Bio-Plex Manager Software 4.1 - Bio-Rad

Bio-Plex Manager Software 4.1 - Bio-Rad
Bio-Plex Manager Software 4.1
TM
User Guide
Life Science
Group
Bio-Rad
Laboratories, Inc.
2000 Alfred Nobel Drive
Hercules, CA 94547
Part no. 10003414
Bio-Rad Technical Support Department
The Bio-Rad Technical Support Department in the United States is open Monday–
Friday, 6:00 a.m. to 5:00 p.m., Pacific Standard Time. Worldwide technical support
is available on the Web at http://www.consult.bio-rad.com/.
Phone:
(800) 424-6723, option 2, option 3
(510) 741-6910, option 2, option 3
Fax:
(510) 741-5802
E-mail:
[email protected] (U.S.)
[email protected] (International)
http://www.consult.bio-rad.com/
Web:
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 damages 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. All other trademarks, including Windows, Cheminert, Tween,
Pentium, Dell, and Titertube are registered trademarks of their respective companies.
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 purchasing a kit containing
Beads.
Copyright © 2001–2005 by Bio-Rad Laboratories. All rights reserved.
ii
Table of Contents
1. Bio-Plex Suspension Array System
Overview.................................................................... 1
Components ......................................................................................... 2
Advantages........................................................................................... 3
For More Information…......................................................................... 4
Bio-Rad Technical Support................................................................... 4
2. Bio-Plex Manager Software Overview .................... 5
Software Editions.................................................................................. 5
Software Licenses ................................................................................ 5
Note on this User Guide ....................................................................... 6
Compatibility with Luminex® IS 2.3 Software ........................................ 6
General Workflow ................................................................................. 7
Types of Files ....................................................................................... 8
Key Software Features ......................................................................... 8
3. Software Installation................................................. 9
System Requirements .......................................................................... 9
Required Screen Resolution................................................................. 9
Installing the Software ........................................................................ 10
Uninstalling ............................................................................................. 12
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Bio-Plex Manager User Guide
4. Getting Started ........................................................13
Starting Bio-Plex Manager .................................................................. 13
Communication to the Array Reader and Microplate Platform ............ 13
Connecting to the Array Reader and Platform ........................................14
Disconnecting and Reconnecting............................................................15
Menu and Toolbars ............................................................................. 15
Status Bar ........................................................................................... 15
Quick Guide ........................................................................................ 16
Sample Needle Adjustment................................................................. 17
5. System Controls .....................................................21
Bio-Plex MCV Plate III......................................................................... 21
Start Up
........................................................................................ 23
Optics Warm Up and Shut Down
Calibration
.................................................. 24
..................................................................................... 25
Opening the Calibration Dialog Box ........................................................26
Calibration Microsphere Control Numbers ..............................................27
Calibration Set Up ...................................................................................29
Performing the Calibration.......................................................................30
Calibration Log ........................................................................................31
Wash Between Plates
Remove Air Bubbles
Unclog
Validation
................................................................... 33
..................................................................... 33
........................................................................................... 34
...................................................................................... 35
Validation Kit ...........................................................................................35
Setting Up a Validation Run ....................................................................36
Performing a Validation Run ...................................................................38
Validation Results....................................................................................39
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Table of Contents
Validation Log ......................................................................................... 40
Platform Heater
............................................................................ 42
Instrument Information
Eject/Retract Plate
.................................................................. 44
......................................................................... 44
Additional Instrument Functions ......................................................... 45
Cancel Operation................................................................................ 46
Shut Down
.................................................................................... 46
Instrument Operations Log ................................................................. 47
6. Preparing a Protocol .............................................. 49
Protocol Files
............................................................................. 49
Creating/Opening Protocol Files......................................................... 49
Saving Protocols................................................................................. 50
Protocol Window................................................................................. 51
Step 1. Describe Protocol
Step 2. Select Analytes
............................................................. 53
................................................................ 53
Selecting Analytes .................................................................................. 54
Customizing Analytes and Panels .......................................................... 55
Combining Panels of Analytes................................................................ 59
Step 3. Format Plate
.................................................................... 59
Plate Formatting...................................................................................... 61
Plate Groupings ...................................................................................... 68
Printing the Plate Format ........................................................................ 71
Step 4. Enter Standards Info
........................................................ 71
Selecting External Standards ................................................................. 73
Entering Standards Information .............................................................. 74
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Bio-Plex Manager User Guide
Step 5. Enter Controls Info
............................................................ 82
Step 6. Enter Sample Info (optional)
7. Running the Protocol
............................................. 85
........................................87
Run Protocol Window.......................................................................... 87
Bead Count ......................................................................................... 89
Sample Timeout .................................................................................. 89
Advanced Settings .............................................................................. 90
Bead Map Selection ................................................................................90
Sample Size ............................................................................................91
Save Options...........................................................................................91
Sampling Errors.......................................................................................92
DD Gates.................................................................................................93
PMT Voltage............................................................................................94
Plate Loading Guidelines .................................................................... 95
Running the Protocol........................................................................... 95
Manually Stopping a Reading ............................................................. 97
Generating Results from a Protocol .................................................... 97
Histogram and Bead Map
............................................................ 98
Histogram ..............................................................................................100
Bead Map ..............................................................................................104
Raw Data Table
.......................................................................... 108
Bead Statistics.......................................................................................109
Sampling Error Codes ...........................................................................110
Raw Data Display Options ....................................................................111
Other Table Formatting .........................................................................113
Copying the Raw Data ..........................................................................113
Rerun/Recovery Mode ...................................................................... 113
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Table of Contents
8. Analyzing the Results .......................................... 115
Results Files
.............................................................................. 115
Opening a Results File ..................................................................... 115
Saving a Results File ........................................................................ 116
Results Window ................................................................................ 117
Viewing/Changing the Protocol Settings........................................... 118
Changing the DD Gate Range.............................................................. 120
Raw Data
.................................................................................... 121
Setting the Plate ID ............................................................................... 122
Exporting the Raw Data ........................................................................ 122
Printing the Raw Data ........................................................................... 123
Report Table
.............................................................................. 123
Report Table Columns .......................................................................... 124
Report Table Options............................................................................ 126
Replicate Groups .................................................................................. 128
Selecting Outliers.................................................................................. 129
Obs/Exp*100 Column ........................................................................... 130
Conc in Range Column......................................................................... 131
Table Formatting................................................................................... 131
Copying the Report Table..................................................................... 132
Exporting the Report Table................................................................... 132
Printing the Report Table...................................................................... 136
Standard Curve
......................................................................... 136
Regression Methods ............................................................................. 138
Linear versus Logistic Regression Methods ......................................... 141
Copying the Standard Curve ................................................................ 142
Printing the Standard Curve ................................................................. 142
XML Export ....................................................................................... 143
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Bio-Plex Manager User Guide
9. Bio-Plex Manager Security Edition......................147
Overview ........................................................................................... 147
Users, Passwords, and User Levels ................................................. 149
Enabling and Disabling Secure Mode ............................................... 150
User Authentication........................................................................... 151
Electronic Records ............................................................................ 152
File Security and Validation...................................................................152
Calibration, Validation, and Instrument Operations Logs......................153
Secure Protocol and Results Files ........................................................153
Audit Trail ..............................................................................................159
Protected Directories.............................................................................163
Locking Bio-Plex Manager Security Edition ...................................... 163
Logging Off........................................................................................ 163
Appendix .....................................................................165
Troubleshooting Guide...................................................................... 165
Glossary ............................................................................................ 171
Table Error Codes............................................................................. 179
Security Edition: User Access by Function ....................................... 181
Basic Concepts ................................................................................. 187
Microsphere Handling ....................................................................... 189
Reporting Problems .......................................................................... 193
Software Warranty ............................................................................ 195
Index ............................................................................197
viii
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 for quantitative analysis of a wide variety of cell biology assays,
including immunoassays, complex genetic assays, and enzymatic assays.
The Bio-Plex suspension array system is fully integrated, and consists of an array
reader and microplate platform, validation and calibration reagents, a selection of
cytokine and phosphoprotein assays, sample preparation reagents, and a computer
running Bio-Plex Manager software.
Figure 1.
Bio-Plex array reader and microplate platform.
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Bio-Plex Manager User Guide
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 and 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, platform, and computer. The
system setup procedure is described in the Bio-Plex Suspension Array System
Hardware Instruction Manual.
The Bio-Plex suspension array system includes the following components:
2
•
Array reader
•
Microplate platform
System Overview
•
Bio-Plex MCV Plate III (required for use with Bio-Plex Manager 4.0 and
later)
•
A Pentium®-class PC, preinstalled with operating system, Microsoft® Excel,
Microsoft® Internet Explorer, Microsoft.net, and Bio-Plex Manager
Workstation software
•
Software package, containing Bio-Plex Manager CD-ROM, user guide, and
a hardware protection key
•
Instrument manual for array reader and microplate platform
•
Sample tube holders (two sizes, large and small)
•
Sheath fluid bottle
•
Sample needles (two long)
•
Protective shield
•
Needle adjustment tool
•
Waste bottle
•
Communications cable to connect the reader to the computer
•
Communications cable to connect the microplate platform to the computer
•
Computer monitor
•
Power cords
•
Computer keyboard
•
Computer mouse
•
HTF (High Throughput Fluidics), if selected
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.
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Bio-Plex Manager User Guide
•
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, compliant with
the Code of Federal Regulations, title 21, Part 11, “Electronic Records;
Electronic Signatures.”
For More Information…
For more information on the principles and concepts of the Bio-Plex suspension
array system, see Basic Concepts on page 187, or visit our Web site at
http://www.bio-rad.com/bio-plex.
Bio-Rad Technical Support
Bio-Rad Technical Support in the United States is open Monday–Friday, 6:00 a.m. to
5:00 p.m., Pacific Standard Time. Worldwide technical support is available on the
Web at http://www.consult.bio-rad.com/.
4
Phone:
(800) 424-6723, option 2, option 3
(510) 741-6910, option 2, option 3
Fax:
(510) 741-5802
E-mail:
[email protected] (U.S.)
[email protected] (International)
Web:
http://www.consult.bio-rad.com/
2. Bio-Plex Manager
Software Overview
Bio-Plex Manager software runs on a computer installed with the Windows® 2000 or
XP 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 (see
page 12). 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: Workstation, Desktop, and Network. These
editions and licenses are described below. The computer included with the Bio-Plex
suspension array system comes preinstalled with a compatible operating system and
Bio-Plex Manager Standard Edition, Workstation 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 users access to different
features of the software and creates a complete electronic audit trail of all
data generation and analysis. 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. Note that Bio-Plex Manager Security
Edition must be installed on the Windows® 2000 or XP Professional
operating system for full security functionality. For more details, see
Chapter 9.
Software Licenses
Bio-Plex Manager software is available with three different licenses:
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Bio-Plex Manager User Guide
•
Workstation license enables the software to control the array reader and
microplate platform and 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 described in Chapters 4, 5, and 7 are not available with this
license.
•
Network license provides Desktop licenses to multiple users over a
computer network. Like the Desktop license, it enables the software to
analyze data files but not control the array reader and platform.
Note on this User Guide
The main body of this user guide assumes that you are using the Standard Edition of
the software with a Workstation license, unless otherwise noted. The Security
Edition of the software and its user levels and restrictions are described in detail in
Chapter 9.
Compatibility with Luminex® IS 2.3
Software
If you install Bio-Plex Manager 4.0 or later on a computer with Luminex® IS 2.3
software or later, note the following:
To avoid communication conflicts with the array reader and platform, do not run
Bio-Plex Manager and Luminex IS 2.3 at the same time.
If you switch between the two software applications, you must re-calibrate the
array reader before acquiring data.
Consult your Luminex documentation for instructions on using Luminex IS 2.3
software.
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Chapter 2. Software Overview
General Workflow
To collect, analyze, and output data using Bio-Plex Manager, you follow the general
steps outlined below:
1.
Start up and calibrate the array reader.
2.
Open or create a protocol file, and specify the analytes that you want to
detect, the layout of the 96-well plate, and information about your
standards.
3.
Run the selected protocol.
4.
Review the results.
5.
Print and/or export the data.
Start up and calibrate array reader
Select analytes, standards, and plate format
Run the Protocol
Generate reports and review results
Print and export the data
Figure 2.
