw Data
You can print the Raw Data table using the Print command
on the main
toolbar or File menu. Print Preview on the File menu displays the table as it will
appear in the printout.
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Report Table
Report Table
The Report Table provides detailed information about the analytes in your
samples.
Click the Report Table button
to open the Report Table window.
Toolbar
The toolbar allows you to make formatting changes to your samples, and to
change your report table in many ways. The figure below shows the toolbar
functions.
Figure 136. Report Table Toolbar
The first six tools allow you to adjust any plate formatting or Protocol settings.
See Viewing/Changing the Protocol Settings on page 148.
The Normalization Settings button allows you to normalize your data.
See Data Normalization, on page 207.
The Export button allows you to export your data to Excel or a word
processing application.
See Exporting the Report Table on page 163.
The Report Table Display Options button accesses the remainder of the
toolbar functions.
See the Display Options section starting on page 154.
The Show/Hide Outliers button allows you to view or hide outlier data.
See Showing/Hiding Outliers on page 161.
The Organize by Type or Group button is discussed on page 156.
The Single or Multiple Analytes button is discussed on page 155.
The Show Replicates button is discussed on page 156.
The Sort button allows you to sort the table data alphanumerically.
See Sorting Report Table Data on page 163.
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Display Options
Click the Report Table Display Options button,
or select Report Table
Options from the Table Options menu, to select columns to display in the
Report Table. This dialog box also contains many other display settings, which
are described below.
Figure 137. Report Table Display Options dialog
SELECTING COLUMNS TO DISPLAY
See Report Table Column Descriptions on page 158 for definitions. Use the
checkboxes to select which columns to display in the Report Table Display
Options dialog.
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PREDEFINED REPORT SCHEMES
These selections display different sets of columns. Select Quantitative or
Qualitative in the Report Scheme field to choose among predefined Report
Table displays. Use the Norm Qualitative column if your data are normalized.
NOTE: The predefined Norm Qualitative column is designed to be used with
gene expression.
SINGLE OR MULTIPLE ANALYTE LAYOUT
You can view the Report Table either in Single Analyte and Multi Analyte view,
and you can also toggle between them.
Click the Single Analyte Layout
in the toolbar, or choose Layout Table
with Single Analyte from the Table Options menu. Select an analyte to review
from the Analyte pulldown list above the table.
Select analyte from the pulldown list
Figure 138. Selecting an analyte for a Single Analyte Report Table
Click the Multiple Analyte Layout button
in the toolbar, or choose Layout
Table with Multiple Analytes from the Table Options menu. The multiple
analyte view shows columns for all analytes. Use the horizontal scroll bar at
the bottom of the window to scroll sideways through analytes.
Figure 139. Multiple analyte table view
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ORGANIZING BY TYPE OR GROUP
Click the Organize by Type button
or go to the Table Options menu and
select Organize Table by Sample Types, to highlight the divisions between the
unknowns, standards, controls, etc. in the report.
Click the Organize by Group button
or go to the Table Options menu and
select Organize Table by Sample Groups, to highlight the divisions between
defined groups (see page 75) in the report.
NOTE: Type and Group selections will override the Sort function. For example,
if you select Type organization, any sorting will be within each type of sample.
EXPAND REPLICATE INFO
If you have defined multiple wells as a replicate group in the Format Plate
window, the wells will be listed together in the Well column, and mean data
values for those wells will be reported in the relevant columns for the selected
analyte (unless the wells have been flagged as outliers; see below).
Click the Expand Replicate Info checkbox in the Report Table Display Options
dialog, to review the data for each well in a replicate group.
Or, click the Show Replicates button
in the toolbar to expand the data for
each well in a replicate group. The mean values, as well as the individual well
values, and are reported for each replicate group.
Figure 140. Hidden vs. expanded data for replicate wells
EXCLUDING/DISPLAYING TABLE ERROR CODES
Error codes such as ***, OOR, ---, or * appear in the Report Table, if data
cannot be measured or calculated, or if a sampling error occurs. To hide error
codes in the table, select the Exclude Table Error Codes checkbox.
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Click the Display Help for Table Legends button
definitions for any error conditions.
on the toolbar, to get
SETTING NUMBER FORMATS
Click the Set Number Format button in the Report Table Display Options
dialog to set the number format for any column in the Report Table. The Set
Column Number Format dialog opens.
Figure 141. Set Column Number Format dialog
In the dialog, select a column from the pulldown list, then specify the number
of decimal places for the value of that column in the Decimal Places field.
Select the Scientific Notation checkbox to display the number in scientific
notation. Select the Same Number Format for All Columns checkbox to use
the selected number format for all columns. Click OK to make the changes, or
Reset Defaults to return all columns to their default number formats.
NOTE: Changing the number format changes only how the numbers are
displayed and printed, and does not affect calculations in any way.
SAVING SETTINGS AS A NEW PROTOCOL
To use the selections in this dialog as the default in Results files generated
from new Protocols, select the Save Settings as Default for New Protocol
checkbox.
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Report Table Column Descriptions
The columns in the table show the analyte data for each well or replicate
group of wells. The available Report Table columns are defined below:
• Type — sample type (unknown, standard, blank, etc.) and number,
based on plate formatting
• Well — well number; all wells are listed for a replicate group (see
next section)
• Outlier — contains a checkbox for flagging the well or group of
wells as an outlier. See page 161
• Description — description you entered for each standard, control,
or sample in the Protocol
• FI — median fluorescent intensity of the analyte
• FI — Bkgd — median fluorescent intensity minus background
(measured using the defined blank wells) of the analyte
• Std Dev — standard deviation of a replicate group of wells
• Std Err — standard deviation of a replicate group of wells divided
by the square root of the number of replicates
• %CV — coefficient of variation of a replicate group of wells. If this
value is high, you may want to review the individual FI values and
select the appropriate member of the replicate group as an outlier
(see next section)
• Norm Ratio — normalized ratio. See Data Normalization on
page 207 for more information on normalizing data
• Norm Ratio Std Dev — standard deviation of the Norm Ratio
values for all the wells within a sample's replicate group
• Norm Ratio Std Err — the normalized standard deviation, Norm
Ratio Std Dev, divided by the square root of the number of replicate
wells within the sample's replicate group
• Norm Ratio %CV — the normalized standard deviation, Norm
Ratio Std Dev, divided by the normalized ratio of the sample's
replicate group
• Norm FI — normalized fluorescent intensity. See Data
Normalization on page 207 for more information on normalizing
data
• Norm FI Std Dev — standard deviation of the Norm FI values for
all the wells within a sample's replicate group
• Norm FI Std Err — the normalized standard deviation, Norm FI
Std Dev, divided by the square root of the number of replicate wells
within the sample's replicate group
• Norm FI %CV — the normalized standard deviation, Norm FI Std
Dev, divided by the normalized FI of the sample's replicate group
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•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Conc in Range — observed concentration that falls within the
range of standard concentrations, given a specified recovery
range. Values outside the range are flagged as ***. See page 160
for how Conc in Range relates to the Obs/Exp * 100 Column
Obs Conc — observed concentration of the analyte, calculated
from its fluorescent intensity and the standard curve. Note that the
observed concentrations of standards are back-calculated from
their fluorescent intensities and the standard curve
Obs Conc Std Dev — observed concentration of the analyte, with
the standard deviation of the replicate group of wells
Obs Conc Std Err — observed concentration of the analyte, with
the standard deviation of a replicate group of wells, divided by the
square root of the number of replicates
Obs Conc % CV — observed concentration of the analyte, with
coefficient of variation of a replicate group of wells. If this value is
high, you may want to review the individual FI values and select the
appropriate member of the replicate group as an outlier
Exp Conc — expected concentration of the analyte standard or
control, as specified in the Enter Standards Info window or Enter
Controls Info window, respectively
Obs/Exp * 100 — recovery rate for the observed versus expected
concentrations of the standard or control (see page 160)
Group — defined group of the selected well (see page 72)
Ratio — a calculation performed when you have grouped samples
in the plate formatting section. This calculation is the ratio of the
member-to-reference measured values, or vice versa. (For more
information, see Ratio (Simple) and the Grouping Function, on
page 226 of the Appendix)
Dilution — specified dilution factor of the analyte
Bead Count — number of beads counted in region for the analyte
Bead Mean — mean fluorescent intensity of the beads counted in
region for the analyte
Bead Std Dev — standard deviation of the bead mean
Bead Std Err — standard error of the bead mean
Bead %CV — coefficient of variation of the bead mean
Trimmed Mean — bead mean, excluding the top 5% and bottom
5% of fluorescent intensities
Trimmed Std Dev — standard deviation of the trimmed mean
Trimmed Std Err — standard error of the trimmed mean
Trimmed %CV — coefficient of variation of the trimmed mean
Sampling Errors — error codes for a reading (see page 105)
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Obs/Exp * 100 Column
Because the standard curve (see page 168) is critical for calculating the
concentrations of unknowns, the Obs/Exp * 100 column in the Report Table
allows you to assess how well the curve fits the standards. This column is also
used with controls (page 96) to determine the overall accuracy of an assay.
The (Obs/Exp) * 100 column shows the recovery rate for the observed vs.
expected concentration of each standard. This number should fall within the
recovery percentage range selected in the Enter Standards Info window. For
example, if you selected a recovery range within 80 to 120%, the observed
(back-calculated) concentration of a standard should be within 80 to 120% of
the expected concentration. Values outside this range are flagged in the Conc
in Range column.
Click Report Table Options
display this column.
and select the (Obs/Exp) * 100 checkbox to
Concentration in Range Column
The concentration in range column (Conc in Range) provides a method for
determining which unknown concentrations are reliable based on the recovery
range of the standards (see the Obs/Exp * 100 section above).
This column shows only observed concentration values that fall within the
range of valid standards; values that do not fall in the range are displayed as
OOR< or OOR>. (The concentration in range values of the back-calculated
standards are also shown, for comparison purposes.)
Back-calculated standard
concentrations within a specified
recovery percentage range of
80–120%.
Concentrations of unknowns
within range
Concentrations of unknowns
outside range
Figure 142. Concentration in Range column
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For example, if only standard concentrations from 1.9 to 8,100 g/ml fall
within the specified recovery range, then only unknown concentrations that
fall within this range are displayed in the Conc in Range column.
Refer to Standard Curve Optimizer on page 199 for information about how the
Conc in Range column can help you optimize your Standard Curve fit.
Showing/Hiding Outliers
In some cases, you may want to exclude a well or replicate group of wells
from statistical calculations (for example, when the %CV of a set of replicates
is high, or the observed concentration of an analyte is out of range). The
Report Table Outlier column allows you to flag a well or replicate group of
wells as an outlier.
Click the Show/Hide Outliers Column button
hide the column.
in the toolbar to show or
Each row in the column contains a checkbox. When you check the checkbox,
the data for the selected analyte is excluded and the table is automatically
recalculated.
Note that flagging a well as an outlier for the selected analyte does not flag it
as an outlier for all analytes. To do this, right click the outlier checkbox for the
well, and select Set Outlier (All Analytes) from the context menu.
Figure 143. Selecting a well as an outlier for all analytes
Now the well data for all analytes are excluded from calculations. To remove
the outlier flag for all analytes, right-click the well and select Clear Outlier (All
Analytes) from the context menu.
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If you flag a replicate group of wells as an outlier, data for the entire group are
excluded. If you click the Show Replicates button
, you can flag each
well in the group as an individual outlier. If you do this, the outlier checkbox for
the entire group appears checked and grayed out, indicating that part of the
group has been flagged.
Group checkbox appears
gray and selected
Figure 144. A single well in a replicate group flagged as an outlier
Resizing the Columns
To automatically size the columns to fit the width of the text, choose Autofit
from the Table Options menu in the toolbar.
Context Menus
There are two context menus available from the Report Table. Right-clicking
within a header cell brings up a menu that allows you to show/hide a column,
abbreviate analyte names, or view the Report Table Options dialog.
Figure 145. Report Table header context menu
Right-clicking within a body cell in the Report Table brings up a context menu
that allows you to set a Plate ID, set a specific analyte as an outlier, or view the
Report Table Options dialog.
Figure 146. Report Table cell context menu
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Sorting Report Table Data
To sort the table data alphanumerically, click the heading of the column that
you want to sort, then click the Sort button
order.
. Clicking twice will invert the
Copying the Report Table
You can copy the contents of the table to the Windows clipboard. Select rows,
columns, cells, or the entire table, then select Copy from the Edit menu. The
copied values can be pasted into other Windows applications.
Printing the Report Table
You can print the Report Table for the selected analyte (or multiple analytes,
when in Multiple Analyte Layout) using the Print command
toolbar or File menu.
on the main
You can print the Report Table for all analytes (when in Single Analyte Layout)
using the Print All Analytes command
on the main toolbar or File menu.
