Bio-Rad Bio-Plex Software Upgrades and Conversions User Manual

Bio-Plex Manager™ Software 6.1

User Guide

Bio-Plex Manager™ Software 6.1 User Guide

Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive,

Hercules, CA 94547 · 1-800-424-6723

10022815 Rev B

Bio-Plex Manager Software 6.1 User Guide

BIO-RAD TECHNICAL SUPPORT DEPARTMENT

The Bio-Rad Technical Support Department in the U.S. is open Monday through Friday, 5:00 a.m. to 5:00 p.m., Pacific Standard Time. Worldwide technical support is available on the Web at www.consult.bio-rad.com.

Phone: 1-800-424-6723, option 2

Fax: 1-510-741-5802

Email: LSG.TechServ.US@Bio-Rad.com (U.S.)

LSG.TechServ.Intl@Bio-Rad.com (International)

Web: www.consult.bio-rad.com

NOTICE

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage or retrieval system, without permission in writing from Bio-Rad.

Bio-Rad reserves the right to modify its products and services at any time. This user guide is subject to change without notice. Although prepared to ensure accuracy, Bio-Rad assumes no liability for errors or omissions, or for any damage resulting from the application or use of this information.

The following are trademarks of Bio-Rad Laboratories: Bio-Rad, Bio-Plex, and Bio-Plex Manager. Luminex, xMAP, and xPONENT are trademarks of Luminex Corporation. Windows is a trademark of Microsoft Corporation. Pentium is a trademark of Intel Corporation.

No rights or licenses under any of Luminex Corporation’s patents are granted by or shall be implied from the sale or acquisition of this Bio-Plex system containing Luminex technology (the “System”) to you, the end-user. By using this System, you agree that (i) the System is sold only for use with fluorescently labeled microsphere beads authorized by Luminex (“Beads”), and (ii) you obtain rights under Luminex’s patents to use this System by registering this System with Bio-Rad in accordance with the instructions accompanying this System and by Bio-Plex Manager Software 6.1 User Guidepurchasing a kit containing Beads.

Bio-Plex Manager Software 6.1 User Guide

Table of Contents

Chapter 1. Bio-Plex Suspension Array System Overview . . . . . 1

Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

For More Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Bio-Rad Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Chapter 2. Bio-Plex Manager™ Software Overview . . . . . . . . . 5

Software Editions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Software Licenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Compatibility with Luminex Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

General Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Quick Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Types of Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Key Software Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Chapter 3. Software Installation . . . . . . . . . . . . . . . . . . . . . . . . 11

System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Required Screen Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Installing the Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Microsoft.NET. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Bioplexdata.mdb File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Luminex LXR Directory and Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Hardware Protection Key (HASP Key). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Uninstalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Chapter 4. Starting the System . . . . . . . . . . . . . . . . . . . . . . . . . 15

Starting Bio-Plex Manager . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Connecting with the Array Reader and Microplate Platform . . . . . . . . 16

Communication between the Array Reader and Microplate Platform . 17

Disconnecting and Reconnecting . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

iii

Bio-Plex Manager Software 6.1 User Guide | Table of Contents

Disconnecting with Sleep Mode. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Menu and Toolbars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Status Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Quick Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Sample Needle Adjustment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Chapter 5. Controlling the System . . . . . . . . . . . . . . . . . . . . . . 25

Bio-Plex MCV Plate IV. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Start Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Optics Warm Up and Shut Down . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Opening the Calibration Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Selecting/Entering Calibration Control Numbers . . . . . . . . . . . . . . . . . 30

Setting Up the Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Performing the Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Logging the Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Start Up and Calibrate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Wash Between Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Remove Air Bubbles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Unclog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Validation Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Validation Kit Control Number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Setting Up a Validation Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Control Number Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Validation Type Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Performing a Validation Run. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Validation Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Validation Log. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Platform Heater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Instrument Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Eject/Retract Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Additional Instrument Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Cancel Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Shut Down . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Instrument Operations Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Bio-Plex Manager Software 6.1 User Guide