Workflow for Bio-Plex Manager
The Quick Guide, which displays automatically at startup, is designed to guide you
through the typical Bio-Plex Manager workflow, from start up and calibration
through shut down. Commands such as Start Up and Calibrate open dialog boxes
that contain step-by-step instructions to guide you through the procedures.
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Bio-Plex Manager User Guide
Types of Files
There are two main types of files created and used by Bio-Plex Manager:
•
Protocol files (see page 49) contain the settings and controls for reading a
microplate.
•
Results files (see page 115) 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 operations information. See page 11 for more details.
Key Software Features
The key features of Bio-Plex Manager software include:
8
•
Graphical user interface
•
Complete control of the array reader and microplate platform
•
Automated maintenance, calibration and validation functions
•
Automatic error detection and logging system
•
Bead map and histogram
•
Raw data reports
•
Data reduction
•
Logistic curve-fitting algorithms
•
Calculation of unknown concentrations and ratios
•
Export to Microsoft® Excel, text files, and XML format
•
Microplate platform heater control
•
Complete electronic audit trail of data generation and analysis (Security
Edition)
•
Different levels of user access (Security Edition)
•
Secure documents—Protocol files, Results files, log files (Security Edition)
3. Software Installation
The Bio-Plex Manager CD can be used as described in the following section to
install, reinstall, or upgrade Bio-Plex Manager software.
System Requirements
Component
Operating system
Minimum
Windows® 2000 or XP
Processor
Pentium III or equivalent,
933 MHz
4 GB
256 MB
1024 × 768
256 colors
2 RS232 serial ports
Hard disk space
System memory
Screen resolution
Screen colors
Ports for connecting
instrument (required for
Workstation license only)
Port for connecting HPK
Other software
1 USB port
Internet Explorer 6.0 or
later
Microsoft Excel 2000 or
later
Recommended
Windows® 2000 or XP
Professional
Pentium III or higher,
1 GHz or higher
40 GB
512 MB
1024 × 768
24-bit True Color
1 RS232 serial port and
1 USB port
1 USB port
Internet Explorer 6.0 or
later
Microsoft Excel 2000 or
later
Required Screen Resolution
Your computer screen resolution must be set to at least 1024 × 768 pixels for correct
display of the Bio-Plex Manager interface. The status bar and some dialog boxes will
not display properly at lower resolution. 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.
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Bio-Plex Manager User Guide
3.
In the Display Properties dialog, select the Settings tab.
4.
Drag the Screen Area slider to the right (More) until you have selected
1024 by 768 pixels. Click on OK to accept the settings.
Figure 3.
Changing the screen settings.
Installing the Software
To install, reinstall, or upgrade the software, insert the Bio-Plex Manager CD into
the CD-ROM drive on your computer.
Before reinstalling Bio-Plex Manager, we recommend that you first uninstall any
existing version of Bio-Plex Manager on your computer.
We recommend that you turn off any anti-virus protection software before
installation. Such software, if active, can greatly slow the progress of the installation.
If you are unable to turn off your anti-virus protection software, allow 15 minutes for
complete installation. Do not cancel the installation during this period.
The Bio-Plex Manager Installation Program will open, displaying a navigation
screen for performing the installation.
10
Chapter 3. Installation and Specifications
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 31.
•
For more information about the Validation Log, see page 40.
•
For more information about the Instrument Operations Log, see page 47.
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 4.1, Security Edition, see page 153.
Note that 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
version 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.
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Bio-Plex Manager User Guide
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 (Workstation, Desktop, or Network)
and the edition (Standard or Security) of Bio-Plex Manager.
Workstation 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 Bio-Plex
Manager.
Uninstalling
To uninstall Bio-Plex Manager from your computer, use the Windows Add/Remove
Programs function. Click on the Windows Start button, select Settings, select
Control Panel, double-click on Add/Remove Programs, and follow the instructions
for removing the program.
12
4. Getting Started
Before starting Bio-Plex Manager, make sure the Hardware Protection Key (see
page 11) is attached to your computer, and switch on the array reader and the
microplate platform. This turns on the optics inside the array reader and enables
communication between the array reader and software.
Avoid using other applications while Bio-Plex Manager is communicating with the
array reader. Running other applications may interrupt communication between BioPlex Manager and the array reader.
Starting Bio-Plex Manager
To start Bio-Plex Manager, click on the application icon on your desktop or select
Bio-Plex Manager 4.1 from the Programs directory on your Windows Start menu.
Figure 4.
Bio-Plex Manager 4.1 desktop icon.
The software will open and connect to the array reader and platform.
Communication to the Array Reader
and Microplate Platform
The computer running Bio-Plex Manager software is connected to the array reader
by a serial or USB cable, and to the microplate platform by a serial cable. See the
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Bio-Plex Manager User Guide
Bio-Plex Suspension Array System Hardware Instruction Manual or the manual sent
with the Luminex 100 IS 2.3 system for a description of the cable connections.
Connecting to the Array Reader and 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 will prompt you to check
the connections.
Figure 5.
Connection Error dialog.
Make sure the instrument is turned on and check the cable connections, then click on
Reconnect in the dialog. If you are still unable to connect, try restarting the
computer, array reader, and platform. If you experience further difficulties, contact
Bio-Rad technical support (see page 4).
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).
14
Chapter 4. Getting Started
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 (e.g., to
troubleshoot the instrument), go 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 will also enable automatic
connection each time you start the software, if it has been disabled (see previous
section).
See the Bio-Plex system hardware manual or the Luminex 100 IS 2.3 system manual
for specific instructions.
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.
The main toolbar includes the major instrument controls, including Start Up, 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 46).
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Bio-Plex Manager User Guide
Figure 6.
Instrument status bar.
When the array reader is performing an operation, the time remaining for the
operation will be shown in the instrument status bar.
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 Chapter 9 for more
information about Bio-Plex Manager 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 is displayed in the upper
right corner of the screen. The Quick Guide is designed to guide you through the
typical workflow when using the system, from start up and calibration through shut
down.
16
Chapter 4. Getting Started
Figure 7.
Bio-Plex Quick Guide.
By default, the Quick Guide opens automatically when you start Bio-Plex Manager.
To disable this feature, deselect the Show at Startup checkbox at the bottom of the
guide.
To open the Quick Guide after it has been closed, select Quick Guide from the View
menu.
Sample Needle Adjustment
See the Bio-Plex Hardware Manual or the Luminex manual for instructions on
installing the sample needle.
The height of the array reader sample needle must be adjusted when (1) the style of
microtiter plate has changed or (2) when the sample needle is replaced. The sample
needle height is the same for flat bottom or filter plates. Note that, for round bottom
plates, the needle must be raised so that it is slightly above the bottom of the round
bottom well.
If you adjust the sample needle for use with round bottom plates, you will not be able
to use the MCV Plate III without readjusting the needle to filter-plate height.
With the array reader and platform switched on, go to the Instrument menu, go to the
Set Up submenu, and select Adjust Needle. The Adjust Needle dialog box will open.
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Bio-Plex Manager User Guide
Figure 8.
Adjust Needle dialog.
Then follow the steps below:
1.
Click on the Eject/Retract button to eject the plate holder, and place the MCV
Plate III on the microplate platform with the arrow pointing toward the array
reader.
2.
While holding the platform door open, click on the Eject/Retract button again
to retract the plate.
3.
Tape the access door of the microplate platform open. You must be able to see
inside the access door.
Warning: DO NOT stick your hands and fingers inside the platform
at any time.
18
4.
Click on the Up/Down button in the Adjust Needle dialog to lower the needle
into the needle adjustment well in the MCV Plate III.
5.
Using the hex key needle adjustment tool (supplied with the array platform),
loosen the screw at the top of the needle so that the needle housing can move up
and down freely.
6.
Move the knurled dial on the needle arm and manually move it downward so
that it just touches the bottom of the needle adjustment well. Use a flashlight to
inspect the needle through the platform door as you adjust it. Move the needle
up and down gently a couple of times to verify that the needle is just touching
the bottom of the well.
Chapter 4. Getting Started
7.
Tighten the needle set screw so that it is no longer possible to manually move
the needle.
8.
Click on the Up/Down button in the dialog a couple of times to make sure that
the needle moves freely up and down. The needle should not push down on the
MCV Plate III.
9.
Finally, remove the tape so that the platform door can open and close normally,
and click on the Close button to close the Adjust Needle dialog.
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Bio-Plex Manager User Guide
20
5. System Controls
This chapter covers the Bio-Plex Manager commands that control basic system
functions, such as instrument start up, warm up, calibration, validation, and shut
down.
The Start Up, Warm Up, and Calibration functions must be performed prior to
running an assay with Bio-Plex Manager, and all together take about 45 minutes.
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 Bio-Plex High-Throughput
Fluidics (HTF; see caution below) 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 (e.g., 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.
Bio-Plex MCV Plate III
Bio-Plex Manager 4.0 and later require the use of the Bio-Plex MCV Plate III (MCV
stands for maintenance, calibration and validation). This plate contains wells marked
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Bio-Plex Manager User Guide
for the different types of fluids used in validation, washing, calibration, and other
functions. The Bio-Plex MCV Plate III has been modified from the previous MCV
plate to work with Bio-Plex Validation Kit 4.0, with its enhanced Reporter and
Classify bead sets. It also includes an open needle well for ease of needle adjustment.
Bio-Plex Manager 4.0 and later require the use of the Bio-Plex MCV Plate III, the
Bio-Plex Validation Kit 4.0, and the Bio-Plex Calibration Kit.
Figure 9.
Bio-Plex MCV Plate III.
When a particular procedure (e.g., Calibration) requires you to add solutions to the
Bio-Plex MCV Plate III, the dialog box describing the procedure will include a
diagram of the MCV Plate III with the wells to be loaded highlighted in blinking
yellow.
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Chapter 5. System Controls
Wells
highlighted in
yellow
Figure 10. Highlighted wells in diagram indicate wells to be filled.
Start Up
Start up is a series of fluidics functions that prepares the array reader to acquire data.
This process requires the Bio-Plex MCV Plate III, distilled water, and 70 percent
isopropanol. Start up takes approximately 10 minutes.
You do not need to wait for the array reader optics to warm up before performing the
start up procedure.
Click on the Start Up button
on the Quick Guide or 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 III.
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Bio-Plex Manager User Guide
Figure 11. Start Up dialog.
Insert the prepared plate into the microplate platform and click on OK to begin the
start up process.
Optics Warm Up and Shut Down
To ensure accurate and reproducible results, the optics (i.e., 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 start up 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.
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 will automatically
power down (though the array reader itself will remain on). Depending on the length
of the shut down, a full 30-minute warm up period may be required before more
readings can be taken.
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Chapter 5. System Controls
To begin warm up after automatic power down, click on the Warm Up button
on the main toolbar or select the command from the Instrument menu.
If you attempt to perform a reading after automatic power down, you will receive a
warning message and the optics will begin warming up.
Calibration
Calibration of the array reader is essential for optimal performance and day-to-day
reproducibility of results. Calibration is required:
•
Each day after the start up procedure is complete and the optics have
warmed up.
•
If the array reader temperature changes by more than 2º C during the course
of the day (if the temperature changes by more than 2º C, a message box
will prompt you to recalibrate).
•
Before data acquisition if you switch between Bio-Plex Manager and
Luminex IS 2.3 software installed on the same computer (see page 6).
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 III, the CAL1
beads and CAL2 beads from the Bio-Plex Calibration Kit, and DI 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 User Guide
Opening the Calibration Dialog Box
To begin calibration, click on the Calibrate button
on the Quick Guide or main
toolbar, or select Calibrate from the Instrument menu. The Calibrate dialog box will
open.
Figure 12. 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 2º C 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.
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Chapter 5. System Controls
Calibration Microsphere Control Numbers
Under Select Control Numbers, you can either select existing control numbers for
your CAL1 and CAL2 microspheres or enter new control numbers.
Select existing control numbers from the pulldown list, and the target values for the
control numbers will 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.
It is critical that you enter the correct target values for your CAL1 and CAL2
calibrators. Entering incorrect values will result in an incorrectly calibrated array
reader, which will adversely impact assay results.
To add a new CAL1 control number, click on the Add button under CAL1 control
number in the Calibrate dialog. The Add New CAL1 Control Number dialog box will
open.
Figure 13. 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 on the pulldown button next
to the field to open the calendar selection box.