Print Preview displays the table as it will appear in the printout.
Exporting the Report Table
To export the data in the Report Table to an Excel workbook, or tab-defined
text file, click the Export Report Table button
Export Options dialog opens.
in the toolbar. The Table
Figure 147. Table Export Options dialog
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Export Format
Select Single Analyte Layout to display all the data for each analyte in
individual worksheets.
Figure 148. Example of an Excel workbook, single analyte layout
Select Multiple Analyte Layout to display one or more columns of data (for
example, FI) for all analytes in a single worksheet.
Figure 149. Example of an Excel workbook, Multiple Analyte Layout
Select 96-Well Plate to display each analyte in a worksheet, laid out like the
wells in a 96-well plate (rows A–H and columns 1–12).
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Note that this selection exports only a single column of data at a time.
Figure 150. Example of an Export worksheet, 96-well layout
Click the Advanced Export Options button to access additional options.
In this section,
choose a subset of data
to export from
the Report Table
These are Preference
Settings governing
where and how your
Report Table
is exported
Figure 151. Report Table Advanced Export Options
Export a Subset of the Data
If you selected 96-Well Plate or Multiple Analyte Layout above, select which
columns you want to export; you can select one column or all visible columns,
by choosing from this pull-down list. You can export only the selected analyte,
or export them all.
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If you have selected Single Analyte Layout, All Analytes is the only choice
available.
Export Preferences
Select Excel Workbook to export the data to a Microsoft Excel file, as
described above, or Text File (Tab Delimited) to export to a tab-delimited text
file (see Figure 152 for an example).
Figure 152. FI — Bkgd data, multiple analytes, 96-well plate format, exported
to a text file
If you are exporting to Excel, Microsoft Excel automatically launches when
you click OK and a worksheet appears.
If you are exporting to a text file, a Save As dialog will open when you click
OK. Select the desired directory and name and click Save to complete the
operation.
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Use Multiple Worksheets
If you selected Excel Workbook and are generating multiple tables of data,
select this checkbox to export each table to a different worksheet within the
workbook. Otherwise, all tables appear on the same worksheet.
Choose from the following settings, based upon how you want your data
presented:
Single Analyte—This choice generates a workbook with one worksheet for
each analyte.
Multiple Analyte—If you check the Use Multiple Worksheets checkbox in the
Advanced Export options dialog, you will generate a workbook with one
worksheet for each column (FI, FI-Bkgd, etc.), with data for all analytes. If you
leave the Use Multiple Worksheets checkbox unchecked, all of the data
appears on one large worksheet, which can be viewed by scrolling with the
elevator bar.
96-well —If you check the Use Multiple Worksheets checkbox in the
Advanced Export options dialog, you will generate a workbook with one
worksheet for each analyte. If you leave the Use Multiple Worksheets
checkbox unchecked, all of the data appears on a single worksheet.
Exclude Standard Curves
Standard curve images are automatically exported to Excel, by default. You
can exclude them from the export by checking the box next to Exclude
Standard Curve.
Exclude Column Headers/Footers
Select the Exclude Column Headers/Footers and Page Headers/Footers
checkbox to exclude this information from the exported file.
Exclude Table Error Codes
See page 234 for a table of possible Report Table Error Codes.
The error code symbols (OOR< >, ***, ----, *) before concentration values in
the Report Table can interfere with macros in some types of spreadsheets,
including Excel. Select this checkbox to exclude these symbols from the
exported file. If this option is selected, table cells with error codes are
exported as empty cells.
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Standard Curve
The Standard Curve window displays the standard curve for each standard
analyte in the Results file, and allows you to change the regression model as
well as various curve display controls for each analyte. (Curve settings are
initially selected in the Enter Standards Info window in a Protocol; see
page 78.)
Click the Standard Curve button
to open the Standard Curve window.
Figure 153. Standard Curve
In the graph, standards defined on the current plate are displayed as blue
squares, while external standards are displayed as red circles. If both
standard curves are displayed on the graph. Outliers and partial outliers will
be displayed on the curve as indicated in the legend below the curve.
The equation for the selected regression method as well as some curve fit
statistics (fit probability, residual variance) will be displayed below the
standard curve. Equations for standards defined on the current plate are
shown in blue, while equations for external standards are displayed in dark
red. Any curve-fitting errors will also be displayed below the curve in red.
Use the Analyte pulldown list to display the curve for a selected analyte.
Above the curve, use the Labels pulldown list to select from a variety of
different labels for the points in the curve. If both external and current
standards are displayed in the graph, labels will only be displayed for the
external standards.
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Use the Error Bars pulldown list to select from several types of error bars
(such as 1-Std Dev, 2-Std Err) to plot for each point in the curve. If there is
only one replicate for a point, the bar will have a value of zero. If both external
and current standards are displayed in the graph, error bars will be displayed
for the external standards only.
Use the Regression Type pulldown list to choose from seven different
regression models to generate the standard curve (see next section).
NOTE: Changing the regression type will automatically recalculate all the data
in the Report Table. If you are unsure whether your data have been
recalculated, select Refresh Calculations from the View menu.
Use the Axis Transformation pulldown list to select the standard curve axis
scale. Four combinations of linear and logarithmic axis scales are available.
NOTE: Changing the axis transformation will only change the display of the
curve—not the calculated data.
You can use a different regression model for each analyte. Deselect the Same
Regression Type for All Analytes checkbox, select a different analyte from the
pulldown list, and select the regression type to use for that analyte.
From the Curve Options menu, select the Swap XY Axes to change the x and
y axes of the curve.
From the Curve Options menu, select the Show Conc Range Lines to display
this range as dotted lines on your graph.
Select the Show Unknown Samples checkbox to display your Unknown
samples as green triangles in your graph.
Select the Show Control Samples checkbox to display your Control Samples
as yellow triangles in your graph.
The Curve Fit area contains automated optimization controls discussed in
greater detail in Standard Curve Optimizer on page 199. You can optimize
individual or all analytes, undo the optimization, and clear outliers from your
display.
To display all the data for each standard sample point in
the curve, position your cursor over the point. A pop-up
box will list the data.
Figure 154. Pop-up data for a standard sample point in
the curve
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Regression Methods
There are seven regression types for calculating the standard curve:
• Logistic-5PL
• Logistic-4PL
• Linear
• Cubic spline
• Point-to-point
• Linear (semi-log)
• Point-to-point (semi-log)
The minimum number of nonzero standards required for each regression
method type is shown in the following table.
Method
Minimum number of standards
Logistic-5PL
6
Logistic-4PL
5
Cubic spline
4
Linear (linear and semi-log)
2
Point to point (linear and semi-log)
2
Figure 155. Example of two Logistic-5PL curves, for standards on the current
plate, and external standards
If you do not have the minimum number of standards for the selected method,
an error message will be displayed in red below the standard curve.
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Standard Curve
LOGISTIC-5PL
This version of the five-parameter logistic equation has been developed by
Brendan Scientific, creator of StatLIA immunoassay software. The Logistic5PL regression type yields the best results for most assays; therefore, this is
the default in Bio-Plex Manager. However, there may be cases in which the
Logistic-4PL equation gives slightly better results.
The Logistic-5PL equation is:
yd
ad
 
1  x b 
c 

g
where:
x is the concentration,
y is the response,
a is the estimated response at infinite concentration,
b is the slope of the tangent at midpoint,
c is the midrange concentration or midpoint,
d is the estimated response at zero concentration, and
g is an asymmetry factor.
LOGISTIC-4PL
In certain cases, in which the curve is a perfectly shaped S, the Logistic 4PL
regression may give better results. However, the Logistic-5PL regression has
been shown to be the most robust routine for most Bio-Plex assays.
The Logistic-4PL equation is:
yd
ad
1 x
c
 
b
where:
x is the concentration,
y is the response,
a is the estimated response at infinite concentration.
b is the slope of the tangent at midpoint,
c is the midrange concentration or midpoint, and
d is the estimated response at zero concentration.
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LINEAR
The Linear equation is:
y  a  bx
where a is the intercept and b is the slope of the line.
The simplest method for determining concentrations from a standard curve is
to construct a plot using the linear portion of the response curve. The R2
integrity value may be used to determine the overall goodness of the linear fit.
A linear regression with an R2 value of greater then 0.99 is considered a very
good fit.
The primary advantage of this method is that it is extremely simple. The
primary disadvantage is that the linear range of concentrations is small
compared to that which may be obtained using nonlinear regression.
The linear curve is available in linear and semi-log versions.
CUBIC SPLINE
Cubic spline curves are smooth curves that go through every data point. The
model is a cubic polynomial on each interval between data points. In some
cases, a spline curve can work well as a standard curve for interpolation.
However, because the curve is calculated individually for every pair of points,
it does not correspond to any single equation.
The cubic spline curve is applicable to a wide range of assays; however, there
are no built-in biochemical restrictions on the curve shape, and values cannot
be extrapolated.1,2
POINT-TO-POINT
No single equation is available for the point-to-point method. The slope of
each segment of the curve between data points is calculated independently.
The Point-to-Point curve is available in linear and semi-log versions.
1
The Immunoassay Handbook. David Wild, Editor. Nature Publishing
Group, New York, NY. (2nd Edition, 2001)
2
The GraphPad Guide to Nonlinear Regression. Harvey Moltusky, PhD.
GraphPad Software, San Diego, CA.
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Standard Curve
Linear vs. Logistic Regression Methods
In terms of performance, the primary difference between linear and logistic
regression models is that the linear range in which concentrations can be
accurately predicted is much smaller. In most cases, the overall response of
the assay is best modeled using a logistic fit and the Logistic-5PL model will
yield the best results. For more information, see Principles of Curve Fitting
from the website (www.bio-rad.com) or from Technical Support.
Nevertheless, in certain cases, you may want to use linear regression to
analyze the data. For most cytokines, the linear portion of the curve is well
within the biological range of concentrations expected for patient samples.
Thus, the linear regression may be useful for serum. The main advantage of a
linear regression is that fewer points (as few as 2) can be used.
Copying the Standard Curve
To copy a bitmap image of the curve to the Windows clipboard, right-click in
the Standard Curve window, and select Copy Graph to Clipboard. You can
then paste this image into your spreadsheets or other documents, using the
Paste command in your other applications.
Figure 156. Copying graph to clipboard
Exporting the Standard Curve
To export a standard curve to Excel, follow the steps below.
1. Select the Export Standard Curve icon
from the toolbar.
2. Select your choice of exporting an individual analyte, or all analytes, in
the Export Standard Curve dialog. Legends, the standard curve
equation, fit probability and residual variance display below the chart.
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You can also copy the regression method equation from below the standard
curve by dragging your cursor over the equation to highlight it, and then
selecting Copy from the Edit menu or right-click Context menu.
Figure 157. Copying regression method equation
Printing the Standard Curve
You can print the standard curve for the selected analyte using the Print
command
on the main toolbar or File menu. You can print the curves for
all analytes using the Print All Analytes command
File menu.
on the main toolbar or
When you select a print command, a dialog box will open, allowing you to
specify the page layout of the printed curve. The layout diagrams show the
number of curves that can be printed on a page. If you are printing the curve
for a single analyte, that curve will be sized based on the layout you select (for
example, the
layout will size the curve at one-quarter the full size of the
page). The regression type will be printed below each curve.
Figure 158. Print Standard Curve dialog.
Select a layout and click OK to display the standard Windows Print dialog.
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Graphing Function
Graphing Function
Click the Graphs button
to display your Results data as graphs. You can
choose one of two default graphs from the Graphs drop-down menu: All
Unknowns & Controls, or All Samples for single or multiple analytes.
The y-axis for each default graph is the FI Result value, but you can change to
a different parameter by choosing from the Result (y-axis) drop-down menu.
Bar graphs represent the mean values displayed with error bars that indicate
one standard deviation above and below the mean.
Figure 159. Unknowns and Controls graph, with FI y-axis
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Figure 160. All Samples graph with Std Dev y-axis
Perform any data normalization functions before displaying your graphs.
If you want to see ratios among samples, or other normalization options, click
the Normalization Settings button
for more information.
176
. See Data Normalization on page 207
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Graphing Function
Choosing Graph Options
VIEW
Select the Graph Options button
to choose the type of graph that best
displays your Results data. These options are in the View area..
Figure 161. Graph Options dialog
SAMPLE LABEL
You can identify samples of the graph either by their Type (X1, C1, S1) or by
their description (which you enter for each sample). Choose either Type or
Description from the Sample area at the bottom of the Graph Options dialog,
shown above.
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Under the Graph Options dialog, you can choose among four basic views, as
shown in the four figures below. Click the appropriate button in the View field
to display your choice.
• Samples across a single analyte—In this view a single analyte is
represented for each sample and the selected result (such as FI,
Norm ratio, etc.) is used as the y-axis parameter.