Chapter 6. Preparing Protocols . . . . . . . . . . . . . . . . . . . . . . . . . 53

Protocol Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Creating/Opening Protocol Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Saving Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Reducing the File Size of Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Multi-Assay Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Uses for Multi-Assay Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Creating a Multi-Assay Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Changing a Protocol from Single to Multi-Assay . . . . . . . . . . . . . . . . . 57

Protocol Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Step 1. Describe Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Step 2. Select Analytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Step 3. Format Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Plate Groupings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Step 4. Enter Standards Info . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Standard Lots. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Step 5. Enter Controls Info (Optional) . . . . . . . . . . . . . . . . . . . . . . . . . 96

Step 6. Enter Sample Info (Optional) . . . . . . . . . . . . . . . . . . . . . . . . . . 98

Chapter 7. Running Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . 99

Run Protocol Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

Bead Count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Sample Timeout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Reservoir Functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

Skip Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

Standard Curve Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

Advanced Run Settings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

Bead Map Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

Sample Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Save Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Sampling Errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Doublet Discriminator Gates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

Plate Loading Guidelines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

Running the Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

Status Bar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

Manually Stopping a Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

Generating Results from a Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

Histogram and Bead Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

Histogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

Bead Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Bio-Plex Manager Software 6.1 User Guide | Table of Contents

Bead Map Display Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Raw Data Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Bead Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

Sampling Error Codes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

Raw Data Display Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

Set Number Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

Other Table Formatting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

Copying the Raw Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

Rerun/Recovery Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Chapter 8. Using the Security Edition . . . . . . . . . . . . . . . . . . . 125

Bio-Plex Manager™ Security Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Background on 21 CFR Part 11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

Standard Mode vs. Secure Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Installing and Starting Bio-Plex Manager Security Edition . . . . . . . . . . . . . . . 127

Users, Passwords, and User Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

User Level Restrictions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

User Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

Enabling and Disabling Secure Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

User Authentication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

Electronic Records . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

File Security and Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Calibration, Validation, and Instrument Operations Logs . . . . . . . . . . . . . . . . 131

Secure Protocol and Results Files . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

Unsigned Protocol and Results Files . . . . . . . . . . . . . . . . . . . . . . . . . 131

Signed Protocol and Results Files . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

Document ID Number and Signature . . . . . . . . . . . . . . . . . . . . . . . . . 134

Adjusting the Number of Unknown Samples in a Protocol . . . . . . . . 135

Generating a Results File from a Protocol File. . . . . . . . . . . . . . . . . . 136

Audit Trail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

Viewing the Audit Trail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Protected Directories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

Locking Bio-Plex Manager . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Logging Off. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Chapter 9. Analyzing the Results . . . . . . . . . . . . . . . . . . . . . . 143

Results Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Opening a Results File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

Saving a Results File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

Bio-Plex Manager Software 6.1 User Guide

Reducing the Size of a Results File . . . . . . . . . . . . . . . . . . . . . . . . . . 144

Results Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

Custom Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

Viewing/Changing the Protocol Settings . . . . . . . . . . . . . . . . . . . . . . 148

Changing the Doublet Discriminator Gate Range . . . . . . . . . . . . . . . 149

Raw Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

Setting the Plate ID. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

Exporting the Raw Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

Raw Data Table Error Codes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

Printing the Raw Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

Report Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

Display Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

Report Table Column Descriptions . . . . . . . . . . . . . . . . . . . . . . . . . . 158

Obs/Exp * 100 Column. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

Concentration in Range Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

Showing/Hiding Outliers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

Resizing the Columns. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

Context Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

Sorting Report Table Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

Copying the Report Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

Printing the Report Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

Exporting the Report Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

Export Format. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

Export a Subset of the Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165

Export Preferences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

Use Multiple Worksheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

Exclude Standard Curves. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

Exclude Column Headers/Footers . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

Exclude Table Error Codes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

Regression Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

Linear vs. Logistic Regression Methods . . . . . . . . . . . . . . . . . . . . . . 173