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Bio-Plex Manager User Guide
Figure 14. Calendar selection box
Scroll through the calendar using the scroll buttons at the top. Select a particular date
by clicking on it.
Next, enter the DD, CL1, and CL2 target values for the control number, as printed on
the bottle, in the appropriate fields.
If you are using a Luminex-type assay from another manufacturer, do not use the DD
target value specified in the manufacturer's manual. Use only the DD target value on
the Bio-Plex CAL1 bottle.
When you are done, click on Add to close the dialog box and save your changes.
To add a new CAL2 control number, click on the Add button under CAL2 control
number in the Calibrate dialog box. The Add New CAL2 Control Number dialog box
will open.
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Chapter 5. System Controls
Figure 15. 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 High RP1
or Low RP1 target value for the control number, as printed on the bottle, in the RP1
field.
The CAL2 calibration bottle label lists two different RP1 target values: Low RP1 and
High RP1. Bio-Plex phosphoprotein assays require calibration using the High RP1
target value, while Bio-Plex cytokine assays may be calibrated with either the Low
or High RP1 target value. Other Luminex-type assays typically require calibration
with the Low RP1 target value. Check with your assay supplier.
When you are done, click on Add to close the dialog box and save your changes.
Your new control numbers will be added to the selection lists in the main Calibrate
dialog.
To delete a particular control number, first select it in the Calibrate dialog box, then
click on the Delete button.
Calibration Set Up
When you have specified your control numbers of calibration microspheres and
specified the type of calibration you want to perform (CAL1 & CAL2, CAL1 only,
or CAL2 only), click on OK in the Calibrate dialog.
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Bio-Plex Manager User Guide
Another dialog box will list step-by-step instructions for preparing the Bio-Plex
MCV Plate III.
Figure 16. Preparing the Bio-Plex MCV Plate III for CAL1 & CAL2
calibration.
When preparing the MCV Plate III, note the following:
•
Important: Before vortexing, remove the calibration beads from 2–8°C
(36–46°F) storage and allow to warm to room temperature. Vortex each
bottle for 30 seconds. Proper resuspension of the microspheres is essential
for efficient calibration.
•
Never dilute the calibration beads, and be careful to limit their exposure to
light. Store at 2–8° C immediately following calibration.
•
Load 5 drops of beads (approximately 200 µl) per reservoir (CAL1 or
CAL2).
•
The DI H2O well of the MCV Plate III holds about 3 ml of distilled water.
Performing the Calibration
Click on the Eject/Retract Plate button to eject the microplate platform plate
carrier, place the MCV Plate III in the carrier with the arrow facing toward the
platform, then retract the plate. Click on OK to start the calibration process.
As calibration proceeds, the status bar at the bottom of the Bio-Plex Manager
window will monitor 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. See the troubleshooting guide at the end of this manual.
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Chapter 5. System Controls
Number of beads per second should be 100 or higher
Figure 17. Status bar during calibration.
Because calibration beads are highly concentrated, calibration is followed by three
wash cycles using distilled water.
An alert box will notify you whether calibration has succeeded or failed. If the
calibration process failed, check the MCV Plate III to verify that the correct beads
were added to the wells. Then perform a fluidics Wash (page 45) and repeat the
above steps. For more information, consult the Troubleshooting guide on page 165.
Calibration Log
The Calibration Log provides a list of past calibration dates and times, results,
instrument settings, and other data obtained by Bio-Plex Manager. This data is stored
in a log file called bioplexdata.mdb, which by default is saved in the main Bio-Plex
Manager application folder on your computer.
Note that 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 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 open the Calibration Log, go to the View menu and select Calibration Log. The
Calibration Log viewer will open.
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Bio-Plex Manager User Guide
Figure 18. 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.
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 on Calibration Detail
to display additional information for
to return to
each calibration, including detector gain. Click on Calibration Log
the default view.
For CAL1-only calibrations, the RP1 column will display a entry of NA (not
applicable). For CAL2-only calibrations, the DD, CL1, and CL2 columns will
display entries of NA.
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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Chapter 5. System Controls
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 on the Wash
Between Plates button
on the Quick Guide or main toolbar, or select the
command from the Instrument menu. A dialog box will guide you through the steps
for preparing the MCV Plate III for a wash procedure.
Figure 19. 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 on OK. This procedure performs a series
of different fluidics operations and will take several minutes. After initial system
pressurization (5–20 seconds), the time remaining in the operation will be displayed
in the Bio-Plex Manager status bar.
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 will stop
and you will be prompted to perform an alcohol wash to force air bubbles out of the
system.
on the main toolbar or select the
Click on the Remove Bubbles button
command from the Instrument menu. A dialog box will guide you through the steps
for preparing the MCV Plate III for the procedure.
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Bio-Plex Manager User Guide
Figure 20.
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 on OK. This procedure performs a series
of different fluidics operations and will take several minutes. After initial system
pressurization (5–20 seconds), the time remaining in the operation will be displayed
in the Bio-Plex Manager status bar.
Unclog
If the array reader detects an unusually low bead count during a reading, you will be
prompted to perform an Unclog operation to remove possible obstructions from the
fluidics lines. Click on the Unclog button
on the main toolbar or select the
command from the Instrument menu. An instruction box will guide you through
preparation of the MCV Plate III.
Figure 21. Steps for unclogging the fluidics system.
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Chapter 5. System Controls
Add 10% bleach solution and distilled water to the appropriate reservoirs, add
5 drops of CAL1 beads to the CAL1 reservoir, insert the plate in the microplate
platform, and click on 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 will take several minutes. After initial system pressurization (5–20 seconds), the
time remaining in the operation will be displayed in the Bio-Plex Manager status bar.
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.
Validation is performed after Start Up 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 III. 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.
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Bio-Plex Manager User Guide
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 manual for more information.
Setting Up a Validation Run
To set up a validation reading, click on the Validation button
on the main
toolbar or select the command from the Instrument menu. The Validate dialog box
will open.
Figure 22. Validate 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 will be listed and grayed out.
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Chapter 5. System Controls
Control Number Selection
Select the Control Number for your specific Bio-Plex Validation Kit from the
Control Number pulldown list. The expiration date for the control number will be
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 on the Add/Remove button.
Figure 23. Add/Remove Control Numbers dialog.
The Add/Remove Control Numbers dialog lists all the available validation Control
Numbers. To make Control Numbers available in the Validate dialog pulldown list,
you must add them to the Selected List. To select all the numbers in the Available
List, click on the Add All>> button. To add control numbers individually, doubleclick on them, or select multiple numbers using Shift + click and Ctrl + click key
combinations and click on 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 on OK, the Control Numbers in the Selected List will be 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 on the Show Specifications button. The Control Number
Specifications dialog box will list the validation specifications.
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Bio-Plex Manager User Guide
Figure 24. 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, Fluidics, Reporter, or Classify.
Performing a Validation Run
When you have made your selections in the Validate dialog, click on OK. The dialog
box for the selected type of validation will open.
Figure 25. Dialog box for performing all validation types.
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Chapter 5. System Controls
See the Bio-Plex Validation Kit manual for information about the bottles in the
Validation Kit, and instructions on loading the Bio-Plex MCV Plate III for
validation. When you have prepared the plate according to the instructions, click on
Eject/Retract Plate in the dialog box, place the plate on the plate carrier, and click
on OK.
Validation Results
After a run, the Validation Results dialog box will automatically open. You can also
view this dialog by going to the View menu and selecting Validation Results.
Figure 26. Validation Results dialog.
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 on 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).
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Bio-Plex Manager User Guide
See the Bio-Plex Validation Kit 4.0 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, which by default is saved in the main
Bio-Plex Manager application folder on your computer.
Note that 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 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 27. Validation Log viewer.
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
40
Chapter 5. System Controls
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 on the Print Report Results button . To print
the Specifications window, click on the Print Specifications button
.
To show or hide the specifications data in the Validation Log viewer, click on the
Show/Hide Specifications Window button
.
To create a spreadsheet report for a particular validation run, click on 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 on
Create Report
. A Microsoft® Excel spreadsheet will be 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.
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Bio-Plex Manager User Guide
Figure 28. Validation Results spreadsheet (Microsoft® Excel).
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 on the button on the main toolbar
42
.
Chapter 5. System Controls
Figure 29. Platform Heater dialog.
In the Platform Heater dialog, click on the Turn Heater on checkbox to turn on the
heater, and set the target temperature within the range 35–60ºC 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 will indicate the current
platform temperature. The status bar will also indicate whether the heater is turned
on.
Figure 30. Status bar with heater on.
If the heater is in the process of warming up or cooling down, the temperature will be
shown in red. If the heater is on and has reached the specified temperature, the
temperature will be shown in green. If the heater is off and is at ambient temperature,
the temperature will be displayed in black.
If you initiate a run before the heater has warmed up or cooled down (i.e., the
temperature is shown in red), you will have the option of waiting until the specified
temperature is reached or proceeding with the run immediately.
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Bio-Plex Manager User Guide
Instrument Information
The array reader continuously monitors its internal temperature, voltage, pressure,
and other systems for diagnostic purposes. You can access this information by
on the main toolbar or selecting
clicking on the Information button
Information from the Instrument menu.
Figure 31. Instrument Information dialog.
Click on the tabs in the dialog to access different 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 (e.g., Start Up, Wash,
Calibrate, etc.) includes an Eject/Retract button in its dialog box.
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Chapter 5. System Controls
Additional Instrument Functions
The Additional Functions submenu under the Instrument menu contains secondary
instrument fluidics functions. Selecting one of these commands will open a dialog
box to guide you through the procedure. Many of these functions require the use of
the Bio-Plex MCV Plate III.
Major fluidics functions such as Wash Between Plates, Remove Bubbles, Unclog,
etc. consist of combinations of the following functions.
Wash
The Wash command performs a single fluidics system wash using DI 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 33)
Select Wash from the Additional Functions submenu of the Instrument menu and
follow the steps in the dialog box.
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 percent bleach and DI water.
Alcohol Wash
This command performs a single fluidics system wash using 70 percent isopropanol.
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Bio-Plex Manager User Guide
Alcohol Flush
This command performs a comprehensive cleaning of the fluidics system using
70 percent 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.
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 on the Cancel button to
cancel the current operation. An alert box will prompt you to confirm the
cancellation.
Figure 32. Cancel button in the status bar.
Shut Down
The shut down procedure consists of a series of functions designed to clean the
fluidics lines and prevent a build up 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.
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Chapter 5. System Controls
Figure 33. 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, which by default is saved in the main Bio-Plex Manager
application folder on your computer.
Note that 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 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 Instrument Operations Log. The
log viewer will open.
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Bio-Plex Manager User Guide
Figure 34. Instrument Operations Log
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 on 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.
48
6. Preparing a Protocol
Protocol Files
A Protocol file contains the parameters of a Bio-Plex Manager 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.
Protocol files also contain the controls for performing the reading. When the reading
is complete, the settings 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 on the New button
on the main toolbar or
Quick Guide or select New from the File menu. A new Protocol window will open.
on the Quick Guide
To open an existing Protocol file, click on the Open button
or main toolbar or select Open from the File menu. Locate and select the file using
the standard Windows® Open dialog.
Standard Protocol files have the file extension *.pbx. Secure Protocol files, created
using Bio-Plex Manager Security Edition, have the file extension *.spbx. For more
information about Secure Protocol files, see page 153.
Secure Protocol files can only be opened as read-only files using the standard (nonSecurity Edition) version of Bio-Plex Manager.
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Bio-Plex Manager User Guide
Protocol files created or modified by Bio-Plex Manager 4.0 or later 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
the next chapter for information about Results files). With the Results file open,
select New Protocol from Results from the File menu. A new Protocol window will
open.
Saving Protocols
To save the settings in a Protocol, click on the Save button
select Save from the File menu.
on the main toolbar or
After you have performed a reading (see Running the Protocol on page 87), the
data from that reading is also saved with the Protocol file. This data is 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 115).
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 megabytes). 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 (e.g., you want 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.
We strongly recommend keeping a copy of the original Protocol file with the raw
bead event data intact. This data is necessary for changing the DD gate range in the
Results file (see page 120), and can also be exported in XML file format from the
Results file (see page 143) for further analysis.
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Chapter 6. Preparing a Protocol
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 will be
preserved.