Figure 162. Samples across a single analyte
•
Samples across multiple analytes—Samples are grouped by
analyte along the x-axis and the result (such as FI, Norm ratio, etc.)
used as the y-axis parameter.
Figure 163. Samples across analytes view
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Graphing Function
•
Analytes across a single sample—In this view a single sample is
represented for each analyte and the selected result (such as FI,
Norm ratio, etc.) used as the y-axis parameter.
Figure 164. Analytes across a single sample view
•
Analytes across multiple samples—Analytes grouped by sample
along the x-axis and the selected result (such as FI, Norm ratio,
etc.) used as the y-axis parameter.
Figure 165. Analytes across multiple samples view
To choose between plain or 3-dimensional graph bars, choose from the Graph
Type drop-down menu.
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To gain room for your graphs, you can click the Hide Legends checkbox from
the Graph Options menu.
To adjust y-axis values so that the data will be comparable throughout a series
of graphs, click the Adjust data to same y-axis scale checkbox.
To change the y-axis display from linear to logarithmic, click the Logarithmic
y-axis checkbox.
To adjust legends to accommodate the number of columns in your graph,
click the x-axis labels orientation drop-down menu from the Graphs Options
dialog. You can select vertical or horizontal legends, or legends angled at 30,
45, or 60 degrees.
Viewing Information within Graphs
To browse through many analyte graphs, click the green arrows to the left and
right of the Analytes field in the toolbar. This displays a series of analyte
graphs without having to search and select from a long list.
Figure 166. Viewing a series of analyte graphs
Analyte names, sample names, user-defined descriptions, and values are
always available for view. Place your cursor over a specific column for a
moment and the data display.
Figure 167. Click to view data from graphs
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Graphing Function
You can zoom into any graph by dragging a marquee around specific bars.
Click the Zoom out button on the toolbar to return to the previous
magnification.
Figure 168. Marquee to zoom into a graph
Right-clicking any results graph displays a context menu to access many
functions described in this chapter.
Figure 169. Right-click context menu
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Adding New Graphs
To create additional graphs, click the Add Graph button
on the toolbar.
Figure 170. Selecting elements from Add Graph dialog
Select samples and analytes by clicking individual checkbox es, or with the
Select All button. You can also click and drag over a range of samples, and
select only those samples, by choosing Select from the right-click context
menu.
Figure 171. Selecting elements from context menu
Click the Graph Options button from the Add Graph dialog to choose a view
and other options for displaying your data, as described on page 177.
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Graphing Function
After you choose options, name and save this graph, it is added to your Saved
Graphs List under the Graph drop-down menu.
Figure 172. Saved graphs list in the Graphs drop-down menu
Editing an Existing Graph
The Edit Graph button
is available only when viewing graphs with
choices created by the user. The button will be grayed out while viewing either
of the default graphs. This dialog displays checked boxes for the elements
already chosen.
Figure 173. Edit Graph dialog
You can edit any graph by choosing it from your Saved Graph List. Add,
delete or change elements by checking or unchecking the sample or analyte
checkbox es.
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Deleting a Graph
Delete a graph from your Saved Graphs List with the Delete Graph button
on the toolbar. You can also delete a graph by right-clicking any graph to bring
up the Context menu.
Exporting Graphs to Other Applications
You can export your data to a spreadsheet application such as Excel, for its
expanded graphing capabilities, or to match existing graphs. Click the Export
to Excel button in the toolbar, or choose Export . . ., under the Graphs menu.
The data open in an Excel spreadsheet.
Figure 174. Exporting graphs to Excel
You can also export the graphs as .bmp files, so they can be used in word
processing, presentation, or other text-based applications. Click the toolbar
File menu or right-click the graph, and a menu appears. Choose Save as
bitmap, and the Save As dialog appears, so that you can name the file and
place it anywhere in your file system.
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Graphing Function
Adjusting Your Graphs
If you open a graph that represents a very large number of data points, you
may see black lines, as in the figure below.
Figure 175. Graph containing many data points
Choose fewer analytes or samples, and your columns will widen, so that the
analyte color codes can be represented.
Zoom into your graph by clicking and dragging over any area of interest, as
shown in Figure 168. To return to the original view, click Zoom Out from the
toolbar.
Another option is to print your graph on multiple pages, which allows the bars
to be much wider. See the Printing Graphs section below.
Printing Graphs
Access the controls to print your graphs from the toolbar, the right-click
context menu, or the File menu.
Figure 176. Print Graph dialog
When the graph containing many data points from Figure 175 is printed onto
four pages, the color-coded columns are visible. To preview how your graph
will look printed over multiple pages, click the Go to Preview button.
The Print Graph dialog allows you to print your graph on a specific number of
pages, to choose a landscape or portrait orientation, and to preview the
printed copy.
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Document Export Options
In addition to the export options available in the raw data and results table
section of the data file, there are a number of document export options. These
options are chosen from the Document Export Properties dialog box shown
below, which is available from the File menu.
In this section you
choose the type of
exported file
In this section you
direct where the file
will be stored
Command line used
to direct the file to an
external application
Figure 177. Document Export Properties dialog box
Here is a brief description of each option.
• Bio-Plex XML —this is a comprehensive export of individual results
files in an .xml format. This format is useful for import into LIMS
(Laboratory Information System) or other database applications
• Output CSV format—this mimics the output of third-party
software, such as IS 2.3 or xPONENT, with common options
• Custom Export using style sheets—customer-developed
stylesheets can extract any of the data exported to the Bio-Plex
XML file. See page 191 for more information. Using stylesheets
allows you to format the data in any way desired, provided you
have experience developing stylesheets. Some default stylesheets
are included for guidance
• Command Line Export—custom export by passing a command
line to external applications. See page 192 for more information
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Document Export Options
All these export options can be set to occur automatically at the end of a run,
by choosing the Auto export after run checkbox from the Run Settings dialog.
The Advanced Run Settings dialog box launches from the Run Protocol
screen, during the process of setting up a Protocol.
Figure 178. Advanced Run Settings
Bio-Plex XML Export
You can export all the information in a Results file as an XML-formatted text
file. This export option generates a comprehensive export of individual results
files in an XML format. This format is useful for import into LIMS databases,
and to perform data analysis using other applications.
NOTE: XML is a self-describing, universal file format compatible with a wide
range of applications.
The XML document generated by Bio-Plex Manager includes all the data
stored in the Results file, including but not limited to:
• Run parameters, including DD gate settings, instrument
temperature settings, data limits, bead regions, etc.
• Run conditions, including time stamps per well, error conditions
during the run, actual temperature during acquisition by well, etc.
• Complete data values and calculations (mean, median, standard
deviation, coefficient of variation, etc.) per well
• Complete protocol information, including analytes selected, plate
formatting, standard values, run settings, etc.
• Raw bead event data (when selected)
• Standard curve information, including mathematical models used,
how well the data fit the curve, etc.
• Instrument and software information, including version numbers
• All logged events associated with the data file for CFR 21 Part 11
compliance
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To export the Results data in XML format:
1. Select Document Export Properties from the File menu.
2. Choose the Bio-Plex XML option.
3. Choose the location to save the file.
XML SCHEMA FILE
An XML schema file named Bio-Plex_5.xsd is included in the main Bio-Plex
Manager application directory, for those who want to write an importer for
Bio-Plex Manager results files. This schema file can be used to automatically
validate the XML file.
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Document Export Options
Output CSV File Format
This option exports an output CSV file similar to those generated by IS 2.3
software. It can be opened in third-party applications designed to analyze
IS 2.3 files. You have the option of changing analyte labels, well labels, and
analyte sort order in the resulting CSV (comma-separated values) file.
There are several export options available in the CSV format Content field.
Figure 179. CSV Content options in the Document Export window
ANALYTE LABELS
Various software packages require different analyte naming conventions, such
as IL-2, which appear in the column heading within the CSV files. Refer to the
software instructions to identify which settings are suitable for your
application.
Name—Only the analyte description is generated in the report (as in IL-2)
Region—Only the region identifier is listed in the column header (as in 18)
Name (Region)—Both name and region ID are exported (as in IL-2 (18))
WELL LABELS
Rows in the CSV file represent well positions. There are two ways to indicate
well position. Well A1 (plate location) is equivalent to Well 1. Well H1 is
equivalent to Well 8. Row H12 is equivalent to well 96 (sequential).
Sequential—(1, 2, 3)
Plate Location—(A1, B1, C1)
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ANALYTE SORT ORDER
You can indicate the left to right sort order in the output CSV file.
None—sorts as the analytes are shown in the panel entry screen of Bio-Plex
Manager
Analyte Name—sorts alphanumerically by analyte name
Analyte Region—sorts by region number
These sort options are shown in the Figure 180.
Both plate location and sequential
Figure 180. Well labels and Analyte labels
190
Analyte Name only
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Document Export Options
Using Stylesheets
Stylesheets allow unlimited customization of exported data. Click the “Use the
following stylesheet” button to navigate to one of the default stylesheets.
Stylesheets describe the format and content of the resulting export.
Using stylesheets allows you to format data in any way desired, provided you
have experience developing stylesheets. Some default stylesheets are
included for guidance.
If you are not familiar with stylesheets, there are contractors who specialize in
stylesheet design. IT professionals who are familiar with XML can often
develop custom stylesheets based on the examples given.
Once a style sheet is identified for document export, it will be used to define
the export file parameters and the resulting file will be placed in the location
indicated in the destination line discussed below.
FILE DESTINATION OPTIONS
Use the default file name and location, or click Choose Folder to specify a
new file name and location. The exported file can be placed on your system or
in a shared file.
Figure 181. Destination field of Document Export Properties
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Command Line Export
This is an advanced option for customers who use custom-designed
applications to analyze their data. You need knowledge of the following to use
this option:
• The term “command argument”
• Writing computer code
• Implementing custom software applications
Select Command Line and click Select Application to locate the software
application you want to automatically launch when the file is generated. This
application must accept a command line filename argument.
After you locate and select the application, the application path name will
appear in the Command Line field enclosed in quotation marks, and the string
"&&XML&&" will be automatically appended to it, as shown in Figure 182.
NOTES:
• If you manually enter the application path name, it must be
enclosed in quotation marks
• The &&XML&& token must also be enclosed in quotation marks
If the application can be found by using the system executable search PATH,
only the application name needs to be entered.
Figure 182. Example of a command line
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Document Export Options
Exporting the Document
After you make the choices described above in the Document Export Options
dialog box, click OK. Choose the Document Export option from the File menu.
The Document Export window uses the properties previously set in the
Document Export Properties dialog and allows you to export. Your choices
can also be changed in this dialog.
Figure 183. Document Export window
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Importing Luminex xPONENT Output Files
From Bio-Plex Manager, you can launch Bio-Plex Results Generator and
import a Luminex xPONENT 3.1 or 4.0 CSV output file into Bio-Plex Manager.
1. From the File menu, select Import from xPONENT Output CSV.
Bio-Plex Results Generator is launched and the Bio-Plex Results
Generator window displays.
NOTE: You must have the Bio-Plex Results Generator software
installed on your machine in order to access this feature in BioPlex Manager.
2. Click Select File(s) and select the file you want to import.
3. Click Select and specify the location for the generated file.
4. Click Generate to convert the CSV file to an .RBX file.
By default, the converted results file is opened in Bio-Plex Manager.
TIP: Click Help in the Bio-Plex Results Generator window to get
more information on using the product.
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Exporting Results Data to Bio-Plex Data Pro™
Exporting Results Data to Bio-Plex Data
Pro™
You can seamlessly export your data from Bio-Plex Manager and import them
into Bio-Plex Data Pro, where you can do an extensive and in-depth analysis
of your results. To use this feature, you must have both products installed on
your computer, though you do not have to have Bio-Plex Data Pro running at
the time. Bio-Plex Manager automatically launches Bio-Plex Data Pro if the
software is not already running.
1. From Bio-Plex Manager, open the Results file that you want to export,
then click Export Results to Bio-Plex Data Pro in the toolbar.
NOTE: Bio-Rad strongly advises that data results are optimized
before exporting to Bio-Plex Data Pro. You may choose to use
the Bio-Plex Manager auto optimization, or you can optimize the
results yourself.
If the data are not optimized, a dialog displays. If the results have been
optimized, click Yes to proceed with the export. If the results are not
optimized, click No to first optimize the data, then proceed with the
export. For more information on curve optimization, see Standard Curve
Optimizer on page 199.
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2. In the Export to Bio-Plex Data Pro dialog, enter a name for the data set
you are exporting.
By default, the name of the results file is displayed.
3. Click Export.
The Bio-Plex Data Pro application is launched if it is not already
running. The Import Data Sets dialog in Bio-Plex Data Pro automatically
appears.
The data set is listed and Bio-Plex Manager is displayed as the source
of the data.