Copying the Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

Exporting the Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

Printing the Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

Graphing Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

Choosing Graph Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

Viewing Information within Graphs. . . . . . . . . . . . . . . . . . . . . . . . . . . 180

Adding New Graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

Editing an Existing Graph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

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Bio-Plex Manager Software 6.1 User Guide | Table of Contents

Deleting a Graph. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

Exporting Graphs to Other Applications . . . . . . . . . . . . . . . . . . . . . . 184

Adjusting Your Graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

Printing Graphs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

Document Export Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

Bio-Plex XML Export . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

Output CSV File Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

Using Stylesheets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

Command Line Export . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

Exporting the Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

Importing Luminex xPONENT Output Files. . . . . . . . . . . . . . . . . . . . . . . . . . . 194

Exporting Results Data to Bio-Plex Data Pro™ . . . . . . . . . . . . . . . . . . . . . . . 195

Chapter 10. Standard Curve Optimizer . . . . . . . . . . . . . . . . . . 199

Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

Standard Curve Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

Concentration in Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

Determining Outliers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

Recommended Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

Standard Curve Optimizer Report . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

Residual Variance and Fit Probability. . . . . . . . . . . . . . . . . . . . . . . . . 204

Logistic Weighting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204

Chapter 11. Data Normalization . . . . . . . . . . . . . . . . . . . . . . . 207

Normalization Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

Selecting Internal Controls/Housekeeping Genes . . . . . . . . . . . . . . . 208

Assigning a Control Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

Report Table Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210

Predefined Normalization Report Schemes . . . . . . . . . . . . . . . . . . . . 211

Normalization Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212

Calculations Based Upon Your Settings . . . . . . . . . . . . . . . . . . . . . . 212

Normalization Factor Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

Security Edition: User Access by Function . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

Normalization Formulas. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

Norm Ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

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Bio-Plex Manager Software 6.1 User Guide

Norm Ratio’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

Norm Ratio’’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

Normalized Fluorescence Values (Norm FI) . . . . . . . . . . . . . . . . . . . . 225

Ratio (Simple) and the Grouping Function . . . . . . . . . . . . . . . . . . . . . 226

Basic Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

Microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

Reporter Fluorochromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

Excitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

Microsphere Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

Microsphere Dispersion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

Probe Sonicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

Bath Sonicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

Enumeration of Microsphere Suspensions . . . . . . . . . . . . . . . . . . . . 230

Microsphere Separation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

Microsphere Agitation During Assay . . . . . . . . . . . . . . . . . . . . . . . . . 231

Microsphere Stability and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

Software Warranty. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232

Reporting Problems to Bio-Rad . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

Report Table Error Codes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234

Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

Bio-Plex Manager Software 6.1 User Guide | Table of Contents

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

Bio-Plex Manager Software 6.1 User Guide | Components

The system also includes validation and calibration reagents, a selection of cytokine and phosphoprotein assays, sample preparation reagents, and a

computer running Bio-Plex Manager phase, diabetes, cancer and isotyping assays have been introduced with this version of the software.

Check www.bio-rad.com/bio-plex/x-plex for newly-available assays and reagents.

The assays contain sets of microscopic color-coded beads, each of which is conjugated with a different reactant.

Reactants can include:

•DNA

• Enzyme substrates

• Receptors

•Antigens

• Antibodies

These can be used to create, for example, a capture sandwich immunoassay.

To perform a multiplex reading, samples are mixed with conjugated microsphere and reactant mixtures; next, fluorescent reporter molecules are added. The assays are loaded into the wells of a 96-well microtiter plate, and the plate is inserted into the microplate platform. The platform and array reader are controlled by a computer running Bio-Plex Manager software.

The detection system in the array reader uses two lasers to analyze the microspheres in a flow stream. The first laser identifies each microsphere and its associated analyte based on the fluorescent signature of the microsphere, and the second measures the amount of analyte, using the reporter molecules attached to the analytes. When the reading is complete, Bio-Plex Manager displays the raw data and generates detailed summary reports.