Select this checkbox to
reduce the file size of a
Protocol containing
reading data
Figure 35. Save As dialog for Protocols.
Note that this compressed file option is not available for Secure Protocol files
generated using the Security Edition of the software (see page 153).
Protocol Window
The Protocol window is divided into a series of subwindows, each containing
different controls and settings. Navigate through the different subwindows using the
buttons along the left side of the Protocol window.
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Bio-Plex Manager User Guide
Click on the
buttons to select
different settings.
Figure 36. Protocol window.
The buttons are numbered to guide you through the selection process:
(see page 53).
1.
Describe Protocol (optional)
2.
Select Analytes
3.
Format Plate
4.
Enter Standards Info
5.
Enter Controls Info
(see page 82).
6.
Enter Samples Info
(see page 84).
7.
Run Protocol
(see page 53).
(see page 59).
(see page 71).
(see page 87).
Click on 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.
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Chapter 6. Preparing a Protocol
Step 1. Describe Protocol
The Protocol description window is displayed when you first open a Protocol.
(Otherwise, click on 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. If you are using the Security Edition, the field contains your
user name. Enter a new name in the field, if desired, and enter a description of the
assay reading in the Description field.
Step 2. Select Analytes
In this step, you select the analytes that you want to detect in the reading.
During a reading, the array reader will always detect all the analytes in the sample,
including any that 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 on the Select Analytes button to view the analyte selection window.
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Bio-Plex Manager User Guide
Figure 37. 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. You can add to these
existing panels of analytes, or create new panels that contain only the analytes that
you use in your experiments.
First you select the panel of analytes that you want to detect, and then you 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 will be displayed in the Available list.
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 on it. Double-click
on an analyte in the Selected list to move it back.
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Chapter 6. Preparing a Protocol
Hold down the Shift or Ctrl key and click on multiple analytes in the Available list to
select them as a group. Then click on 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.
1. Select the panel of analytes.
2. Select the
analytes in
the Available
column.
3. Use the buttons to move your
selections to the Selected column.
Figure 38. Selecting the analytes to be detected.
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.
If you have assay kits for analytes that are not included in Bio-Plex Manager, you
will need to add these analytes using the commands described in this section.
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Bio-Plex Manager User Guide
Creating a New Panel of Analytes
Click on the Add Panel button
in the toolbar to create a new panel of analytes.
Figure 39. Creating a new panel of analytes.
Enter a name for the panel in the top field, then click on Add to add analytes to the
panel. The Add Analyte dialog box will open.
Figure 40. 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.
The bead region number must be correct for proper detection of analytes. Confirm
that this number is correct before proceeding.
Click on Add Continue to add the analyte to the panel and continue adding more
analytes. When you have entered your last analyte, click on the Add button to add it
to the list and close the Add Analyte dialog.
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Chapter 6. Preparing a Protocol
After you have added 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. Click on an analyte
in the list to select it, then click on the appropriate arrow to move it up or down in
the list.
Sort buttons
Figure 41. Sorting analytes.
You can also remove an analyte from the list by selecting it then clicking on the
Remove button, or remove all analytes by clicking on the Remove All button.
To edit an analyte in the list, select it, then click on the Edit button. A dialog box
will open in which you can change the analyte region and name.
When you are finished creating the panel, click on OK to save your changes and
return to the Protocol window.
Editing a Panel of Analytes
To edit an existing panel of analytes, first select it from the Panel name pulldown
menu, then click on the Edit Panel button
. The Edit Panel dialog box will open.
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Bio-Plex Manager User Guide
Figure 42. Editing a panel.
Use the New, Edit, Remove, Remove All, and Sort buttons as described above to
change the analytes in the panel.
If you also change the Panel Name, the changes will be saved under the new name,
and the old panel will not be overwritten.
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 on OK.
Deleting and Renaming Custom Panels
To delete a custom panel of analytes, first select it from the Panel name pulldown
menu, then click on the Remove Panel button
.
To rename a custom panel of analytes, first select it from the Panel name pulldown
menu, then click on the Rename Panel button
box, and click on OK.
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. Enter a new name in the pop-up
Chapter 6. Preparing a Protocol
Combining Panels of Analytes
To combine entire panels of analytes (e.g., 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.
Step 3. Format Plate
After you have selected the 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.
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 on the Format Plate button in the Protocol window to display the plate
template and the formatting controls.
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Bio-Plex Manager User Guide
Figure 43. 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 these views using the tabs above the plate
diagram. As you toggle, the plate template will remain the same, but the available
commands will 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.
Plate formatting is required to perform a reading, because only formatted wells will
be read by the array reader, whereas plate groupings are optional and can be defined
later.
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Chapter 6. Preparing a Protocol
Plate Formatting
To format the well types on the plate, make sure that the Plate Formatting tab is
selected. 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 44. Plate formatting tools (Plate Formatting view).
The different well types are:
•
Unknown sample
•
Standard
•
Control
•
Blank
•
Undefined*
*Remember that all undefined wells will not be read by the array reader.
Defining Unknown Sample Wells
To define wells containing unknown samples, first click on the Unknown Sample
button
, then click or drag on 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.
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Bio-Plex Manager User Guide
Figure 45. Defining unknown sample wells.
Note that the cursor changes to an Unknown Sample cursor as you move it over the
template. As you click on each well, the wells are 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 (only available in the Security
Edition of the software) is described on page 157.
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 on them
or Autofill Down
individually, or select Autofill Across
over the wells to number them sequentially.
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and drag the cursor
Chapter 6. Preparing a Protocol
Figure 46. Defining multiple sample wells, left to right.
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.
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 will be labeled with the same number.
Figure 47. 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.
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Bio-Plex Manager User Guide
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 a well number up to 10 from the pulldown
menu.)
Figure 48. Entering the number of replicate wells in an autofill sequence.
Now when you drag your cursor, the sequential sets of replicate wells will be
defined.
Figure 49. 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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Chapter 6. Preparing a Protocol
Changing a Replicate Group
To change the replicate group of a well or group of wells:
1.
Click on the Select tool , and click or drag on the wells that you want to
change. The wells will appear highlighted.
2.
Right-click on 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 on OK. The selection will be changed to the specified group number.
Figure 50. 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
will be renumbered sequentially, based on whether you have select Autofill Across
or Autofill Down. This is a cosmetic change, and will 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 fluorescent intensity versus analyte concentration. The regression
equation for the curve is then used to calculate the concentrations of your unknowns.
To define the wells containing standards in your microtiter plate, click on the
Standard button
, then click or drag on the wells in the template. Use the
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Bio-Plex Manager User Guide
Autofill buttons as described on page 62 for defining multiple standard wells and/or
replicate groups of standards.
Figure 51. Defining a row of standard wells.
Note that 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 71.
Defining Control Wells
Control wells contain samples of known concentration; the expected versus 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 on the Control
, then click or drag on the wells in the template. Use the Autofill buttons
button
as described on page 62 for defining multiple control wells and/or replicate sets of
controls.
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Chapter 6. Preparing a Protocol
Figure 52. Defining a row of control wells.
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.
After you have defined the control wells, you are ready to enter the concentrations of
the controls as described on page 82.
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 fluorescent intensity values of the wells containing standards,
controls, and unknowns.
To format blank wells in the plate template, click on the Blank button
, then click
or drag on the blank well(s) in the template. Note that the cursor changes to a Blank
cursor as you move it over the template.
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Bio-Plex Manager User Guide
Figure 53. Defining a row of blank wells.
Blank wells are marked in the template with a diamond shape containing a 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 on the well(s). To clear the
formatting from all the 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.
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 fluorescent intensity to
the fluorescent intensity of its Reference. This type of analysis is similar to that
performed in Western blotting.
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Chapter 6. Preparing a Protocol
Click on the Plate Groupings tab to display the plate groupings tools.
Figure 54. 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 55. Plate groupings tools (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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Bio-Plex Manager User Guide
Defining a Group
To define an assay group, click on the Group button
the defined wells in the assay. Note that:
and drag the cursor across
•
Wells must be defined as Samples, Standards, Controls, or Blanks before
they can be grouped.
•
You can group across well types (e.g., a single group can include Standards,
Samples, Controls, and/or Blanks).
•
Replicate wells will be automatically placed in the same group (even if you
drag over only one well in the replicate set).
Figure 56. Defining a group of wells.
The grouped wells will change color to indicate their grouping, and the first member
of the group will be 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 on 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.
Defining the Reference
To change the Reference member of the group, click on the Reference button
and click on the desired Reference member in the group. For replicates, all replicate
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Chapter 6. Preparing a Protocol
wells of the same number within the group will be 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 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
on the main toolbar. For a preview of the printed format,
on the Print button
select Print Preview from the File menu.
Step 4. Enter Standards Info
Standards are analytes of known concentration, which can be used to generate a
standard curve of values using one of the several regression methods built into
Bio-Plex Manager. This curve can be used to calculate the concentrations of your
unknowns.
The standards you select will depend on the performance characteristics of your
assay as well as 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 Bio-Plex cytokine assay manual for
recommendations of specific standard concentrations.
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 53. If your
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Bio-Plex Manager User Guide
current plate contains your standards, you must also identify the standard wells as
described on page 65.
Click on the Enter Standards Info button in the Protocol window to enter
information about your standards.
Figure 57. 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.
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.
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Chapter 6. Preparing a Protocol
Selecting External Standards
This section describes how to select a Results file from which to import external
standards data. If you are not using external standards, you can skip this section and
proceed to Entering Standards Information on page 74.
Click on the Select External Standards tab. In the window, click on 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 will only display
Results files.)
Figure 58. Selecting the Results file containing your external standards.
The Available External Standards list will be populated with the standards from the
selected file. To add additional standards to the list, repeat the procedure, selecting a
different file.
To delete all standards from the Available list, click on the Clear Available List
button.
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Bio-Plex Manager User Guide
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 select all the
standards in the Available column, click on the Add All>> button. To add available
standards individually, double-click on them, or select multiple analytes using
Shift + click and Ctrl + click key combinations and click on the Add>> button. (Use
the <<Remove and <<Remove All buttons to unselect them.)
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, and 34 in the Select
Analytes window (page 53), you can only select 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 list should list all the analytes that you want to
use as external standards, and their source file(s).
Figure 59. Selected external standards.
When you are done, click on the External Standards Info tab to enter additional
information about your external standards.
Entering Standards Information
This section describes how to enter information about your standards. To enter
information for standard wells defined on the current plate, make sure that the
Standards Info tab is selected. To enter information for external standards, first select
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Chapter 6. Preparing a Protocol
the external standards as described in the previous section, and then click on the
External Standards Info tab.
The fields under these tabs are the same, except that you cannot alter the
concentrations under the External Standards Info tab (they will appear grayed out),
as shown in Figure 60.
Figure 60. Standards Info window (top) and External Standards Info window
(bottom) (external standards concentrations cannot be changed).
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Bio-Plex Manager User Guide
The analytes that you selected in the Select Analytes window will be available under
the Analyte pulldown list. Select the first analyte that you will be using as a
standard.
Figure 61. Selecting a standard analyte.
For the settings described on the following pages, you can select different settings
for each analyte, or select the same settings for all analytes.
Entering Concentrations (for Current Standards Only)
After you have selected an analyte, you are ready to enter the concentrations of the
standards on your current plate. If you are using external standards, the
concentrations will be automatically entered, and you can skip to the next section on
selecting concentration units (page 78). To enter concentrations, make sure the
Standards Info tab is selected.
To enter the concentrations manually, select Enter Manually. To calculate them
automatically from a dilution series, select Enter Automatically.
Entering Concentrations Manually
If you selected the Enter Manually option button, the table will list your standard
wells (S1, S2, S3, etc.). These are the wells that you defined in the Format Plate
window (page 59). For the selected analyte, enter the concentration for each standard
in the concentration column.
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Chapter 6. Preparing a Protocol
Selected analyte
Entered
concentrations
Figure 62. Entering standard concentrations manually.
If the Same conc. values for all analytes checkbox is selected, the concentration
values you enter will be 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.
You can use the Cut, Copy and Paste commands on the Edit menu to copy your
descriptions and concentrations between analytes. You can also copy and paste from
and to other applications, such as Microsoft® Word or Excel. Click on 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.
Calculating Concentrations Automatically
If a dilution series was created for the standards, the software can automatically
calculate the concentrations of each standard. Select the analyte, and then select the
Enter Automatically option button.