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Exporting Results Data to Bio-Plex Data Pro™
4. Select an existing project from the Import Into drop-down list or create
a new project into which to import the data.
Additional data sets can be added to the Data Set Files list by using the
Browse button. You may also return to Bio-Plex Manager to export
another data set.
5. Click Import.
When the data have been imported, the Status displays Done. If the
same data were previously imported into the project, Bio-Plex Data Pro
does not import the data a second time, and the Status displays Exists.
If a file with the same name was previously imported but the data are
different, or the same data are exported twice from different versions of
Bio-Plex Manager, the data set is imported and the Status displays
Renamed.
6. Click Close.
NOTE: You start this procedure from Bio-Plex Manager. However,
at the end of the procedure, you are left in Bio-Plex Data Pro
because the assumption is that you will want to proceed with
analyzing your results in Bio-Plex Data Pro.
For more information on importing data in Bio-Plex Data Pro, see Bio-Plex
Data Pro User Guide.
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Bio-Plex Manager Software 6.1 User Guide
10 Standard Curve
Optimizer
Bio-Plex Manager™ 6.1 software contains a new function to optimize the fit of
standard curves. A standard practice used to improve the fit is detecting and
removing outliers by using nonlinear logistic (4PL or 5PL) equations when
fitting data points. Bio-Plex Manager uses the percent recovery of individual
standard points to assess standard curve recovery and assess the goodness
of fit.
The optimizer tools are bordered in red on the Standard Curve figure below.
Figure 184. Standard Curve Fit Optimizer tools
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Bio-Plex Manager Software 6.1 User Guide | Background
Refer to Determining Outliers on page 201 for more information and
instructions.
Background
Standard Curve Recovery
Bio-Plex Manager calculates an observed concentration for each standard
point from the standard curve. This is divided by the expected concentration
(entered by the user in the Enter Standards Info window) and multiplied by 100
to give a recovery percentage. These calculations are automatically performed
by the software. The reported recovery of the standard curve points are in the
Obs/Exp * 100 column pictured below.
Figure 185. Obs/Exp * 100 column reports Standard Curve Recovery
The recovery percentages of the standard curve points indicate the overall
accuracy of the assay. The user can specify more or less stringent ranges for
individual assays.
Use the Acceptable Recovery Range drop-down menu in the Enter Standards
Info window to select an acceptable recovery range. To set different recovery
ranges for different analytes, uncheck the box next to Same recovery range
for all analytes. For more information on this feature, see Recovery
Percentage Range on page 95.
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Determining Outliers
Concentration in Range
Concentration in range (Conc in Range) reports which standards, controls,
and unknown concentrations are reliable, based on the recovery range of the
standards. The Conc in Range column shows only observed concentration
values that fall within the range of valid standards, as defined by acceptable
recovery ranges. Values that do not fall in the range are displayed as < OOR or
OOR >.
Figure 186. Concentration in Range and Observed Concentration columns
Determining Outliers
The Standard Curve Optimizer maximizes the usable range of an analyte's
standard curve, based on the percent recovery range selected (the
recommended recovery range is 70-130%). The optimization program
evaluates several parameters, including tests for bead count, CVs of
replicates hook effect, recovery value and points in the noise region of the
curve (flatness at the low end).
Additionally, the optimization program will favor the fit at the low or high end of
the curve, depending upon the number of unknowns falling in the respective
regions.
Recommended Use
We recommend you review the standard curve fit optimizer report after
running an optimization, and review the Results and Comments material
outlined below. These are designed to help you review your data quickly for
issues that may concern you. We also recommend that you pay particular
attention to those analytes with comments, or those that return a Check Data
state.
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Bio-Plex Manager Software 6.1 User Guide | Determining Outliers
Standard Curve Optimizer Report
Once a data set has been optimized, a report is generated that will indicate
any standard curves which should be reviewed by the user.
Figure 187. Standard Curve Optimization Report
Two types of information presented for each analyte; Results and Comments.
RESULTS
This column presents the optimization event results, reported as three
different outcomes.
• Successful —The algorithm was successful. It was able to improve
the range of the assay, or no changes were implemented.
• Not Optimized—No changes were identified which could improve
the range of the assay. It is possible that manual optimization could
improve the fit.
• Check Data—There were significant challenges associated with
this data file, and it is recommended that the data be reviewed.
Review the included comments, in any case. Possible comments are
described below.
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Determining Outliers
COMMENTS
Comments are included to point out particular issues that may be of interest.
• Low Bead Counts Detected (Low Bead)—This message will be
presented if fewer than 20 beads are detected. This suggests one
of the following problems:
- Incorrect gates are set
- Incorrect region is set
- Insufficient agitation of plate before reading
- Perforated well (if using a filter plate)
- Some other contributor to bead loss
• Standard Concentrations are not set (Stand Conc)—Standards
have either no value of 0 value entered. Correct this by applying the
appropriate concentration to your standards, or renaming your 0
standard as a blank (it is inappropriate to have a standard of 0
value).
• One or more standard has a CV higher than 20% (High % CV) - If
one or more standard has a CV higher than 20%, you may want to
check your data to identify systemic loading issues. Also make
sure you agree with the algorithm decisions to mark certain
standards as outliers.
• More than one standard (all replicates) has been marked as an
outlier (>1 Std Removed) - This message will be displayed if all
replicates of more than one standard point has been completely
removed (that is, marked as an outlier). You may want to review the
data for such standard curves.
• The upper range of your curve is limited because 2 or more high
concentration standards have poor recovery.
By default, the report displays at the end of an optimization. You may disable
the automatic display by deselecting the checkbox next to Show report after
optimization. This report can also be viewed at any time, by choosing the
button below the optimizer controls.
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Bio-Plex Manager Software 6.1 User Guide | Determining Outliers
Residual Variance and Fit Probability
Other statistical parameters that can be used to evaluate and adjust goodness
of fit include spiked recovery, residual variance, fit probability and weighting.
Residual variance measures how well a curve fits the individual data points. Fit
probability provides statistical evaluation of residual variance to determine
acceptability. The values of fit probability range from 1.0 (perfect fit) to
0 (no fit).
These fit probability and residual variance values are reported by Bio-Plex
Manager, as shown in this figure.
Figure 188. Fit probability and residual variance
Logistic Weighting
If you are using Logistic 5PL or Logistic 4PL, Bio-Plex Manager will
automatically weight the points in the curve. There are eight models for
calculating the variance in the logistic weighting algorithm.
The default setting for Variance Model is Power Law Variance. Weighting
coefficient a will be automatically calculated.
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Determining Outliers
Alternatively, users can specify a different coefficient. Click the Curve Options
menu and select Logistic Weighting to display the dialog and deselect the
Auto calculate coefficient a checkbox. Weighting coefficient b has a default
value of 1.8. Users can enter a different value to adjust curve fitting.
Figure 189. Logistic weighting
Under default settings, data points that cannot be measured or calculated
according to pre-defined specifications display as error codes.
Figure 190 tabulates the most commonly encountered error codes and their
corresponding causes. The non-numeric code symbols in the Report table
(OOR<>,***, or * before concentration values) may interfere with macros in
some types of spreadsheets, including Excel.
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Bio-Plex Manager Software 6.1 User Guide | Determining Outliers
These symbols can be excluded from the exported file by checking the box
for Exclude table error codes, as shown in the figure below. Sampling errors
related to instrument, reagent and sample preparation can also be monitored
by checking the box for Sampling Errors under Report Display Options.
Figure 190. Excluding error codes
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Bio-Plex Manager Software 6.1 User Guide
11 Data Normalization
There are two main reasons to use data normalization:
• You may need to account for differences in the amount of material
loaded in each sample. This is accomplished using analytes that
will not vary from sample to sample (for example, housekeeping
genes, reference genes, and internal controls)
• You may want to express all other samples' measured values
relative to an assigned control sample. The normalized control
sample has a value of 1 and other samples have measured values
relative to the control (for example, 5.57-fold higher mRNA
compared to the control, or 2-fold higher protein level compared to
the control)
Normalization Settings
Normalization is off by default. Click the Normalization Settings
button
on the toolbar, or choose View> Normalization Settings, to enable data
normalization from any view of the experiment results. The dialog allows you
to specify how you want to normalize data.
The Normalization Settings dialog box provides the following options:
• Internal Control/Housekeeping Genes (None, Single, and Multiple)
• Control Sample (always None or Single)
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Bio-Plex Manager Software 6.1 User Guide | Data Normalization
Selecting Internal Controls/Housekeeping
Genes
The Internal Control/Housekeeping Gene field in this dialog allows you to
account for differences in the amount of sample material. This function
requires that the sample contain analytes whose concentrations will not vary
from sample to sample (for example, housekeeping genes, reference genes,
and internal controls).
To select a single internal control/housekeeping gene, click the Single radio
button and select a housekeeping gene from the pulldown list.
Figure 191. Selecting a Single Internal Control
To select multiple internal controls/housekeeping genes, click the Multiple
radio button, and select which housekeeping genes to use from the following
dialog box.
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.
Figure 192. Selecting Multiple Internal Controls
Double-click an analyte, or select and click Add. The housekeeping genes
populate the Selected field of the dialog box. Bio-Plex Manager™ allows you
to add up to 10 housekeeping genes.
NOTE: The normalized value for each sample is calculated by dividing the
mean fluorescence intensity (MFI) of the analyte of interest by the MFI of the
analyte used for normalization. When multiple analytes are used for
normalization, the geometric mean of the normalization analytes' MFI is used.
Assigning a Control Sample
The Control Sample field in this dialog allows you to express all gene sample
measured values relative to an assigned control. The control sample has a
value of 1 and other samples are expressed as ratios (for example, 5.57-fold
higher mRNA compared to the control, or 2-fold higher protein level compared
to the control).
Brackets around analytes in any list indicate they are used as internal controls.
To assign a control sample, click the Single button in the Control Sample field.
Click None to disable your choice.
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Bio-Plex Manager Software 6.1 User Guide | Data Normalization
Figure 193. Assigning a Control Sample
NOTE: For the formulas used to calculate control sample ratios, see
Normalization Formulas on page 223.
Report Table Options
To display normalization results, click the Report Table Options
button,
and select the columns you want to display from the Report Table Display
Options dialog.
210
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Figure 194. Report Table Display Options dialog
The Report Table displays columns for data normalization. The column titles
associated with the normalization calculations begin with Norm Ratio or Norm
FI.
Predefined Normalization Report Schemes
Select Norm Qualitative in the Report Scheme field. The Norm Qualitative
column is designed to be used with gene expression.
When selected in Report Table Display Options, the following data
normalization columns appear in the Report Table.
Figure 195. Normalization Columns in the Report Table
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Bio-Plex Manager Software 6.1 User Guide | Data Normalization
Normalization Definitions
The following columns and their associated calculations are provided when
performing data normalization. Here are definitions for these columns:
• Norm Ratio—normalized ratio. See Normalization Formulas on
page 223, for details about the formulas used for this calculation
• Norm Ratio Std Dev—standard deviation of the Norm Ratio values
for all the wells within a sample's replicate group
• Norm Ratio Std Err—the normalized standard deviation, Norm
Ratio Std Dev, divided by the square root of the number of replicate
wells within the sample's replicate group
• Norm Ratio %CV—the normalized standard deviation, Norm Ratio
Std Dev, divided by the normalized ratio of the sample's replicate
group
• Norm FI—normalized fluorescent intensity. See Normalization
Formulas on page 223, for details about the formulas used for this
calculation
• Norm FI Std Dev—standard deviation of the Norm FI values for all
the wells within a sample's replicate group
• Norm FI Std Err—the normalized standard deviation, Norm FI Std
Dev, divided by the square root of the number of replicate wells
within the sample's replicate group
• Norm FI %CV—the normalized standard deviation, Norm FI Std
Dev, divided by the normalized FI of the sample's replicate group
Calculations Based Upon Your Settings
Norm (Ratio)—Normalized (FI—Bkgd) ratio.
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The following table is based on the selections made in Normalization Options.
It also describes how the Norm (Ratio) is calculated:
Case
Internal
Controls
Control
How Norm (Ratio) is Calculated
Sample
A
Single
None
B
Multiple
None
Geometric mean of the normalized (FI - Bkgd)
ratios for each housekeeping gene
C
Single
Single
Normalized (FI - Bkgd) ratio/(FI - Bkgd) control
ratio for the control (as defined in Case A):
Ratio of (FI-Bkgd) for the analyte and the
housekeeping gene:
Norm (Ratio) = (FI-Bkgd)/(FIhk-Bkgdhk)
Norm (Ratio) = Norm (Ratio) single gene/Norm
(Ratio) control
D
Multiple
Single
(Ratio) multiple internal control divided by the
Norm (Ratio) multiple control (as defined in
Case B) for the control:
Norm (Ratio) = Norm (Ratio) multiple internal/
Norm (Ratio) multiple…
E
None
Single
Ratio of (FI-Bkgd) for the analyte and the
control:
Norm (Ratio) = (FI-Bkgd)/(FI controlBkgdcontrol)
NOTES:
• Replicates are done at the plate level. See the section on plates to
define replicates
• When using multiple replicates, the Norm (Ratio) for each replicate
is calculated as described in Case A, B, C, D, and E. For each
sample type, the replicates' average is the Norm (Ratio)
• For information about the formulas Bio-Plex Manager uses to
calculate normalization values, see Normalization Formulas on
page 223
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Bio-Plex Manager Software 6.1 User Guide | Data Normalization
Normalization Factor Calculations
Normalization factors are determined in the following ways.