™

6.1 software. New angiogenesis, acute

Components

A certified Bio-Rad service engineer will install the complete Bio-Plex suspension array system at your site, including the array reader, microplate platform, and computer. The system setup procedure is described in “Bio-Plex 200 System Hardware Instruction Manual”, available for download from the Bio-Rad website.

The Bio-Plex suspension array system includes the following components:

• Array reader

• Microplate platform

• Bio-Plex MCV Plate IV (required for Bio-Plex Manager 5.0 and later)

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”

Bio-Plex Manager Software 6.1 User Guide | For More Information

For More Information

For more on the principles and concepts of the Bio-Plex suspension array system, see Basic Concepts in the Appendix on page 227.

Bio-Rad Technical Support

Bio-Rad Technical Support in the United States is open Monday through Friday, 5:00 a.m. to 5:00 p.m., Pacific Time. Worldwide technical support is available on the Web at www.consult.bio-rad.com.

Phone: (800) 424-6723, option 2

Fax: (510) 741-5802

E-mail:LSG.TechServ.US@Bio-Rad.com (U.S.)

LSG.TechServ.Intl@Bio-Rad.com (International)

Web: http://www.consult.bio-rad.com

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

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.

ANALYZING LUMINEX DATA IN BIO-PLEX MANAGER

Start up and calibrate array reader

Create a protocol by specifying analytes, plate, and standards

Run the protocol

Review results and generate reports

Print and/or export the data

Bio-Plex Results Generator 3.0 converts CSV Output files from xPONENT 3.1 and 4.0 into Bio-Plex Manager data files. You can analyze the converted results on any computer running Bio-Plex Manager 4.0 or later. You can make the files available to an entire workgroup by placing them on a public server.

With earlier versions of Bio-Plex Manager, you had to separately install Bio-Plex Results Generator. Beginning with Bio-Plex Manager 6.1, Bio-Plex Results Generator is automatically included in the Bio-Plex Manager installation.

The Bio-Plex Results Generator 3.0 application (RBXGenerator_3.exe) is included as a separate product on the Bio-Plex Manager 6.1 software CD. This allows you to install it on a machine that does not have a Bio-Plex Manager installation. There is also a help file (Bio-Plex Results Generator 3.0 Online Help) to walk you through installing the Results Generator and setting up Luminex protocols for conversion.

General Workflow

To collect, analyze, and output data using Bio-Plex Manager software, follow the general steps outlined below. .

General Workflow

Figure 2. Bio-Plex Manager software workflow

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

Types of Files

There are two main types of files created and used by Bio-Plex Manager:

• Protocol files (see page 53) contain the settings and controls for reading a microplate

• Results files (see page 143) contain the data from each reading, and tools and reports for analyzing that data

The application database file (bioplexdata.mdb) is installed on your hard drive or file server when you install the software. This database contains calibration, validation, and instrument operation information. See page 13 for more detail.

Key Software Features

Bio-Plex Manager 6.1 is a single integrated software package that has:

Comprehensive instrument functions

• Data collection

• Maintenance

• Validation

• Automatic error detection and logging

Comprehensive data display

• Raw data presentation including tables, bead maps and histograms

• Customizable tabular presentations

• Customizable standard curve and bar graph displays

Comprehensive Calculations

• Data normalization (internal controls for gene expression analysis)

• Auto calculation and display of usable assay range

• Multiple standard curve fitting options, including Brendan Scientific, StatLIA, Logistic 5PL.

Comprehensive and versatile automated export options

• Excel (now including optional standard curve graphs)

• Comprehensive .xml file

• New Luminex CSV (comma-separated values) output

• New customizable reports through style sheets

• New custom report for export to custom macros

CFR 21 Part 11 compliance

• User access management

• Comprehensive audit trail and instrument logs

• Secure documents (results files and protocols)

Types of Files

Bio-Plex Manager Software 6.1 User Guide | Key Software Features

Bio-Plex Manager Software 6.1 User Guide

3 Software Installation

Install, or reinstall, Bio-Plex Manager™ 6.1 software as described in the following section. If you need to upgrade your software from a previous version, see the “Bio-Plex Manager 6.1 Software Upgrade and Configuration Guide” to determine the part number of your upgrade kit.