First, select the appropriate Most Concentrated Standard well number. (The most
concentrated standard must be in either the first standard well or the last.)
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Bio-Plex Manager User Guide
Figure 63. Entering standard concentrations automatically.
Then 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.
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.
Click on the Calculate button when all the information is entered. The
concentrations in the table will be automatically updated.
If the Same conc. values for all analytes checkbox below the concentrations table is
selected, the concentration values will be applied to all the analyte standards.
Deselect the checkbox if you want to enter different concentrations for each analyte
standard, and repeat the procedure.
Concentration Units
You can enter the units of concentration in the Concentration Units field (note that
units are not required and will not affect calculations). These units will appear in
your reports. If all your analytes have the same concentration units, select the Same
units for all analytes checkbox.
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Chapter 6. Preparing a Protocol
Regression Method
Using the Std Regression Curve pulldown list, choose from among seven different
regression methods to generate the standard curve (page 136):
•
Logistic-5 PL
•
Logistic-4 PL
•
Linear
•
Cubic spline
•
Point-to-point
•
Linear (Semi-log)
•
Point-to-point (Semi-log)
Figure 64. Selecting a regression method.
You can change your selection later in the Standard Curve window of the Results
file. See page 136 for a more detailed discussion of the different regression methods.
You can also select the regression curve axis scale from four different combinations
of linear and logarithmic. Select the axis scale from the Axis Transformation
pulldown list.
Figure 65. Selecting a regression curve axis transformation.
Note that the axis transformation only changes how the curve is displayed, not the
calculated values.
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The regression method and axis transformation you select in the Protocol window
will be used to calculate your initial results. However, these settings will 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.
Logistic Weighting
If you select a logistic curve fitting method (Logistic 5PL or Logistic 4PL), the
software will perform automatic weighting of the points in the curve. Click on the
Logistic Weighting button to access the weighting algorithm controls.
Figure 66. Logistic Weighting dialog.
There are eight models for calculating the variance in the logistic weighting
algorithm:
80
•
Linear Variance
•
Logarithmic Variance
•
Exponential Variance
•
Power Law Variance (default)
•
Linear CV
•
Logarithmic CV
Chapter 6. Preparing a Protocol
•
Exponential CV
•
Power Law CV
Coefficient a
Weighting coefficient a will be automatically calculated 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.
Recovery Percentage Range
Because the standard curve (page 136) 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.
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 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 82) 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.
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Figure 67. Selecting the recovery percentage range.
For example, if you select a range of 70–130%, the observed concentration should be
within 70–130% 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 130). Concentrations that fall outside this range
are flagged as *** in the Conc in Range column of the Report table.
Step 5. Enter Controls Info
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 that you will be analyzing in the reading as described on page 53,
and identify the plate well(s) containing your controls as described on page 66.
Only formatted wells will be read by the array reader. Be sure to define all your
control wells before running a Protocol.
Click on the Enter Controls Info button in the Protocol window to enter
information about your controls.
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Figure 68. Enter Controls Info dialog.
The analytes that you selected in the Select Analytes window will be available under
the Analyte pulldown menu. Select the first analyte that you will be using as a
control.
The table will list your control wells (C1, C2, C3, etc.). These are the wells that you
defined in the Format Plate window (page 59). Enter the concentration of the
selected analyte in the Conc column for each well.
Selected analyte
Entered
concentrations
Figure 69. Entering concentrations of the controls.
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Bio-Plex Manager User Guide
If the Same conc. values for all analytes checkbox is selected, the concentration
values you enter will be 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 the performance parallelism studies,
whereby a control diluted by different dilution factors is analyzed to verify that the
final concentration is the same for all of the samples.
Figure 70. 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 on the Set All Dilution Factors button. The dilution factor
you enter for one analyte is automatically applied to all analytes.
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
on a cell, row, or column in the table to copy and paste.
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Chapter 6. Preparing a Protocol
Step 6. Enter Sample Info (optional)
Before entering information about your unknown samples, select the analytes that
you will be analyzing in the reading as described on page 53, and then identify the
plate well(s) containing your unknown(s) as described on page 61.
Only formatted wells will be read by the array reader. Be sure to define all your
unknown sample wells before running a Protocol.
Click on the Enter Sample Info button in the Protocol window to enter information
about your unknown samples.
Figure 71. Enter Sample Info dialog.
The information table will list all the unknown sample wells as defined in the plate
template for the reading (page 61).
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 on the Set All Dilution Factors button.
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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 on a cell, row, or column in the table to copy and paste.
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7. Running the Protocol
After you have prepared the Protocol as described in the previous chapter, you are
ready to run the Protocol. Click on the Run Protocol button
window to select the final settings and run the Protocol.
in the Protocol
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 (see caution below). 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 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 (e.g., 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.
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 on 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).
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Figure 72. Run Protocol window—Raw Data table view.
Figure 73. Run Protocol window—Histogram/bead map view.
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Chapter 7. Running the Protocol
The bead map and histogram (see page 98) provide graphical data about a run, and
are continually updated throughout the run. The Raw Data table (see page 108)
displays the raw data of the reading after it is complete.
The controls for performing the run are described in the following sections.
Bead Count
The Beads field 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 pull-down menu 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 100 beads per region. This ensures that at least 100
beads in each region will be acquired before sample acquisition is complete.
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 underrepresented in the final tally.
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 (i.e., 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.
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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.
Advanced Settings
Click on the Advanced Settings button to access additional reader settings and
controls.
Figure 74. Advanced Run Settings dialog.
Bead Map Selection
Bio-Plex Manager 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 104). The
default bead map is the 100-region map, which is appropriate for assays that use
xMAP microspheres (e.g., Bio-Plex assays and Bio-Plex COOH beads). A 50-region
map and 25-region map are also available. The 50-region map includes alternating
regions from the default 100-region map. The 25-region map also includes select
regions from the default 100-region map. The result is that the regions are larger and
more spread out.
Consult your bead assay manual for instructions on selecting the appropriate map for
your beads.
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If you select the 50- or 25-region maps, the software will automatically verify
whether the analytes selected in the protocol can be accommodated within the
selected regions. If not, you will receive a warning message, and you must change
either your analyte selections or Bead Map field selection before you can run the
protocol.
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.
To avoid air uptake, make sure that the sample needle is properly aligned (see
page 17) and 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 on Start to begin a reading, you
will be prompted to enter a name for the Results file.
In Bio-Plex Manager Security Edition, Secure Mode, this checkbox is selected and
cannot be deselected (see page 159).
If the Auto Save After Run checkbox is selected, you can also select the Auto
XML Export After Run checkbox. With this checkbox selected, Bio-Plex Manager
will automatically generate an XML file and save it to a folder or open it in an
application after the reading (these options are specified in the XML Export
Properties dialog). The XML file will have the same name as the Results file. For
more information on XML files, see page 143.
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Sampling Errors
During a reading, the system will continuously monitor 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.
Select Pause Run If Error Occurs to select all the possible error conditions, or
select/deselect the individual error condition checkboxes. By default, these are not
selected (i.e., the run will not pause for any error condition).
Figure 75. Error condition checkboxes
All errors will be logged in the Raw Data and Report tables, whether or not the
checkboxes are selected (see page 110).
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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 Guide on page 165 for a detailed list of reading errors,
causes, and suggested solutions.
With the checkboxes selected, the reading will stop and an alert box will describe the
problem and suggest steps for correcting it. After you have performed the suggested
steps, you can rerun the reading.
If you are rerunning wells in Rerun/Recovery Mode, you may want to disable the
Low bead number checkbox, since wells that have already been read once contain
fewer beads.
DD 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 that are 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 100-region
and 50-region bead maps (Low = 4335, High = 10000) is designed to accommodate
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the standard 5.5-micron beads distributed by Bio-Rad. If you are using differentsized beads, 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 101).
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 will 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 120). If you change the DD gate range in the Results
file, a record of the range during the reading will be preserved.
You can only set the gates on 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 re-analyze the data.
PMT Voltage
Users who want to manually set the PMT voltage on the reporter detector can select
the Override calibration checkbox in the Advanced Run Settings dialog and enter a
new voltage in the PMT Volts field.
Note that the PMT voltage is set during calibration, and should only be adjusted by
users who know the precise PMT value that they want to specify. If you change the
PMT, you will no longer have day-to-day and unit-to-unit reproducibility of results.
The PMT voltage will be reset to the calibrated values after the plate is read and the
results generated.
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Plate Loading Guidelines
Before starting the run, note the following plate handling guidelines and 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 µl 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
will be sucked into the sample loop and injected into the flow
chamber. As a result, bubbles will form in the cuvette and interfere
with the analysis. If this happens, perform a Remove Bubbles
procedure (page 33) and rerun the Protocol.
Shake the microplate on a plate shaker for 30 seconds prior to
performing a reading.
Running the Protocol
When you have selected and reviewed all your Protocol options and prepared your
microplate, click on the Start button. The Run Protocol dialog will open.
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Figure 76. Run Protocol dialog.
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.
If your microplate has a barcode number or other type of ID number, enter it in the
Plate ID field.
Click on Eject/Retract Plate to insert the plate into the reader, and click on OK to
begin the reading.
If the Auto Save After Run checkbox is selected, you will be prompted to enter a
name for the Results file to be generated.
The following conditions may delay the start of a reading:
•
If the array reader optics have not warmed up, the reading will be delayed
until warm up is complete.
•
If the pressure is too low in the fluidics system, you will 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 change.
When the reading begins, the sample needle will lower, draw sample from a plate
well, and retract to its original position. The sample will be drawn from the needle
into the sample loop and the reading will begin.
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Chapter 7. Running the Protocol
After initial system pressurization (5–20 seconds), a typical well reading may take up
to 2 minutes; in most cases, individual wells are read in a matter of seconds.
Status Bar
As the sample is analyzed, the status of the reading will be displayed in the status bar
at the bottom of the Bio-Plex Manager window.
Figure 77. 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 54).
•
The Gate fields monitor all beads that pass an internal discriminator "gate"
designed to eliminate particles that are 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 will change to a Stop button in the
Protocol window. You can click on Stop to cancel the reading. An alert box will
notify you that you can generate a report with the incomplete results or rerun the
Protocol as described on page 113.
Generating Results from a Protocol
The reading stops when you click on Stop, when an error stops the reading, or when
all the samples have been analyzed. At the end of the reading, the sample needle
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lowers and the syringe pump purges the sample loop and needle with sheath fluid,
sending a fraction of sheath fluid (approximately 160 µl) back into each well.
At the end of a complete reading, the Results will be automatically calculated and
displayed.
Note that the Results file will be automatically saved if you selected Auto save after
run (page 91) in the Protocol. Otherwise, you must save your Results as a separate
step (see the following chapter).
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 (see page
108). If the histogram and bead map are not visible, click on the Show/Hide
Histogram/Bead Map button
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to display them.
Chapter 7. Running the Protocol
Well number
selected
Figure 78. Histogram and bead map.
You can see some of the 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 different wells to display by selecting rows
in the table below the graphs.
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 (see
page 93). Make your selection using the pulldown menu above the histogram.
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Figure 79. Selecting the analyte(s) to display.
To print the histogram and/or bead map, click on 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 a particle such as a bead or
aggregated beads passes through the path of the lasers. Each event generates signals
in different channels:
•
The fluorochromes embedded in each bead generates 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 is the
events, and the X-axis is the channel numbers from 1 to 32766 for the selected
channel type.
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DD Gate Range
The DD Gates range specified under the Run Protocol Advanced Settings (see page
93) is shown on the histogram as two red lines perpendicular to the X-axis. 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. Note that this will also change the
range as listed in the Advanced Settings dialog.
Figure 80. 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 on the Restore Default Gates button
above the histogram.
Selecting the Channel Type
You can change the histogram to display different channel types. Click on the
histogram with the right mouse button and select X-Axis from the context menu.
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Figure 81. 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.
•
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 that changing the channel type will change the channel numbers and the
histogram display.
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Chapter 7. Running the Protocol
Scaling the Histogram Data
If the histogram is too big or too small to fit comfortably within the display range,
click on the Auto Scale button
on the histogram toolbar. The height of the Y-axis
will change to better fit the data. You can also select this command from the rightclick 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.