Table 1.
Case
Factor
Definition
A
Control sample
present for
normalization
Average of (FI-Bkgd) for the external control
for all analytes.
Factor = Average [
(FIcontrol-Bkgdcontrol) analyte1,
(FIcontrol-Bkgdcontrol) analyte2,
(FIcontrol-Bkgdcontrol) analyte3, etc.]
B
No control sample Average of (FIhk-Bkgdhk) for all wells with this
and single
housekeeping gene. Data are taken from raw
housekeeping gene data table (average replicate values for each
sample type from report table are not
included).
Factor = Average [
(FIhk-Bkgdhk) well1,
(FIhk-Bkgdhk) well2,
(FIhk-Bkgdhk) well3, etc.]
C
214
No control sample
and multiple
housekeeping
genes
Factor = Geometric Mean (Factorhk1,
Factorhk2, Factorhk3, etc.)
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Bio-Plex Manager Software 6.1 User Guide
Troubleshooting
Follow these suggestions to troubleshoot your Bio-Plex system.
Message/Problem
Cause
“Bio-Plex Manager 6.1 More Likely:
software has detected Too few beads in the assay
a problem with low
(2,500 per region recommended).
bead number”
Plate not shaken for 30 seconds
before analysis.
Solution
Check bead number calculations in
assay.
Remove plate from array reader and
shake for 30 seconds.
Buffer volume in wells is too low
(125 μl is recommended).
Resuspend wells in 125 μl. Perform
Remove Bubbles.
Microbubble in cuvette.
Perform Remove Bubbles. Perform
Unclog to verify fluidics integrity.
Low/no sheath fluid.
Refill sheath fluid, check sheath
connections. Perform Start Up.
Possible clog.
Perform Unclog and rerun. If
unsuccessful, repeat. If still
unsuccessful, contact Technical
Support.
Less Likely:
“Bio-Plex Manager has
detected a problem
with selection of
regions”
Incorrect needle height.
Adjust needle height.
Incompatible plate type used.
Replace with flat bottom or filter plate
and adjust needle height.
Vacuum system not calibrated.
Calibrate vacuum system (see hardware
manual).
Red laser failure.
Contact Technical Support.
Filter plate not flat.
Check filter plate flatness (see hardware
manual).
Incompatible suspension buffer
used.
Check buffer compatibility (see
hardware manual).
Incorrect bead regions were
selected in the Protocol.
Compare bead regions in the assay
with those selected in the Protocol.
Incorrect regions were selected
when preparing the assay.
Verify regions chosen during assay
preparation.
Too few beads in the assay in
one or more regions.
Verify regions chosen during assay
preparation.
Too few beads in the assay in
one or more regions.
Verify that the correct number of beads
was used by referring to the specific
assay preparation procedure.
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Bio-Plex Manager Software 6.1 User Guide | Troubleshooting
Message/Problem
Cause
“Bio-Plex Manager has
detected a possible
problem due to
microbubbles in the
cuvette or
photobleached beads”
More likely:
Solution
Calibration was performed
before the array reader was
warmed up.
Perform 30-minute warm up and
recalibrate.
Calibrated with incorrect CL1 or
CL2 target values.
Check that the target values of the CAL
beads match values entered in the
software. If different, recalibrate.
Array reader was calibrated with Clean MCV Plate IV and recalibrate.
a dirty MCV Plate IV.
Misalignment of optics.
Perform Optics Validation. Contact
Technical Support if values are not
within range.
Less likely:
Microbubbles present in cuvette. Perform Remove Bubbles.
Calibration beads are
Recalibrate with new CAL1 beads.
photobleached (do not expose to
light for more than 1 hour).
Needle stuck in down
position
Optics, Fluidics,
Reporter, or Classify
Validation Procedure
fails due to low bead
number
Protective assay plate covering
was not removed.
See hardware manual for procedure for
raising needle stuck in down position.
Then remove cover from assay plate.
Needle guide is not screwed all
the way in.
Tighten needle guide by turning tube
clockwise until tight.
Sample needle is bent.
Replace bent needle with a new needle
(see hardware manual for procedure).
Beads not resuspended.
Resuspend beads and repeat validation
procedure. If procedure still fails,
contact Technical Support.
Clog in instrument.
Perform Unclog operation and repeat
validation procedure. If procedure still
fails, contact Technical Support.
“Bio-Plex Manager has Incorrect DD gate values used.
detected a problem
with bead
aggregation”
Clumped beads present.
216
View histogram in Run Protocol window
and make sure DD peak falls within the
gate range. If necessary, adjust gates.
Vortex plate at 900 rpm for 11 minutes.
Sheath reservoir is empty.
Refill sheath reservoir, then perform
Start Up.
Waste reservoir is overfilled.
Empty waste and reconnect.
Incompatible suspension buffer
used.
Check hardware manual for buffer
compatibility.
BioPlex_6.book Page 217 Friday, September 23, 2011 11:55 AM
Message/Problem
Cause
“Check Link” in status Array reader or microplate
bar of software
platform is not turned on.
Software is not communicating
with array reader.
Solution
Turn on array reader and microplate
platform.
Close and restart Bio-Plex Manager.
Cables between computer and
Check cables for proper connections.
array reader or microplate platform See System cable connections on
are loose/not connected.
page 16.
“Pressurizing” in
software status bar
Leak in sheath bottle or cap.
Tighten sheath cap.
Platform heater not at
target temperature
Insufficient warm-up time
allowed.
Allow at least 15 minutes for platform
heater to reach target temperature. If
heater does not reach target in 30
minutes, contact Technical Support.
Platform heater not at
ambient temperature
Platform heater not functioning
properly.
Contact Technical Support.
Insufficient cool-down time
allowed.
Open platform door to facilitate cooling.
An ice pack can also be placed on the
platform plate holder to facilitate
cooling.
No Assay Signal
Detected
More likely:
Sheath reservoir cap not on
securely.
Tighten sheath cap. Message should
disappear within two minutes.
Sheath bottle lines are not
connected properly.
Make sure that all hoses are connected
to the appropriate ports, and that they
have clicked into place.
Less likely:
Bio-Plex Manager has
detected a change in
array reader temperature. Please calibrate
before running an
assay to ensure
accurate results.
Sheath fluid level above the AIR
port on the sheath container.
Adjust sheath fluid level so that sheath
fluid is below the AIR port of sheath
bottle.
Sheath bottle has a leak.
Try new sheath bottle. Call Technical
Support for further assistance.
High-throughput fluidics (HTF)
system not turned on.
Turn on HTF and check to ensure that
message disappears.
Air Compressor not working.
List for air pump to turn on when Warm
Up is selected. Contact Technical
Support for further assistance.
Room temperature has changed. Calibrate array reader.
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Bio-Plex Manager Software 6.1 User Guide | Troubleshooting
Message/Problem
Cause
Solution
The calibration was
unsuccessful. Please
repeat calibration. If
calibration fails a
second time, follow
these steps.
Calibration procedure failed.
1. Make sure CAL1 beads and CAL2
beads are placed in the appropriate
wells (CAL1 in red well and CAL2 in
green well).
2. Make sure the calibration target
values match the target values printed
on the CAL1 and CAL2 bottles.
3. Replace or clean the needle.
4. Adjust needle height.
5. Run Unclog until it passes.
(If it doesn't pass by the 5th attempt,
call Technical Support.)
6. Be sure to calibrate with freshly
vortexed beads and a clean MCV IV
plate.
7. Repeat calibration and record bead
flow (optimum flow should be > 200
beads/second)
If problem persists, record any
additional error messages (such as
temperature problem, pressure
fluctuation, warm-up cancellation)
before calling tech support.
Or
Or
The calibration was
unsuccessful. Bio-Plex Calibration procedure failed due
Manager has detected to low bead number.
a problem with low
bead number. Please
repeat calibration.
Optics Validation
Procedure shows
value(s) outside of
acceptable range(s).
Problem with optical component Repeat validation procedure. If values
of array reader.
are still out of range, contact Technical
Support.
Fluidics Validation
Procedure shows
value(s) outside of
acceptable range(s).
Problem with fluidics component Repeat validation procedure. If values
of array reader.
are still out of range, contact Technical
Support.
Reporter Validation
Procedure shows
value(s) outside of
acceptable range(s).
Problem with optical component Repeat validation procedure. If values
of array reader.
are still out of range, contact Technical
Support.
Classify Validation
Procedure shows
value(s) outside of
acceptable range(s).
Problem with calibration or
optical component of array
reader.
218
Repeat validation procedure. If values
are still out of range, contact Technical
Support.
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Bio-Plex Manager Software 6.1 User Guide
Appendix
This Appendix contains:
• Security Edition: User Access by Function (below)
• Normalization Formulas (see page 223)
• Basic Concepts of the Bio-Plex suspension array system (see
page 227)
• Microsphere Handling (see page 229)
• Software Warranty (see page 232)
• Reporting Problems to Bio-Rad (see page 233)
• Report Table Error Codes (see page 234)
Security Edition: User Access by Function
The following table lists the functions and user levels in Bio-Plex Manager™
6.1 Security Edition software. An X indicates that the user level has access to
the function.
Function
Admin Supervisor Service Clinician 2 Clinician 1 Reviewer
Functions that Apply to Whole Application
Opening Files
X
X
X
X
X
View All Screens (No Modify)
X
X
X
X
X
View Audit Trail
X
X
X
X
X
Print
X
X
X
X
X
Save
X
X
X
X
Save As
X
X
X
X
Export
X
X
X
X
X
Electronically Sign Document
X
X
X
X
X
Functions that Apply to Instrument Control
Start Up
X
X
X
X
Shut Down
X
X
X
X
Warm Up
X
X
X
X
Remove Bubbles
X
X
X
X
Wash Between Plates
X
X
X
X
Run Calibration
X
X
X
X
Enter Calibration Lot/Exp Date
X
X
X
X
Run Validation
X
X
X
X
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Bio-Plex Manager Software 6.1 User Guide | Appendix
Function
Admin Supervisor Service Clinician 2 Clinician 1 Reviewer
Enter Validation Lot/Exp Date
X
X
X
X
Instrument - Set Up
X
X
X
X
Instrument - Reconnect
X
X
X
X
X
Instrument Info
X
X
X
View Operation Log
X
X
X
X
X
X
View Calibration Log
X
X
X
X
X
X
View Validation Log
X
X
X
X
X
X
X
X
Functions that Apply to Protocol File—General
New Protocol File
X
X
New Results from Protocol
X
X
Functions that Apply to Protocol File, Describe Protocol View
Author Field
X
X
Description Field
X
X
Functions that Apply to Protocol File, Select Analytes View
Select/Remove Analytes
X
X
Add/Delete/Rename/Edit Panel
X
X
Functions that Apply to Protocol File, Formal Plate View
Format Wells
X
X
Change Number of Unknown
Samples
X
X
X
Functions that Apply to Protocol File, Enter Standards Info View
Enter Standards Info (Description
& Concentration)
X
X
Enter Std Curve Regression
Method
X
X
Enter Std Curve Adv Settings
X
X
Enter Std Concentration Units
X
X
Enter Recovery Range
X
X
Select External Standards
X
X
Functions that Apply to Protocol File, Enter Sample Info View
Enter Sample Info (Sample
Description)
X
X
X
Enter Sample Info (Sample
Dilution)
X
X
X
Functions that Apply to Protocol File, Enter Controls Info View
Enter Controls Info
X
X
Functions that Apply to Protocol File, Run Protocol View
Start a Run
220
X
X
X
X
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Security Edition: User Access by Function
Function
Admin Supervisor Service Clinician 2 Clinician 1 Reviewer
Rerun/Recovery Mode
X
X
Select Beads to Count
X
X
X
X
Select Bead Count Method (Per
Well/Per Analyte)
X
X
Set Sample Timeout
X
X
Advanced Settings
X
X
Change Raw Data Display
Options
X
X
X
X
X
Print Histogram & Bead Map
X
X
X
X
X
Histogram—Restore Default
Gates
X
X
Histogram—Adjust DD Gates
X
X
Histogram—Autoscale
X
X
X
X
X
Histogram—Log/Linear View
X
X
X
X
X
Histogram—Zoom
X
X
X
X
X
Histogram—Max/Restore
X
X
X
X
X
Bead Map—Log/Lin
X
X
X
X
X
Bead Map—Zoom
X
X
X
X
X
Bead Map—Max/Restore
X
X
X
X
X
X
X
X
Functions that Apply to Results File—General
New Protocol from Results
X
X
Change DD Gates
X
X
Refresh Calculations
X
X
Modify Plate ID
X
X
Functions that Apply to Results File, Describe Protocol View
Author Field
X
X
Description Field
X
X
Functions that Apply to Results File, Select Analytes View
Select/Remove Analytes
X
X
Add/Delete/Rename/Edit Panel
X
X
Functions that Apply to Results File, Format Plate View
Formatting Wells
X
X
Functions that Apply to Results File, Enter Standards Info View
Enter Standards Info (Description
& Concentration)
X
X
Enter Std Curve Reg Method
X
X
Enter Std Curve Advanced
Settings
X
X
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Bio-Plex Manager Software 6.1 User Guide | Appendix
Function
Admin Supervisor Service Clinician 2 Clinician 1 Reviewer
Enter Standard Concentration
Units
X
X
Enter Recovery Range
X
X
Select External Standards
X
X
Functions that Apply to Results File, Enter Sample Info View
Enter Sample Info (Desc)
X
X
Enter Sample Info (Dilution)
X
X
Functions that Apply to Results File, Enter Controls Info View
Enter Controls Info (Desc, Conc,
& Dilution)
X
X
Functions that Apply to Results File, Raw Data View
Change Display Options
X
X
Set/Clear Outliers
X
X
X
X
X
Functions that Apply to Results File, Report Data View
Set/Clear Outliers
X
X
Show/Hide Outliers
X
X
X
X
X
Report Table Options
X
X
X
X
X
Organize by Type
X
X
X
X
X
Organize by Group
X
X
X
X
X
Show Replicates
X
X
X
X
X
Sort
X
X
X
X
X
Show Help for Table Legend
X
X
X
X
X
Select Analytes to View
X
X
X
X
X
Functions that Apply to Results File, Standard Curve View
Regression Type
X
X
Same Reg Type—All Analytes
X
X
Logistic Weighting Options
X
X
Labels
X
X
X
X
X
Error Bars
X
X
X
X
X
Analyte
X
X
X
X
X
Axis Transformation
X
X
X
X
X
Swap XY Axes
X
X
X
X
X
Turning On and Off Secure Mode
Turn On/Off Secure Mode
Unlock Application Override
X
X
NOTE: System Administrator creates Groups and assigns Users to Groups.