System Requirements

Component Minimum Recommended

Operating system Windows XP (XP Professional required

for running Security Edition) or

Windows 7 (32-bit) Processor Pentium 4 or equivalent, 2.8 GHz Core 2, 2.6 GHz or higher Hard disk space 80 GB 160 GB System memory 1 GB 2 GB Screen resolution 1024 x 768 (Windows XP)

1280 x 1024 (Windows 7) Screen colors 256 colors 24-bit True Color Ports for connecting instrument (required for Instrument Control

license only) Port for connecting

the HASP key Other software Internet Explorer 6.0 or later

NOTE: For Windows 7, the Instrument Control version of Bio-Plex Manager 6.1 can be run only on 32-bit; it is not compatible with 64-bit.

1 RS232 serial port and

1 USB port

1 USB port 1 USB 2.0 port

Microsoft Excel 2003 or later

Windows XP Professional

1280 × 1024

1 RS232 serial port and 1 USB 2.0 port

Internet Explorer 8.0 Microsoft Excel 2007

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

Installing the Software

To install or reinstall the software, insert the Bio-Plex Manager CD into the CD-ROM drive on your computer.

NOTES

• Before installing Bio-Plex Manager 6.1, we recommend that you first uninstall any existing version of Bio-Plex Manager on your computer

• We recommend that you turn off any antivirus protection software before installation. Such software, if active, can greatly slow the progress of the installation. If you are unable to turn off your antivirus protection software, allow 15 minutes for complete installation. Do not cancel the installation during this period

The Bio-Plex Manager Installation Program opens, displaying a navigation screen for performing the installation.

Microsoft.NET

Microsoft.NET is automatically installed on your computer when you install Bio-Plex Manager. It is required to support the logistic curve-fitting features of Bio-Plex Manager.

Installing the Software

Bioplexdata.mdb File

During installation, you will be prompted to save the application database file (bioplexdata.mdb) to a location on your hard drive or a file server. This database file contains logs of calibration, validation, and instrument operations activity for your instrument.

• For more information about the Calibration Log, see page 34

• For more information about the Validation Log, see page 45

• For more information about the Instrument Operations Log, see page 51

You can save the bioplexdata.mdb file to any folder on your computer. The default location is the Bio-Plex Manager application folder. If your computer is connected to multiple instruments, each instrument must have a separate bioplexdata.mdb file saved in a different folder.

For more information about the calibration, validation, and instrument operations logs in Bio-Plex Manager 6.1 Security Edition, see Controlling the System on page 25.

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.

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.

Bio-Plex Manager Software 6.1 User Guide | Starting Bio-Plex Manager

Microplate Platform

Power

Serial port

Array Reader

Power

P1 P2

HTF

Computer

Serial

port

Power

Serial port Power

USB1

USB2

Connecting with the Array Reader and Microplate Platform

The computer running Bio-Plex Manager software is connected to the array reader by a USB cable, and to the microplate platform by a serial cable. See the figure below for a diagram of the cable connections.

Figure 5. System cable connections

Starting Bio-Plex Manager

Communication between the Array Reader and Microplate Platform

Communication between the computer and the array reader and microplate platform is automatically established when you open Bio-Plex Manager. If Bio-Plex Manager is unable to communicate with the instrument, a dialog box prompts you to check the cable connections.

Figure 6. Connection Error dialog

Make sure the instrument is turned on, check the cable connections, and then click Reconnect in the dialog. If you are still unable to connect, try restarting the computer, array reader, and platform. You can also choose the Run Diagnostic option found under the Instrument menu. This produces a report which can help Bio-Rad Technical Support determine the cause of the problem.

Figure 7. Run Diagnostics Report

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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