The default view of the data is log scale. From the toolbar, click on the Log/Linear
to change the display from log to linear. Click again to return to log.
button
Magnifying and Resizing the Histogram
Click on the Zoom button
on the histogram toolbar to magnify the display. With
when you position it
Zoom selected, the cursor will change to a “hand” symbol
over the histogram. Drag the hand cursor to view different magnified regions of the
histogram.
to maximize the histogram portion of the window
Click on the Maximize button
within the larger Protocol window. Click on the Restore button
to return the
histogram to its default state.
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Figure 82. 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 (100, 50, or 25) is listed
above the map, and is based on the selection in the Advanced Settings dialog (see
page 90).
In the default bead map, the X-axis is the Classification 1 channel and the Y-axis 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
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.
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 will appear in a
pop-up. The data point clusters should fall within these regions.
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Chapter 7. Running the Protocol
Each cluster of dots
that falls within a
white region
represents a unique
bead set (analyte)
within an assay
Figure 83. Bead map, with pop-up indicating the analyte region under the
cursor.
If one or more clusters do not fall within the white areas, it may indicate 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 0 on page 165 for guidance with troubleshooting.
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 on 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.
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•
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.
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 84. Changing the X-axis.
Scaling the Bead Map Data
The default view of the data is log scale. From the toolbar, click on the Log/Linear
to change the display from log to linear. Click again to return to log.
button
Magnifying and Resizing the Bead Map
Click on the Zoom button
in the bead map toolbar to magnify the display. With
when you position it
Zoom selected, the cursor will change to a “hand” symbol
over the map. Drag the hand cursor to view different magnified regions of the bead
map.
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Chapter 7. Running the Protocol
Click on the Maximize button
to maximize the bead map portion of the window
within the larger Protocol window. Click on the Restore button
to return the bead
map to its default state.
Bead Map Display Options
To access additional Bead Map display settings, right-click on the bead map and
select Options.
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 between
different numbers of events. You can adjust the display so that data points with low
numbers of events are not shown, or a certain number of events is required before the
data point color changes.
Figure 85. Bead map display options.
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 will change at 2 events, 4 events, 8
events, 16 events, etc.
If you want to filter lower numbers of events from the display, select the Filter
Levels checkbox and use the arrow keys next to Display above level to select the
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exponent level below which no data points will be displayed. For example, if the
Level Multiplier is set to 2, and Display above level 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 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.
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 that the instrument hardware and software information previously included in
the Run Info window 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. See page 121.)
The first few lines of this table are visible below the bead map and histogram.
Alternatively, hide the bead map/histogram display by clicking on the Show/Hide
Histogram/Bead Map button
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to maximize the table.
Chapter 7. Running the Protocol
Figure 86. 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 is displayed.
In the table, the fluorescent intensities (FI) of the analytes you selected are listed for
each well number.
During a reading, the array reader will always detect all the analytes in the sample,
including any that you have not selected. However, analytes that were not selected in
the Protocol will not be 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 on-line Help information about the Raw Data table, click on the Display Help
for Table Legend button .
Bead Statistics
To display statistical data for the individual beads as well as the entire well, click on
on the table toolbar. The
the Show/Hide Bead Statistics Columns button
number of beads collected for each analyte will be displayed in parentheses after the
analyte fluorescent intensity.
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Fluorescent intensity
Bead count
Figure 87. 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 93).
•
% Agg Beads—Percentage of aggregated beads in the well
(100 – (Gate/Total*100)).
Figure 88. Well data.
The number in the Region column will be 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, the bead count, the bead regions, and the
platform temperature are continuously monitored. The Sampling Errors column
reports errors in any of these areas for each analyte. These errors will be reported in
the table even if you chose not to pause the run due to an error condition (see
page 92).
The sampling error codes in the column are listed in the following table.
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Error Code: Indicates:
1
2
3
4
5
Low bead number detected in the well
Aggregated beads detected in the well
Bead classification efficiency problem detected in the well
Region selection problem detected in the well
Platform temperature problem detected
Raw Data Display Options
To select the columns to display in the Raw Data table, as well as other display
settings, click on the Raw Data Display Options button
from the Table Options menu.
or select the command
Figure 89. 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
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Standard or Diagnostic in the Report Scheme field; these selections will 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.
Set Number Format
In the Raw Data Display Options dialog, click on the Set Number Format button to
select the number format for each column in the Raw Data table.
Figure 90. 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 in 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 that changing the number format only changes how the numbers are displayed
and printed, and does not affect calculations in any way.
Click on OK to make the changes, or Reset Defaults to return all columns to their
default number formats.
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Other Table Formatting
To display the description of each sample in the table, click on the Show/Hide
Description Column button above the table. The Description column will be
displayed.
To automatically size the columns to fit the width of the text, click on 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.
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 will appear next to each well
number in the Run Protocol table. Select the checkboxes next to the wells that you
want to read again.
Figure 91. Selecting wells to rerun.
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To rerun wells in a filter plate, you must first vacuum the excess fluid from the wells
and resuspend the remaining beads in 125 µl. (The beads will remain on top of each
filter plug after vacuuming.)
Vacuum the wells of the filter plate using the vacuum filter apparatus and resuspend
the beads in each well with 125 µl of resuspension buffer. 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 89). Also,
click on the Advanced Settings button and deselect the Low bead number error
condition checkbox (page 90). Otherwise, a low bead error may stop the reading.
When you are ready, click on Start to begin the reading.
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Results Files
After a reading is complete, a Results file is generated and automatically opens. 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 only change the parameters of wells that
have already been read, and you can only calculate results based on the analytes that
were present in the sample.
Note that the Results file is automatically saved after a reading if you selected Auto
save after run in the Protocol window. 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
from the Run Protocol toolbar or the File menu.
Opening a Results File
To open an existing Results file, click on the Open button
on the Quick Guide or
main toolbar or select Open from the File menu. Locate and select the file using the
standard Windows Open dialog.
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Standard Results files have the file extension *.rbx. Secure Results files, created
using Bio-Plex Manager Security Edition, have the extension. *.srbx. See page 153
for more information about Secure files.
Secure Results files can only be opened as read-only files using the Standard Edition
of Bio-Plex Manager.
Results files created or modified by Bio-Plex Manager 4.0 or later cannot be opened
by earlier versions of the software.
Saving a Results File
To save a Results file, with the file open click on 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 megabytes). 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 (e.g., 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.
We strongly recommend keeping a copy of the original Results file with the raw
bead event data intact. This data is necessary for changing the DD gate range in the
Results file (see page 120), and can also be exported in XML file format (see
page 143) for further analysis.
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 will be
preserved.
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Select this checkbox
to reduce the size of
the Results file
Figure 92. Save As dialog for Results files.
Note that this compressed file option is not available for Secure Protocol files
generated using the Security Edition of the software (see page 153).
Results Window
The Results window is divided into subwindows, each containing different options
for viewing, formatting, and calculating your data.
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Click on these buttons to review/change the original Protocol settings.
Click on these
buttons to view
and calculate
results.
Figure 93. Results window.
Navigate among the different subwindows using the buttons along the left side of the
Results window:
•
Raw Data
•
Report Table
•
Standard Curve
(see page 121).
(see page 123).
(see page 136).
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.
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Any changes that you make in the Results file will not change the original Protocol
file or the data from the reading. They will only change how that data is displayed in
the Results file tables.
Click on the Describe Protocol button
in the Results toolbar to review or
change the description of the Protocol, as described on page 53.
in the Results toolbar to review the analyte
Click on the Select Analytes button
selection or select different analytes, as described on page 53.
During a reading, the array reader will always detect all the analytes in the sample,
including any that 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 correct your analyte selections, and the detected analytes will appear in
the tables.
Click on the Format Plate button
in the Results toolbar to review or change the
formatting of the plate. Plate formatting is described on page 59.
Only formatted wells are read by the array reader. You must define all the wells that
you want to read before running a Protocol. However, you can change the formatting
of a defined well (e.g., from unknown to standard) after a reading, and the analysis of
that well will change.
Click on the Enter Standards Info button
in the Results toolbar to review or
change the concentrations of the standards, as described on page 71.
in the Results toolbar to review or
Click on the Enter Controls Info button
change the concentrations of the controls, as described on page 82.
Click on the Enter Sample Info button
in the Results toolbar to review or
change the sample information, as described on page 85.
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Changing the DD Gate Range
You can change the DD gate range (see page 93) in the Results file and recalculate
the data based on the new range. Note the original DD gate range set in the Protocol
file and used during the reading will be 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 will open,
displaying the histogram and bead map with controls for changing the DD gate
range. (See page 98 for a complete description of other histogram and bead map
controls.)
Figure 94. Change DD Gates dialog.
In the Change DD Gates dialog, first select the well that 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 and click
on the Update Gates button.
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When you have made your changes, click on OK. The data in the Raw Data table
(see next section) will be recalculated based on the new settings.
Raw Data
The Raw Data window in the Results file includes the same histogram and bead map
and numerical Raw Data table as in the Run Protocol window. These features are
described in the previous chapter on the following pages:
•
Histogram and bead map—page 98.
•
Raw Data table—page 108.
Figure 95. 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.
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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 select Set Plate ID from the context menu.
Figure 96. Set Plate ID dialog.
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 on 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 on the Export to
Excel button
in the toolbar or select the command from the File menu. This
command will automatically launch Excel and display a spreadsheet containing your
data.
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Figure 97. 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 will open a Save As
dialog box in which you can specify a file name and location for the text file.
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.
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.
Report Table
The Report table provides detailed information about each of the analytes in your
samples. Click on the Report Table button
to open the Report table window.
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Select analyte from the pulldown list
Figure 98. Report table.
First, select the analyte that you want to review from the Analyte pulldown list
above the table. All the data for that analyte in each defined well of the plate will be
displayed in the table.
For on-line Help information about the Report table, click on the Display Help for
Table Legends button
.
Report Table Columns
The columns in the table show the data for the selected analyte for each well or
replicate group of wells.
The available Report table columns are listed below:
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•
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 129.
Chapter 8. Analyzing the Results
•
Description—The description you entered for each standard, control, or
sample in the Protocol. Click on the Show/Hide Description Columns
button
to view or hide this column.
•
FI—The median fluorescent intensity of the selected analyte.
•
FI – Bkgd—The median fluorescent intensity minus background (measured
using the defined blank wells) of the selected analyte.
•
Std Dev—The standard deviation of a replicate group of wells.
•
Std Err— The standard deviation of a replicate group of wells divided by
the square root of the number of replicates.
•
%CV— The 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).
•
Obs Conc—The observed concentration of the selected 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.
•
Exp Conc—The expected concentration of the selected analyte standard or
control, as specified in the Enter Standards Info window or Enter Controls
Info window, respectively.
•
(Obs/Exp)*100— The recovery rate for the observed versus expected
concentrations of the standard or control. See page 130.
•
Conc in Range—An observed concentration (see above) that falls within
the range of standard concentrations given a specified recovery range (see
page 80). Values outside the range are flagged as ***.
•
Group—The defined group of the selected well (see page 68).
•
Ratio—The ratio of the FI values of a member/reference or
reference/member in a group (see page 68).
•
Dilution—The specified dilution factor of the selected analyte.
•
Bead Count—The number of beads counted in region for the selected
analyte.
•
Bead Mean—The mean fluorescent intensity of the beads counted in region
for the selected analyte.
•
Bead Std Dev—The standard deviation of the bead mean.
•
Bead Std Err—The standard error of the bead mean.
•
Bead %CV—The coefficient of variation of the bead mean.
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•
Trimmed Mean—The bead mean excluding the top 5% and bottom 5% of
fluorescent intensities.
•
Trimmed Std Dev—The standard deviation of the trimmed mean.
•
Trimmed Std Err—The standard error of the trimmed mean.
•
Trimmed %CV—The coefficient of variation of the trimmed mean.
•
Sampling Errors—Error codes for a reading. See page 110.
Report Table Options
To select the columns to display in the Report table, as well as other display settings,
click on the Report Table Options button
Table Options menu.
or select the command from the
Figure 99. Report Table Display Options dialog.
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In the Report Table Display Options dialog, select the columns to display using the
checkboxes. You can also choose between predefined report settings by selecting
Quantitative or Qualitative in the Report Scheme field; these selections will
display different sets of columns.
The predefined set of Quantitative columns are designed to be used with cytokine
assays, while the Qualitative columns are designed to be used with phosphoprotein
assays.