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Normalization Formulas
Normalization Formulas
Bio-Plex Manager uses the following formulas to calculate normalization
values.
• All calculations are done first on a per well basis
• All fluorescence values are adjusted mean fluorescence
(Fl – Background), unless no blanks are provided in the experiment
• All calculations are done on a per gene level, except where
indicated
• The following equations are written for gene expression studies.
You can substitute “gene” with “analyte” and use the same
calculations for normalizing protein concentrations with internal/
endogenous controls
For these calculations:
Gene of Interest
=
Analyte of Interest
Housekeeping Gene
=
Endogenous Control Protein
Variables below are
HKG = Housekeeping Gene
FI = Fluorescence Intensity
n = number of housekeeping genes
Average FI(sample) = FIwell1, FIwell2 . . . FIwelln
Norm Ratio is the software’s term for normalized expression values. It is
calculated three different ways, depending on the options chosen in the
Normalization Settings dialog. These are outlined below as Norm Ratio, Norm
Ratio’ and Norm Ratio’’.
A control sample may be assigned in order to:
• Compare results across multiple plates (control sample must be
the same biological sample on each plate)
• Simply express all results on the same plate, relative to the controls
sample
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Bio-Plex Manager Software 6.1 User Guide | Appendix
Norm Ratio
The calculation is a simple division of each sample’s calculated value by the
control sample’s calculated value.
When internal controls are not chosen:
Norm Ratio (sample) =
When internal controls are chosen:
Norm Ratio’’(sample x)=
Norm Ratio’
When internal controls are chosen and no control samples are identified:
Norm Ratio’(well)
=
To figure Norm Ratio’ per sample, use the average normalized value of each
sample.
Norm Ratio’ (sample)
=
Norm Ratio’’
Calculating Norm Ratio” allows the researcher to express all data relative to
the control, so that data across experiments using the same control samples
can be compared.
When control sample and housekeeping genes (internal controls) are chosen:
Norm Ratio’’ (sample x)
224
=
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Normalization Formulas
Normalized Fluorescence Values (Norm FI)
(This calculation requires internal controls to be chosen.)
Normalized Fluorescence Values (Norm FI) are fluorescence values adjusted
to account for experimental sample to sample variations (loading differences).
Norm FI can be used to compare results across experiments to get a clearer
picture of experimental variability. It is left to the researcher to decide the
appropriateness of this calculation for a particular experiment.
Norm FI is calculated as follows when housekeeping genes (internal controls)
are chosen.
Norm FI (well) = FI Gene of Interest *
where Avg All HKG FI (ALL wells) = the average FI for all housekeeping genes in
every well of the experiment.
This number serves as a mean factor around which all others will fall and
converts a “unitless” ratio back into FI.
The next calculation averages per well data to arrive at the per sample
normalized fluorescence value (Norm FI (sample)).
Norm FI (sample) =
When control is chosen, Norm FI is calculated as follows.
Norm FI (sample x) = Norm ratio (sample x) * Average FI (control sample)
Average FI (control sample) serves as a mean factor around which all others will
fall and converts a “unitless” ratio back into FI.
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Bio-Plex Manager Software 6.1 User Guide | Appendix
Ratio (Simple) and the Grouping Function
If you are not using the grouping function present in the plate formatting
section of the software, this does not apply to you.
When using this function, remember that the Ratio value presented is only
relevant for making comparisons within a group. Outside the group, these
numbers are arbitrary and meaningless (unless the reference sample is
another instance of the exact same biological sample in every group).
Since data outside of groups cannot be compared, the grouping function in
the plate formatting section of the software defeats any inter- and many intraexperiment normalization. Relative differences among samples within the
same group are still valid.
If you are normalizing your data, ratio calculation is a simple ratio of the
calculated value of a reference sample of the group, compared to the
calculated value of each member of that group. Data can be presented as
member over reference, or as reference over member.
Ratio =
If you are not normalizing your data, the ratio is calculated using FI values.
Ratio =
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Basic Concepts
Basic Concepts
The following is a brief overview of the basic principles of the Bio-Plex
suspension array system. For more information, see "Practical Flow
Cytometry", 3rd edition, by Howard M. Shapiro, M.D. (New York: Wiley-Liss
Inc., 1995).
Microspheres
xMAP microspheres are highly uniform, 5.5-micron polystyrene beads that
have been crosslinked during polymerization for physical and thermal stability.
Microspheres are grouped into sets; each set is then embedded with specific
quantities of two fluorescent dyes. The ratio of these two dyes gives each set
of microspheres a unique spectral address.
Each set of microspheres is then conjugated with a different reactant specific
for a particular target analyte. Reactants can include enzyme substrates,
receptors, antigens, and antibodies. A conjugated bead-reactant mixture can
be mixed with a sample to create, for example, a capture sandwich
immunoassay.
NOTE: To ensure the stability of the spectral address, it is essential to protect
the microspheres from light. Do not subject microspheres to prolonged high
temperatures, and protect them from freezing and thawing.
Reporter Fluorochromes
When you mix your samples with the microspheres, the target analytes bind to
the reactants on the surface of the beads. The amount of analyte that binds to
the beads is then quantitated through the use of a fluorochrome, Rphycoerythrin. The intensity of this fluorochrome as detected by the array
reader is directly proportional to the amount of analyte present. The excitation
wavelength of the array reader reporter channel is 532 nm; the emission
wavelength is 575 + 12 nm. Specific information on R-phycoerythrin is shown
in the following table.
R-Phycoerythrin Specifications
Formula weight (Daltons)
240,000
Absorbance max (nm)
480, 546, 565
Extinction max (M-1cm-1)
1,960,000
Emission max (nm)
578
Quantum yield
0.82
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Bio-Plex Manager Software 6.1 User Guide | Appendix
Fluidics
There are two fluidic paths in the array reader. The first path includes a
syringe-driven mechanism that controls the sample uptake. This mechanism
permits small sample uptake volumes from small reaction volumes. The
syringe-driven system transports a user-specified volume of sample from a
microplate well to the cuvette. The sample is injected into the cuvette at a
steady rate, and the microspheres in the sample are aligned in a single file
through the path of the two lasers.
Following analysis, the first sample path is automatically purged with sheath
buffer fluid via the second fluidics path. This process effectively removes
residual sample in the tubing, valves, and sample needle.
Excitation
There are two lasers in the array reader: one for classifying each microsphere
and its associated analyte, and the other for quantitating the amount of
analyte bound to each microsphere. The first laser, the classification or "red"
laser, excites the dyes embedded in each microsphere; the fluorescent signal
is discriminated with selective emission filters and converted into intensity
units by fluorescence detectors and a digital signal processor, and the
microsphere is classified. The second laser, the reporter or "green" laser,
excites the fluorochromes bound to the target analytes on the surface of the
microspheres; again, the fluorescent signal is discriminated with selective
emission filters and converted into intensity units by fluorescence detectors
and a digital signal processor, and the amount of analyte is quantitated.
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Microsphere Handling
Microsphere Handling
Microsphere Dispersion
xMAP microspheres will settle and aggregate if left undisturbed. You should
always ensure that they are evenly distributed before dispensing. Gentle
vortexing is the most effective method of mixing for most coated microsphere
preparations; however, sonication can also be used to separate aggregated
microspheres prior to coupling. After sonication, microspheres generally
remain dispersed for about 1 hour. Sonication is most effective if applied to a
centrifuged pellet of microspheres. In order to maintain bead concentration,
we suggest you centrifuge your stock beads before sonication.
The following two indirect sonication methods are effective at separating
aggregates in closed containers of microsphere preparations. Inserting a
sonicator probe into preparations is not recommended.
Probe Sonicator
To disperse a microsphere pellet using a probe sonicator:
1. Place probe tip in a small bath of water.
2. Insert the end of the microsphere tube near the tip of the sonicator
probe, without touching it. Do not immerse the tube closure.
3. Adjust sonication for optimal disruption and pulse sonicate until the
microsphere pellet is dispersed.
Bath Sonicator
To disperse a microsphere pellet using a bath sonicator:
1. Turn on the bath sonicator and examine the surface for an area of
maximum disruption.
2. Insert the end of the tube into the sonicator bath near the area of
maximum disruption.
3. Sonicate until the microsphere pellet is dispersed.
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Bio-Plex Manager Software 6.1 User Guide | Appendix
Enumeration of Microsphere Suspensions
xMAP microspheres are provided at standard concentrations. The microsphere
yield after a coupling process may be less than the starting concentration due to
loss during wash steps. The microsphere loss can vary according to operator
technique, coupled reactant properties, and scale of coupling.
It is not advisable to construct an assay without defining the concentration of
the subject-coupled microsphere preparation. The total surface area (total
number of microspheres) represented in an assay is a critical variable, so the
optimization and control of this variable begins with manipulation of known
microsphere numbers. Microspheres can be counted with a hemocytometer.
Please follow counting and calculation methods outlined in the instructions for
your hemocytometer.
Microsphere Separation Methods
Bio-Plex assay development often requires separation of microspheres from
an aqueous matrix. This can be done using either centrifugation or vacuum
methods.
CENTRIFUGATION
Microsphere coupling protocols require separation of microspheres from
reaction mixtures or wash buffers by means of centrifugation. Generally, a
tabletop microcentrifuge that can spin 1.5 ml microcentrifuge tubes can pellet
microspheres in 1-2 minutes. The g-forces vary in these instruments from
approximately 6,000 to 13,000 X g.
VACUUM
A vacuum separation method can be used for routine high-throughput
washing required for washed assay procedures. In this case, the microsphere
reactions are performed in microtiter filter-bottom 96-well plates. A vacuum is
applied to the plate, allowing the liquid to filter through while retaining the
microspheres on the filter. Resuspension is accomplished by adding adequate
fluid to each well and by repetitive vigorous pipetting.
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Microsphere Handling
Microsphere Agitation During Assay
Agitation of Bio-Plex assays with a plate shaker during incubation steps
prevents microspheres from settling during extended incubation periods. The
benefit of agitation may not be discernible for some assay conditions and
should be evaluated according to the requirements of a given application. It is
important to resuspend the beads in an assay for 30 seconds before
performing a reading.
Microsphere Stability and Storage
xMAP microspheres are light sensitive and should be protected from light
during all stages of storage and usage. In addition, freezing conditions and
organic solvents should be avoided.
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Bio-Plex Manager Software 6.1 User Guide | Appendix
Software Warranty
Bio-Rad Laboratories warrants that Bio-Plex Manager™ software shall
substantially conform, in all operational features, to Bio-Rad's current
specifications as published in Bio-Rad's user and installation guides and that,
when properly installed, it will be free of material defects which affect system
performance.
The Purchaser must notify Bio-Rad in writing, within 30 days of delivery of the
software (not including delivery of any subsequent modifications to the software),
of any defect. If the software is found to be defective by Bio-Rad, Bio-Rad's sole
obligation under this warranty is to remedy the defect in a manner consistent with
Bio-Rad's regular business practices. For a defect which adversely affects the
performance of the software, Bio-Rad shall use its best efforts to cure such defect
as soon as reasonably practicable after receipt of Purchaser's notice. For minor
defects, Bio-Rad shall use its best efforts to correct such minor defects in the next
release of its software. If, however, Bio-Rad is unable to cure a major defect within
90 days of receipt of Purchaser's notice, Purchaser shall have the option to cancel
this agreement, whereupon Bio-Rad shall refund only the software fees paid.
The warranties set forth in this agreement are in lieu of all other representations
and warranties, expressed or implied, including warranties of merchantability and
fitness for a particular purpose and any other statutory or common-law warranty.
Bio-Rad on its own behalf expressly disclaims and excludes any and all such
other representations and warranties. Liability of Bio-Rad to Purchaser, if any, for
breach of warranty, or any other claim relating to this agreement, shall be limited to
the total amount of software fees paid by Purchaser to Bio-Rad. In no event shall
Bio-Rad be liable for incidental or consequential damages, loss of business or
profits, special or indirect damages of any nature whatsoever. No amendment,
waiver, or other alteration of the warranties in this agreement may be made except
by mutual agreement in writing.
Purchaser agrees that Bio-Rad's liability arising out of contract, negligence, strict
liability in tort or warranty shall not exceed the amount of software license fees
paid by Purchaser.
This manual and the software (computer program) described in it are copyright
Bio-Rad Laboratories, Inc. with all rights reserved worldwide. Under the copyright
laws, this manual and the software program contained herein may not be copied,
in whole or in part, without the prior written consent of Bio-Rad, except in the
normal use of the software or to make a backup copy. This exception does not
allow copies to be made for others, whether or not sold, but all of the materials
purchased (with all backup copies) may be sold, given or loaned to another
person. Under the law, copying includes translating into another language or
format.
A multiuse license may be purchased to allow the software to be used on more
than one computer owned by the Purchaser, including a shared disk system.
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Reporting Problems to Bio-Rad
Reporting Problems to Bio-Rad
Included in your Bio-Plex Manager installation is a program called Solobug.
You can use this program to request features and design changes, or report
noncritical problems.
NOTE: For critical problems, contact Bio-Rad Technical Support at the
numbers listed on the inside front cover of this manual.
To use Solobug, select Solobug from the Start menu, enter your information
and a description of the request in the Solobug window, and click Save. This
will create a report that you can attach to an email and send to Bio-Rad.
Please send your Solobug files to Bio-Rad at:
[email protected] (in the U.S.)
[email protected] (International)
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Bio-Plex Manager Software 6.1 User Guide | Appendix
Report Table Error Codes
Certain data that cannot be measured or calculated will be flagged in the
Report Table.
Columns and their possible error codes are listed below.
Column
FI
FI - Background
Error Code Cause
***
No data present
---
Marked as an outlier
***
No data present
(negative values, if any will be displayed
---
Marked as an outlier
SD
***
No data present
CV%
***
No data present
Obs Conc (4PL/
5PL)
***
Out of Range - Above asymptote of equation
OOR<
Out of Range - Below asymptote of equation
OOR
*Value
Obs Conc (Linear)
234
No data present
OOR>
***
Out of Range - Math Error
Above or below standards FI
No data present
OOR
Negative extrapolated data
*Value
Positive extrapolated data or
Data above/below the standards FI
Exp Conc
***
No data entry - this value is input by user
OBS/Exp * 100
***
OOR displayed in Obs Conc column
Conc in Range
OOR< or
OOR>
Sampling Error
1
Low bead number detected in the well
2
Aggregated beads detected in the well
3
Classify efficiency problem detected in the well
4
Region selection problem detected in the well
5
Platform temperature problem detected in the well
Obs Conc is not within the specified recovery range
of standards conc
(the recovery range is specified by the user)
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Bio-Plex Manager Software 6.1 User Guide
Glossary
Term
Definition
Accuracy
The closeness of a measured value to the actual value or the difference
between expected and measured values. Using the Reporter Validation
Kit, accuracy is defined as the percent difference between the measured
regression curve and the expected or actual data points.
Aggregated
microspheres
Microspheres that have associated. Two microspheres that have
associated are called a doublet.
Analyte
The substance being measured, identified, quantitated, or otherwise
analyzed in an experiment. In the Bio-Plex system, analytes are
molecules that are being quantitated.
Antibody
Immunological protein capable of binding a unique antigen via a specific
binding site or epitope. Used in immunological assays to quantitate the
amount of a specific analyte present.
Assay
The bead and reagent mixture that, when combined with sample, is
loaded into microplate wells and read by the array reader.
Background (noise)
Fluorescent intensity measured by the reporter channel that is due to
non-specific binding of a fluorochrome and the electronic noise of the
array reader. If you include blank wells in your microplates, you can
calculate the average background intensity and subtract it from the
intensity readings of your standards and unknowns.
Bead
A microsphere.
Calibration
A process that uses microspheres (calibrators) with a stable fluorescent
intensity to adjust the gain settings in the array reader's detectors for
optimal and consistent microsphere classifications and reporter
readings, over time and across different array readers.
Calibrators
Microspheres with stable fluorescent intensities used to standardize the
signal output of the array reader.
Capture antibody
An antibody used in sandwich immunoassays that binds or "captures" a
specific analyte for quantitation.
Carboxylated
microspheres
Microspheres with chemically modified surfaces covered with carboxyl
functional groups (COOH) that allow covalent biomolecular attachment.
Classification channels
Fluorescent detection channels of the array reader that detect the
fluorescent intensities of two dyes embedded in each microsphere. If the
signals from the two dyes place the bead within a specified classification
region, the bead is identified as a unique analyte in a sample.
Classification regions
Defined regions in a dot plot bead map that are used to identify beads in
an assay based on the fluorescent signals from their embedded dyes.
Classify validation
A validation procedure for measuring the ability of the array reader to
efficiently classify assay beads into specified regions.
Clumping
The aggregation of two or more microspheres.
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Bio-Plex Manager Software 6.1 User Guide | Glossary
Term
Definition
Concentration in range
A measure of unknown, standard, and control concentrations that are
reliable, based on the recovery range of valid standards. Values outside
the range are flagged as >OOR or <OOR.
Conjugated
microsphere
A microsphere set that has been chemically modified so that its surfaces
are covered with molecules that bind with the target analytes in an assay
system.
Cytokine
An immunological protein that can signal a response in the immune
system.
Detection antibody
An antibody used in capture sandwich immunoassays that binds a
captured analyte to a fluorochrome, allowing for quantitation of the
protein. Detection antibodies may be biotinylated, which allows for
addition of a streptavidin-bound fluorochrome, or the detection antibody
may be directly labeled with a fluorochrome.
Doublet
Two microspheres that have associated. The resulting signal of the
associated microspheres is twice that of a single microsphere (singlet).
Doublet discriminator
A channel that measures the amount of light scatter from particles that
flow past the red laser. Light scatter is directly proportional to particle
size, and the channel is designed to identify particles that are smaller or
larger than a single microsphere, including microspheres that are
clumped or aggregated.
Dynamic range
The calculated number of decades covered by the reporter channel
scale of the array reader. Range is calculated using the Reporter
Validation Kit.
Emission spectrum
The wavelength range of light emitted by an excited fluorochrome when
its electrons fall from a higher to a lower energy state. Expressed in
nanometers (nm).
Ex/Em
A common way of reporting a molecule's excitation and emission
wavelengths (for example, Ex/Em = 488/520 nm).
Excitation spectrum
The wavelength range that excites a molecule's electrons to a higher
energy state. Expressed in nanometers (nm).
External standard
A standard that has been imported from a different file.
Fit probability
A statistical measure of goodness of fit that incorporates the residual
SSE (sum of the square of the error). A fit probability of 1 indicates a
perfect fit, while a fit probability of 0 indicates no fit.
Fluidics validation
The process of verifying the well-to-well carryover of beads in the array
reader.
Fluorescence
The emission of light that occurs when the electrons of a fluorochrome
drop to a lower energy state.
Fluorochrome
A fluorescent molecule.
Gate
An electronic method of discriminating or eliminating aggregated
microspheres in the analysis of an assay. A gate separates singlet beads
from aggregated beads.
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Term
Definition
Group
A group of wells defined on a plate. You can calculate the ratio of the
fluorescent intensities of the group's member wells to the fluorescent
intensity of a Reference well in the group.
Immunofluorescence
A technique that uses a covalently linked fluorochrome-antibody
complex to detect or quantify a particular antigen.
Instrument threshold
The instrument threshold is a measure of system noise, and is the
reporter channel signal from a blank bead that contains no reporter dye.
Kinase
An enzyme that functions to phosphorylate specific proteins. Kinases are
responsible for the activation of a variety of proteins through the process
of phosphorylation.
Laser
A highly purified source of light used to excite fluorochrome electrons.
An acronym for Light Amplification by Stimulated Emission of Radiation.
Linearity
A measurement of the coefficient of determination, or R2 value, of a set
of standard beads. An R2 value of 0.995 or greater is an acceptable
linearity value using the Bio-Plex Validation Kit.
Logistic regression
A regression model for binary (dichotomous) outcomes. The data are
assumed to follow binomial distributions with probabilities that depend
on the independent variables.
Lower limits of
Quantitation
For any given assay, this is the lowest value Bio-Plex Manager will report
in the Concentration in Range column of the data table. Values below
this range are considered less trustworthy based on either the poor
recovery values of standards below this range or background of the
assay. For more details, review the definition of Concentration in Range.
Median fluorescent
intensity
A relative measurement of fluorescent intensity based on the median, a
robust statistical measure.
Microparticle
A solid substance with a diameter in the micrometer range. Often used
as a synonym for a microsphere.
Microsphere set
A set of multianalyte microspheres containing a unique mixture of two
distinct fluorochromes to distinguish them from other multianalyte
microspheres.
Microspheres
Latex spheres with a diameter in the micrometer range. Also called
beads.
Multianalyte or multiplex An analysis of several assays or tests performed simultaneously in the
same reaction container.
Operational qualification The process of determining that an instrument is fit for its intended use.
The Bio-Plex Validation Kit is a tool for operational qualification.
Optics validation
The process for verifying the alignment of the optics assembly of the
array reader.
Outlier
A well or replicate group of wells whose measured value is outside the
normal range. You can exclude outliers from statistical calculations.
Panel
A group of related bead sets. For example, the Human Cytokine panel
includes bead sets for Human IL-2, Human IL-4, Human IFN gamma,
and other human cytokines. You can select multiple bead sets from the
same panel or sets from different panels in the same multiplex assay.
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Bio-Plex Manager Software 6.1 User Guide | Glossary
Term
Definition
PE
See phycoerythrin.
Phosphoprotein
An enzyme that functions to phosphorylate specific proteins.
Phosphoproteins are responsible for the activation of a variety of
proteins through the process of phosphorylation.
Photobleaching
A chemical reaction caused by exposure to light, in which a
fluorochrome is converted into a differently fluorescent or nonfluorescent compound.
Photomultiplier tube
A light detector typically used in fluorescence detection systems,
designed to convert a fluorescent signal into an electronic signal that can
be quantitated. PMT settings display as an RP1 number during
calibration, when using Bio-Plex Manager.
Phycoerythrin (PE)
The fluorochrome used as the reporter molecule in Bio-Plex assays.
Excitation = 546 nm, emission = 575 nm.
PMT
See photomultiplier tube
Precision
A measure of the reproducibility of replicate readings, usually
represented by the coefficient of variation (%CV).
Protocol file (*.pbx or
*.spbx)
A file containing the settings of a reading, including the microplate wells
to read, the analytes to detect, sample size, etc. To perform a reading,
you open a Protocol file, select the settings, and then run the Protocol.
You can then save the Protocol settings and reuse or modify them. After
a reading, a Results file is created containing the settings from the
Protocol as well as the data results. The raw data from the most recent
reading are also stored in the Protocol file. In Bio-Plex Manager Security
Edition, Protocol files may be Secure files, in which case they have the
file extension *.spbx.