Under Organize samples by, you can group the rows in the Report table by Type
(standards, controls, unknowns, etc.), Group (if defined), or None. Note that Type
and Group selections will override the Sort function in the Report table (see page
131). For example, if you select Type organization, any sorting you do will be within
each type.
To review the data for each well in a replicate group (see page 126), select the
Expand Replicate Info option.
To hide error codes in the table such as OOR, ***, and ----, as well as * before
concentration values, select the Exclude table error codes checkbox.
Select the Save Settings as Default for New Protocol checkbox to use the
selections in this dialog as the default in Results files generated from new Protocols.
Set Number Format
In the Report Table Display Options dialog, click on the Set Number Format button
to select the number format for each column in the Report Table.
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Figure 100. 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 in 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 that changing the number format only changes how the numbers are displayed
and printed, and does not affect calculations in any way.
Click on OK to make the changes, or Reset Defaults to return all columns to their
default number formats.
Replicate Groups
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 page 129).
Figure 101. Example of a standard with two replicate wells (A11 and A12).
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Chapter 8. Analyzing the Results
To review the data for each well in a replicate group, click on the Show Replicates
button
.
Figure 102. Expanded data for replicate wells.
The mean values as well as the values for the individual wells will be reported for
each replicate group.
Selecting Outliers
In some cases, you may want to exclude a well or replicate group of wells from
statistical calculations (e.g., when the CV% of a set of replicates is high, or the
observed concentration of an analyte is out of range). The Outlier column in the
Report table window allows you to flag a well or replicate group of wells as an
outlier.
Click on the Show/Hide Outliers Column button
the column.
in the toolbar to show or hide
Each row in the column contains a checkbox. When you select the checkbox, the
data for the selected analyte in the well 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 on the outlier checkbox for the well,
and select Set Outlier (All Analytes) from the context menu.
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Figure 103. Selecting a well as an outlier for all analytes.
Now the well data for all analytes will be excluded from calculations. To remove the
outlier flag for all analytes, right-click on the well select Clear Outlier (All
Analytes) from the context menu.
If you flag a replicate group of wells as an outlier, data for the entire group is
excluded. If you click on the Expand Replicates Info button
, you can flag each
well in the group as an individual outlier. If you do this, the outlier checkbox for the
entire group will appear checked and grayed out, indicating that part of the group has
been flagged.
Group checkbox
appears gray
and selected
Figure 104. Flagging a single well in a replicate group as an outlier.
Obs/Exp*100 Column
Because the standard curve (page 136) 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 82) to
determine the overall accuracy of an assay.
To display this column, click on Report Table Options
(Obs/Exp)*100 checkbox.
and select the
The (Obs/Exp)*100 column shows the recovery rate for the observed versus
expected concentration of each standard. This number should fall within the recovery
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Chapter 8. Analyzing the Results
percentage range selected in the Enter Standards Info window (see page 81). For
example, if you selected a recovery range of 80–120%, the observed (backcalculated) concentration of a standard should be within 80–120% of the expected
concentration. Values outside this range are flagged in the Conc in Range column.
Conc 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 previous section). 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 ***. (The concentration in range values of the backcalculated standards are also shown, for comparison purposes.)
Back-calculated standard
concentrations within a specified
recovery percentage range of 80–120%
Concentrations of unknowns within the
range of valid back-calculated standard
concentrations
Concentrations of unknowns outside the
range of valid back-calculated standard
concentrations
Figure 105. Conc in Range column.
For example, if only standard concentrations from 1.9 to 8,100 ρg/ml fall within the
specified recovery range, then only unknown concentrations that fall within this
range will be displayed in the Conc in Range column.
Table Formatting
To sort the data in the table alphanumerically, click on the heading of the column
that you want to sort by, then click on the Sort button
the order.
. Clicking twice will invert
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To highlight the divisions between the unknowns, standards, controls, etc. in the
report, click on the Organize by Type button
sorting you do will be within each type.
. With this button selected, any
To highlight the divisions between defined groups (see page 68) in the report, click
on the Organize by Group button
will be within each group.
. With this button selected, any sorting you do
To automatically size the columns to fit the width of the text, click on the Autofit
button
on the toolbar.
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.
Exporting the Report Table
To export the data in the Report table to an Excel spreadsheet or workbook, or tabdefined text file, click on the Export Report Table button
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in the toolbar.
Chapter 8. Analyzing the Results
Figure 106. Table Export Options dialog.
The Table Export Options dialog will open.
Export Format
Select Table per Analyte to display all the data for each analyte on an individual
spreadsheet.
Select Table per Column to display one column of data (e.g., FI) for all the analytes
on an individual spreadsheet.
Select 96-Well Plate to display one type of data for each analyte in a spreadsheet,
laid out like the wells in a 96-well plate (rows A–H and columns 1–12). Note that
this selection will export only one type of data at a time.
For additional export options, click on the Advanced Export Options button.
Advanced Export Options
If you selected 96-Well Plate or Table per Column above, select the data to export
under the Report Table Column to Export pulldown list.
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If you selected 96-Well Plate or Table per Analyte above, under Export Source,
select Selected Analyte to export only the data for the currently selected analyte in
the Report table or All Analytes to export the data for all analytes in the table.
Under Export Destination, select Excel Workbook to export the data to a
Microsoft® Excel file, or Text File (Tab Delimited) to export it to a tab-delimited
text file (see Figure 107 for an example).
If you selected Excel Workbook and will be generating multiple tables of data,
select the Use Multiple Worksheets checkbox to export each table to a different
worksheet within the Excel workbook. Otherwise, all tables will appear on the same
worksheet.
Figure 107. FI - Bkgd data for multiple analytes exported to a tab-delimited
text file, 96-well plate format.
Select the Exclude Column Headers/Footers and Page Headers/Footers to
exclude this information from the exported file.
The error codes symbols in the Report table (OOR< >, ***, ----, * before
concentration values) can interfere with macros in some types of spreadsheets,
including Excel spreadsheets. Select the Exclude Table Error Codes checkbox to
exclude these symbols from the exported file. If this option is selected, table cells
with error codes will be exported as empty cells.
If you are exporting to Excel, Microsoft® Excel will automatically launch when you
click on OK and a spreadsheet will be created.
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Chapter 8. Analyzing the Results
Figure 108. Example of an Excel spreadsheet, Table per Analyte format.
Figure 109. Example of an Excel spreadsheet, Table per Column format.
If you are exporting to a text file, a Save As dialog will open when you click on OK.
Select the desired directory and name and click on Save to complete the operation.
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Printing the Report Table
You can print the Report table for the selected analyte using the Print command
on the main toolbar or File menu. You can print the Report table for all analytes
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.
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 71.)
Click on the Standard Curve button
to open the Standard Curve window.
Figure 110. Standard curve.
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Chapter 8. Analyzing the Results
In the graph, standards defined on the current plate will be displayed as blue squares,
while external standards will be displayed as red circles. If both types of standards
are used in the Results file, both will be displayed on the curve. 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
(R2, fit probability, residual variance) will be displayed below the standard curve, if
possible. 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.
Use the Error Bars pulldown list to select from several different error bars (e.g.,
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 only be displayed for the external
standards.
Use the Regression Type pulldown list to choose from seven different regression
models to generate the standard curve (see next section).
Changing the regression type will automatically recalculate all the data in the Report
table. If you are unsure whether your data has been recalculated, select Refresh
Calculations from the View menu.
Use the Axis Transformation pulldown list to select the standard curve axis scale.
Four different combinations of linear and logarithmic are available.
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.
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Select the Swap XY Axes checkbox to change the X and Y axes of the curve.
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 111. Pop-up data for a standard sample point in the curve.
Regression Methods
There are seven regression methods for calculating the standard curve:
•
Logistic-5 PL
•
Logistic-4 PL
•
Linear
•
Cubic spline
•
Point-to-point
•
Linear (semi-log)
•
Point-to-point (semi-log)
The minimum number of non-zero standards required for using each type of
regression method is shown in the following table:
Method
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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)
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Chapter 8. Analyzing the Results
Figure 112. Example of Logistic-5PL curves for standards on the current
plate and external standards.
If you do not have enough points for the selected method, an error message will be
displayed in red below the standard curve.
Logistic-5PL
This version of the five parameter logistic equation has been developed by Brendan
Scientific, creator of StatLIA immunoassay software. The Logistic-5PL regression
type yields the best results for most assays; therefore, this is the default in Bio-Plex
Manager. However, there may be cases where 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,
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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, where 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
( c)
1+ x
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,
Linear
The Linear equation is:
y = a + bx
Where a is the intercept and b is the slope of the line.
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Chapter 8. Analyzing the Results
The simplest method for determining concentrations from a standard curve is to
construct a plot using the linear portion of the response curve. The R-squared value
may be used to determine the overall goodness of the linear fit. A linear regression
with an R-squared 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 relatively 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
A single equation is not 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.
Linear versus 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
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, Ph.D. GraphPad Software, San
Diego, CA.
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modeled using a logistic fit and the Logistic-5PL model will yield the best results.
For more information, see Bio-Rad bulletin 2861, available on our Web site
(www.bio-rad.com) or from Technical Support (see page 4).
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, for serum the linear
regression may be useful. 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, click in the
Standard Curve window and select Copy from the Edit menu. You can then paste
this image into your spreadsheets or other documents using the Paste command in
your other applications.
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.
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 curve for all analytes using
the Print All Analytes command
on the main toolbar or File menu.
When you select a print command, a dialog box will open allowing you to specify
the page layout of the printed curve.
Figure 113. Print Standard Curve dialog.
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Chapter 8. Analyzing the Results
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 (e.g., the
layout will size the curve at one-quarter the full size
of the page). The regression type will be printed below each curve.
Select a layout and click on OK to display the standard Windows® Print dialog.
XML Export
You can export all the information in a Results file as an XML-formatted text file for
data sharing, import into Laboratory Information System databases, and data analysis
using other applications.
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
To export the Results data in XML format, go to the File menu and select XML
Document Export.
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Figure 114. XML Export dialog.
In the XML Export dialog, select the Exclude Raw Bead Event Data checkbox to
exclude this information from the export file.
Under Destination, specify whether you want to send the data to a file or display the
data using another software application.
If you select File, you can use the default file name and location, or click on Change
File to specify a new file name and location.
If you select Command Line, click on Select Application to locate the software
application that 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 pathname 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 115.
Note the following:
144
•
If you manually enter the application pathname, it must be enclosed in
quotation marks, as shown in Figure 115.
•
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.
Chapter 8. Analyzing the Results
Figure 115. Example of a command line.
When you have made your selections, click on OK.
Figure 116. Example of an XML document.
You can set the default XML export settings using the XML Export Properties
dialog. Go to the File menu and select XML Export Properties to open this dialog.
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Figure 117. XML Export Properties dialog.
This dialog is similar to the XML Export dialog, except that it sets the default XML
export settings and default folder where XML files will be saved. These settings are
used if you select the Auto XML Export After Run checkbox in the Advanced Run
Settings dialog (see page 91).
XML Schema File
An XML schema file named Bio-Plex_2.xsd is included in the main Bio-Plex
Manager application directory. This schema file can be used to automatically
validate the XML file.
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9. Bio-Plex Manager
Security Edition
Overview
Bio-Plex Manager Security Edition works in conjunction with the built-in security
features of the Windows® 2000 and Windows® XP Professional 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).
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 100 IS 2.3
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
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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)).
Note that 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 Versus Secure Mode
Bio-Plex Manager Security Edition can run in Standard Mode, in which all the
security and audit trail features are disabled and the 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 that you are running the software in
Secure Mode, unless otherwise noted.
When you are in Secure Mode, the status bar displays a “locked” symbol . When
you are in Standard Mode, the status bar displays an “unlocked” symbol .
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® 2000 or XP Professional 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 next section) enables Secure Mode.
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Chapter 9. Security Edition
When you start the Bio-Plex Manager in Secure Mode, you will be prompted to log
in with your user name and password.
User names and passwords must be set up on your computer or computer network as
described in the Bio-Plex Manager Security Edition Configuration Setup Guide
before you can use the application in Secure Mode. This guide is provided with your
Security Edition license.
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
Security Edition Configuration Setup Guide.
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 in 0. 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.
•
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
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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 will receive a pop-up warning. In addition, many fields and settings that
are restricted by your user level will 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 on the user name displayed in the application status bar.