Quality control
Procedures and guidelines that determine conformity to requirements.
Recovery percentage
A mechanism for assessing the fit of the standard curve to the actual
standards. For each analyte standard, an observed concentration is
back-calculated from the standard curve and the fluorescent intensity.
This is divided by the expected concentration and multiplied by 100 to
give the recovery percentage.
Recovery range
The range of acceptable recovery percentages (see above). For
example, a recovery range of 80–120% means that the observed
concentration of a standard should be within 80–120% of the expected
concentration. Concentrations of standards and unknowns that fall
outside this range are flagged as unreliable.
Reference
A well within a group of wells defined on a plate that is used as a
reference. You can calculate the ratio of the fluorescent intensity of the
Reference well to the fluorescent intensities of the other wells in the
group.
Region
The region of a fluorescent color map used to identify a particular bead
set. Each bead set is embedded with specific quantities of two
fluorescent dyes; the combination of these fluorochromes, as detected
by the array reader, places the bead set within a unique region on the
color map, thereby identifying the set and its associated analyte.
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Term
Definition
Reporter
A fluorescent molecule that is incorporated into an assay in such a way
that the fluorescence intensity is directly proportional to the amount of
analyte in an assay.
Reporter channel
The channel of the array reader that detects the fluorescent signal of the
reporter molecule (phycoerythrin).
Reporter system
Any combination of molecules in solution that serves to quantitate the
analytes in a particular assay.
Reporter validation
A standard method for verifying the performance of the reporter channel
of the array reader. The five primary parameters that verify accurate
performance of the reporter channel include linearity, dynamic range,
sensitivity, accuracy, and slope of the response.
Residual variance
A statistical measure of a good fit. This is the weighted sum of the
squared errors between the observed response and the response
predicted by the fitted curve (residual SSE), divided by the number of
degrees of freedom. A small residual variance indicates a good curve fit.
Results file (*.rbx or
*.srbx)
A file containing the results of a reading, including the raw data, the
settings information from the Protocol, and tools for analyzing and
exporting the data (tables, a standard curve, export functions, etc.). Each
reading generates a new Results file. In Bio-Plex Manager Security
Edition, Results files may be Secure files, in which case they have the file
extension *.srbx.
Sample
A solution of analyte standards or unknowns that, when added to an
assay, will bind to the microspheres in that assay and be identified and
quantitated by the array reader.
Sampling error
A measure of the degree to which a sample differs from the entire
population.
Sandwich immunoassay Immunoassay for the quantitation of a specific analyte through the use of
monoclonal antibodies (Ab). A sandwich is formed when an analyte of
interest binds to a capture Ab, and then a second antibody (detection
Ab) binds to a different epitope of the protein.
Secure file
A Protocol or Results file that has been electronically "signed" by a user.
Once signed, this file is a controlled document that cannot be
overwritten by Bio-Plex Manager and that preserves a built-in audit trail
of all saved changes. If you make changes to a Secure file and save it as
a new file, the new file will not be signed; however, the audit trail will be
preserved in the new file and the file will remain a Secure file.
Sensitivity
The lowest detectable signal above instrument noise. Noise may be
attributed to the lasers, the detectors and the amplification electronics of
the array reader.
Signal
The detectable measurement unit of a reporter molecule.
Signal-to-noise ratio
The ratio of a specific assay signal to the underlying noise in the assay.
Signal to noise is typically used to measure the sensitivity of an assay.
Singlet(s)
A single microsphere or a population of single microspheres. Compare
to doublets which are two microspheres associated together.
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Bio-Plex Manager Software 6.1 User Guide | Glossary
Term
Definition
Slope
Defined as the rise over the run when plotting a series of points to form a
line. Using the Reporter Validation Kit, the slope of the response is
related to the dynamic range of the array reader and yields information
about the response of the photomultiplier tube (PMT).
Spectral address
The unique fluorescent emission spectra of a microsphere.
Standard deviation
The spread in individual data points (for example, in a replicate group) to
reflect the uncertainty of a single measurement.
Standard error
The standard deviation of a set of replicates divided by the square root
of the number of replicates.
Unconjugated
microspheres
A microsphere set that has been chemically modified so that its surfaces
are covered with some functional group, but that has not been
chemically prepared for use as a component in an assay system. These
base microspheres do not carry biomolecular reactant.
Uniformity
The reproducibility of a signal over a series of replicate measurements.
Upper Limit of
Quantitation
For any given assay, this is the highest value Bio-Plex Manager will
report in the Concentration in the Range column of the data table.
Values above this range are considered less trustworthy based on either
the poor recovery values of standards above this range or saturation of
the assay. For more details, review the definition of Concentration in
Range.
Validation
A formal process for documenting that an instrument is fit for its
intended use and is kept in an appropriate state of maintenance and
calibration.
Validation kit
A set of reagents and procedures designed to verify the performance of
the array reader apart from an assay. Bio-Plex Validation Kit 4.0
assesses the alignment of the optics assembly, measures the
performance of the reporter channel, evaluates the fluidics system, and
verifies the classify efficiency of the array reader.
Variance
The mean square deviation of a variable around the average value.
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Bio-Plex Manager Software 6.1 User Guide
Index
Numerics
B
21 CFR Part 11 Rule 168
Back flush 44
Basic Concepts 219
Excitation 220
Fluidics 220
Microspheres 219
Reporter Fluorochromes 219
Bead Map 108
Density Filtering 111
Display Options 110
Logarithmic/Linear Display 111
Magnifying and Resizing 110
Scaling the Bead Map Data 110
Bio-Plex assay and reagent website 2
Bio-Plex Manager
General Workflow 7
Installing 11
Main toolbar 16
Menu bar 16
Quick Guide 17
Required screen resolution 10
Status Bar 17
System requirements 9
Bio-Plex Manager software Editions 5
Bio-Plex MCV Plate IV 22
A
Adjusting Your Graphs 158
Advanced Run Settings 98
Bead Map Selection 98
Doublet Discriminator Gates 100
Sample Size 98
Sampling Errors 99
Save Options 99
Alcohol Flush 44
Alcohol Wash 44
Autofilling Replicate Groups 65
Autofilling Well Numbers 64
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Bio-Plex Manager Software 6.1 User Guide | Index
Bio-Plex suspension array system
Advantages 3
Components 2
Description 1
Disconnecting 14
Reconnecting 14
Bio-Rad Technical Support
Contact information
Website address 4
Defining a Replicate Group 65
Defining Blank Wells 68
Defining Control Wells 67
Defining Standard Wells 66
Defining the Reference 71
Defining Unknown Sample Wells 63
Deleting and Renaming Custom Panels 61
Deleting Well Formatting 68
Doublet Discriminator Gate Range
Changing 106
Drain 44
C
Calculating Concentrations Automatically
86
Calibration
Logging the Calibration 30
Performing the Calibration 30
Setting Up the Calibration 29
Changing a Replicate Group 66
Changing the Doublet Discriminator Gate
Range 123
Changing Well Formatting 68
Choosing Graph Options 150
Combining Panels of Analytes 61
Compatibility with Luminex Software 6
Creating a New Panel of Analytes 56
Customizing Analytes and Panels 55
D
Data Normalization 193
Assigning a Control Sample 195
Calculations Based Upon Your
Settings 198
Factor Calculations 199
Predefined Schemes 197
Report Table Options 196
Selecting Internal Control(s) 194
Defining a Group 70
242
E
Editing a Panel of Analytes 60
Entering Concentration Units 87
Entering Concentrations 85
F
Fluidics Functions 43
G
Generating Results from a Protocol 103
Glossary 205
Graphing
Adding 155
Analytes across a single sample 152
Analytes across multiple samples 152
Deleting 157
Editing 156
Exporting to Other Applications 157
Samples across a single analyte 151
Samples across multiple analytes 151
Viewing Information 153
Graphing Function 149
BioPlex_6.book Page 243 Friday, September 23, 2011 11:55 AM
H
N
Hardware protection key use in USB port
12
Histogram
Magnifying 107
Resizing the Histogram 107
Scaling the Histogram Data 107
Selecting the Channel Type 106
Histogram and Bead Map 104
Normalization Definitions 197
Normalization Formulas 215
Normalization Settings 193
Normalized Fluorescence Values (Norm FI)
217
O
Obs/Exp * 100 Column 134
I
Instrument Information 43
Instrument Operations Log 45
L
Logistic Weighting 88
Luminex xPONENT software
compatibility with Bio-Plex Manager 6
M
Manually Stopping a Reading 103
Microsphere Handling 221
Agitation During Assay 223
Bath Sonicator 221
Dispersion 221
Enumeration of Microsphere
Suspensions 222
Separation Methods 222
Stability and Storage 223
P
Performing a Validation Run 38
Plate Formatting 63
Plate Groupings 69
Plate Loading Guidelines 101
Predefined Normalization Schemes 197
Preparing Protocols
Step 1. Describe Protocol 53
Step 2. Select Analytes 53
Step 3. Format Plate 62
Step 4. Enter Standards Info 72
Step 5. Enter Controls Info 90
Step 6. Enter Sample Info 92
Prime 44
Printing Graphs 158
Printing the Plate Format 71
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Bio-Plex Manager Software 6.1 User Guide | Index
R
S
Raw Data
Exporting 125
Printing 126
Setting the Plate ID 124
Table Error Codes 126
Raw Data Table 112
Bead Statistics 113
Copying the Raw Data 115
Display Options 114
Formatting 115
Sampling Error Codes 113
Set Number Format 115
Recovery Percentage Range 89
Regression Methods 87
Report Table
Concentration in Range Column 134
Context Menus 136
Copying 137
Display Options 128
Displaying Table Error Codes 130
Exclude Table Error Codes 141
Expand Replicate Info 130
Exporting 137
Organizing by Type or Group 130
Predefined Report Schemes 129
Printing 137
Resizing the Columns 136
Saving Settings 131
Selecting Columns to Display 128
Setting Number Formats 131
Showing/Hiding Outliers 135
Single or Multiple Analyte Layout 129
Toolbar 127
Report Table Column Descriptions 132
Reporting Problems to Bio-Rad 225
Resequencing Well Numbers 66
Reservoir Functions 96
Run Protocol Window 94
Bead Count 95
Sample Timeout 95
Running Protocols 93
Running the Protocol 102
Status Bar 103
Sample Needle Adjustment 18
Sanitize 44
Security Edition
21 CFR Part 11 168
Audit Trail 179
Calibration Logs 173
Document ID Number 176
Electronic Records 172
Enabling Secure Mode 171
Features 167
File Security and Validation 172
Installing 169
Instrument Operations Logs 173
Locked symbol shows Secure Mode
17
Locking Bio-Plex Manager 183
Logging Off 183
Secure Protocol and Results Files 173
Signed vs. Unsigned Files 173
Standard Mode vs. Secure Mode 168
System Requirements 169
Unlocked symbol shows Standard
Mode 17
User Access by Function 211
User Authentication 171
User Information 170
User Level Restrictions 170
Users, Passwords, and User Levels
169
Validation Logs 173
Selecting Analytes 54
Selecting External Standards 81
Selecting/Entering Calibration Control
Numbers 26
Software Editions
Security Edition 5
Standard 5
Software Licenses
Desktop 6
Instrument Control 6
Network 6
Software Warranty 224
Solobug
Reporting Problems 225
244
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Sorting Report Table Data 137
Standard Concentration Lots 73
Standard Curve 142
Copying 147
Linear vs. Logistic Regression
Methods 147
Printing 148
Regression Methods 144
Standard Curve Optimizer 185
Starting Bio-Plex Manager 13
System cable connections 14
System Controls
Eject/Retract Plate 43
Instrument Information 43
Optics Warm Up and Shut Down 24
Platform Heater 42
Remove Air Bubbles 33
Start Up 23
Unclog 34
Validation 35
Wash Between Plates 32
V
Validation
Setting Up a Validation Run 36
Validation Kit 35
Validation Log 39
Validation Type Selection 37
W
Wash 44
Well Numbering and Replicate Groups 64
U
Uninstalling Bio-Plex Manager 12
Using the Security Edition 167
245
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Bio-Plex Manager Software 6.1 User Guide | Index
246
Bio-Rad
Laboratories, Inc.
Life Science
Group
10022815 Rev B
Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01
Belgium 09 385 55 11 Brazil 55 31 3689 6600 Canada 905 364 3435 China 86 21 6169 8500
Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65
Germany 089 31 884 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 459 6100
India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460
Malaysia 60 3 2117 5260 Mexico 52 555 488 7670 The Netherlands 0318 540666
New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700
Russia 7 495 721 14 04 Singapore 65 6415 3170 South Africa 27 861 246 723 Spain 34 91 590 5200
Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 Thailand 66 2 6518311
United Kingdom 020 8328 2000
11-1432
0911
Sig 0211
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