Figure 118. User Information dialog.
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.
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When Secure Mode is enabled, the status bar will display the locked symbol.
Figure 119. Status bar showing disabled (left) and enabled (right) Secure
Mode status
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 that only users set up on the domain you specify will be able to use the
software.
When you click on OK, you will be prompted to sign in as an Administrator-level
user to authenticate the change. You will 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 120. Log In dialog
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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 different operations. For example, a Clinician 1level 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 into 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 only accessible 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 will check the integrity and validity of a
file each time the file 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
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.
If the application is in Secure Mode, each event in a log will include the name and
access level of the user who performed/authorized the action. If the application is in
Standard Mode, the event will be flagged as having occurred outside Secure Mode
and the access level will be 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 that 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 31.
For more information about the Validation Log, see page 40.
For more information about the Instrument Operations Log, see page 47.
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 by 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
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previous versions of the software or created in Standard Mode are also unsigned,
uncontrolled documents, and you will be 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.
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 (page 159) will be preserved
in the new file and the file will remain a Secure file.
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 Protocol files have the file extension *.spbx. Secure 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. (Note that this dialog will open
automatically when a Results file is generated from a Protocol file.)
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Figure 121. Electronic Signature dialog.
In the dialog, the signing party enters their user name and password in the
appropriate fields. (Note that 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.
When you click on OK, a Save As dialog will open.
Figure 122. Save As dialog for Secure Protocol file.
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Enter a name for the file or use the default name and click on 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 159.
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 will be flagged as read-only and
any subsequent changes must be saved under a new file name.
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 123. Document Properties dialog.
The Document Properties dialog also provides information about the signature.
The full name of the user who has signed the document is displayed in the title bar of
the file window.
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Figure 124. Signed and read-only status of file shown in title bar
All printouts and exports from a signed document also include the signature and
Document ID number.
Figure 125. Example of a Document ID and signature in an Excel export file
Clinician 2 Users: 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, and then a Clinician 2level user can 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.
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Clinician 2-level users do not have access to the other plate formatting tools in
Protocols. See pages 61–65 for information on defining unknown sample wells and
replicates in Protocols.
With the Protocol file open and the Format Plate window displayed, click on the Set
in the formatting toolbar or select the
Number of Unknown Samples button
command from the Format Options menu.
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 126. Set Number of 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 on
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 that you can only specify 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.
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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 91) is
automatically selected and cannot be deselected. You will be prompted to sign and
save the Results file at the start of each reading.
In Secure Mode, each reading, including reruns of plate wells, will generate a new
Results file that cannot be overwritten.
Figure 127. Electronic Signature required for a Results file before a reading
You will also be prompted to sign and save the Results file if you select the New
Results from Protocol command from the File menu (see page 115).
Audit Trail
Any changes made or actions performed after a Protocol or Results file has been
signed will automatically generate 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
will be 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 will be preserved in the
Results file generated from that Protocol.
All major actions and changes are audited. Examples of auditable actions include:
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•
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 the display only, such as selecting different columns in the
Report Table or changing the axis transformation in the Standard Curve, are not
audited.
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 between
subwindows in the Protocol or Results file.
Figure 128. Reason for Change dialog, used to generate the audit trail
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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 on the
Finished button.
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 129. Audit trail for a Protocol file
Figure 130. Audit trail for a Results file
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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.
The audit trail also notes each time the file has been signed. In the example in Figure
131, note that each time the file was signed and made read-only, the audit trail was
continued in the new file.
New file:
Human_Cytokines_002.spbx
New file:
Human_Cytokines_001.spbx
New file:
Human_Cytokines.spbx
Figure 131. Example of a Protocol audit trail with signatures
To display the complete contents of the Description and Reason cells in the table,
you can hold your cursor over a table cell and the information for that cell will be
displayed in a context pop-up box. Or you can click on the Expand Audit Entries
button
in the audit trail window toolbar to expand all the cells.
Figure 132. Example of a pop-up information box in the audit trail
To copy the contents of the audit trail to the Windows® clipboard, click on the Copy
to Clipboard button
in the window toolbar. To print the audit trail, click on the
Print button .
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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 BioPlex Manager user name and password). Consult your system administrator for
information about protected directories.
Locking Bio-Plex Manager Security
Edition
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.
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 on OK.
If a Supervisor-level user unlocks the application, that Supervisor-level user will
then be logged in as the current user.
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.
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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.
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Appendix
Troubleshooting Guide
Message/Problem
"Bio-Plex Manager
has detected a
problem with low
bead number"
Cause
Solution
Most Likely:
Too few beads in the assay
(5,000 per region recommended).
Check bead number calculations in
assay.
Plate not shaken for 30 seconds
before analysis.
Remove plate from array reader and
shake for 30 seconds.
Buffer volume in wells is too low
(must be at least 125µl).
Re-suspend wells in 125µl. 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:
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).
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Message/Problem
"Bio-Plex Manager
has detected a
problem with
selection of
regions"
" Bio-Plex Manager
has detected a
possible problem
due to microbubbles
in the cuvette or
photobleached
beads "
Cause
Solution
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 that the correct number of
beads was used by referring to the
specific assay preparation procedure.
Most likely:
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
a dirty MCV Plate III.
Clean MCV Plate III and recalibrate.
Misalignment of optics.
Perform OPTICS VALIDATION.
Contact technical support if values
are not within range.
Less likely:
166
Microbubbles present in cuvette.
Perform REMOVE BUBBLES.
Calibration beads are
photobleached (do not expose to
light for more than 1 hour).
Recalibrate with new CAL1 beads.
Appendix: Troubleshooting Guide
Message/Problem
"Bio-Plex Manager
has detected a
problem with bead
aggregation"
Cause
Solution
Incorrect DD gate values used.
View histogram in Run Protocol
window and make sure DD peak falls
within the gate range. If necessary,
adjust gates (see page 101).
Incorrect DD target value used for
calibration.
View histogram in Run Protocol
window and make sure that the DD
peak falls within the gate range.
If running a Luminex-type assay from
another manufacturer, use only
Bio-Plex DD settings (targets and
gates).
Clumped beads present.
Vortex plate at 900 rpm for 1 minute.
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.
Array reader or microplate
platform is not turned on.
Turn on array reader and microplate
platform.
Software is not communicating
with array reader.
Close and restart Bio-Plex Manager.
Cables between computer and
array reader or microplate
platform are loose/not connected.
Check cables for proper connections.
"Pressurizing" in
status bar of
software
Leak in sheath bottle or cap.
Tighten sheath cap.
Needle stuck in
down position
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).
"Check Link" in
status bar of
software
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Message/Problem
No Assay Signal
Detected
Cause
Solution
Most likely:
Error in assay preparation.
Make sure that streptavidin and
cytokine standards have been added.
Consult assay preparation procedure.
Incorrect target values used in
calibration.
Check that target values in the
Calibrate dialog match the values on
CAL1 and CAL2 bottles.
Waste container overfilled.
Empty waste. Reconnect waste.
Perform UNCLOG.
Sheath reservoir low or empty.
Refill sheath reservoir. Perform
START UP. Proceed with run.
Less likely:
"Bio-Plex Manager
has detected a
change in sheath
pressure"
Waste line not connected
properly.
Check waste line connection. Make
sure that it clicks into place.
Laser not functioning.
Perform calibration. If no signal
detected, call technical support.
Most 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:
168
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.
Listen for air pump to turn on when
WARM UP is selected. Contact
technical support for further
assistance.
Appendix: Troubleshooting Guide
Message/Problem
Cause
Solution
Bio-Plex Manager
has detected a
change in the
temperature of the
array reader. Please
calibrate before
running an assay to
ensure accurate
results.
Room temperature has changed.
Calibrate array reader.
The calibration was
unsuccessful.
Please repeat
calibration. If
calibration fails a
second time,
consult
Troubleshooting
Guide.
Calibration procedure failed.
Make sure CAL1 beads and CAL2
beads are placed in the appropriate
wells (CAL1 in red well and CAL2 in
green well). Repeat calibration.
The calibration was
unsuccessful.
Bio-Plex Manager
has detected a
problem with low
bead number.
Please repeat
calibration.
Calibration procedure failed due
to low bead number.
Run UNCLOG procedure, then repeat
calibration. If problem persists,
contact technical support.
Optics Validation
Procedure shows
value(s) outside of
acceptable range(s).
Problem with optical component
of array reader.
Repeat validation procedure. If values
are still out of range, contact technical
support.
Fluidics Validation
Procedure shows
value(s) outside of
acceptable range(s).
Problem with fluidics component
of array reader.
Repeat validation procedure. If values
are still out of range, contact technical
support.
Reporter Validation
Procedure shows
value(s) outside of
acceptable range(s).
Problem with optical component
of array reader.
Repeat validation procedure. If values
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.
Repeat validation procedure. If values
are still out of range, contact technical
support.
If problem persists, contact technical
support.
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Bio-Plex Manager User Guide
Message/Problem
Cause
Solution
Optics, Fluidics,
Reporter, or Classify
Validation
Procedure fails due
to low bead number
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.
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 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.
Platform heater not
at ambient
temperature
170
Glossary
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 is 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.
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Carboxylated
microspheres
Microspheres with chemically modified surfaces such that
they are 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.
Conjugated
microspheres
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.
Coupled microsphere
See conjugated microsphere.
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 streptavidinbound 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
(singlet) microsphere.
172
Appendix: Glossary
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. 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.
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 fluorochromeantibody complex to detect or quantify a particular antigen.
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Bio-Plex Manager User Guide
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.
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.
174
Appendix: Glossary
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.
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
non-fluorescent 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.
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 is also stored in the Protocol file. In BioPlex 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.
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Bio-Plex Manager User Guide
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.
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.
176
Appendix: Glossary
Residual variance
A statistical measure of goodness of 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 by 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.
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Singlet(s)
A single microsphere or a population of single microspheres.
Compare to doublets which are two microspheres associated
together.
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 (e.g., 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.
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. BioPlex 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.
178
Table Error Codes
Certain data that cannot be measured or calculated will be flagged in the Raw Data and
Report tables. Columns and their possible error codes are listed below:
Column
FI
FI – Bkgd
SD
CV%
Obs Conc
(4PL/5PL)
Obs Conc
(Linear))
Error Code
***
--***
--***
***
***
Cause
No data present
Marked as an outlier
No data present (negative values, if any, will be
displayed)
Marked as an outlier
No data present
No data present
No data present
OOR>
OOR<
OOR
*Value
***
Out of Range - Above asymptote of equation
Out of Range - Below asymptote of equation
Out of Range - Math Error
Above or below standards FI
No data present
OOR
*Value
Negative extrapolated data
Positive extrapolated data or
Data above/below the standards FI
No data entry - this value is input by user
OOR displayed in Obs Conc column
Obs Conc is not within the specified recovery
range of standards conc (the recovery range is
specified by the user; see page 81)
Low bead number detected in the well
Aggregated beads detected in the well
Classify efficiency problem detected in the well
Region selection problem detected in the well
Platform temperature problem detected in the
well
Exp Conc
Obs/Exp*100
Conc In Range
***
***
***
Sampling Error
1
2
3
4
5
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180
Security Edition: User Access by
Function
The following table lists the functions and user levels in Bio-Plex Manager Security
Edition. 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
Viewing All Screens (Not
Modify)
X
X
X
X
X
View Audit Trail
X
X
X
X
X
Printing
X
X
X
X
X
Save
X
X
X
X
Save As
X
X
X
X
X
Exporting
X
X
X
X
X
Electronically Sign Document
X
X
X
X
X
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
Enter Validation Lot/Exp Date
X
X
X
X
Functions that Apply to Instrument Control
Instrument - Set Up
X
X
X
X
Instrument – Reconnect
X
X
X
X
Instrument Info
View Operation Log
X
X
X
X
X
X
X
X
X
X
181
Bio-Plex Manager User Guide
Function
Admin Supervisor Service Clinician 2 Clinician 1 Reviewer
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, Format Plate View
Formatting 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
X
X
X
X
Rerun/Recovery Mode
X
X
X
X
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Appendix: Security Edition: User Access by Function
Function
Admin Supervisor Service Clinician 2 Clinician 1 Reviewer
Select Beads to Count
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
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Function
Admin Supervisor Service Clinician 2 Clinician 1 Reviewer
Enter Std Curve Regression
Method
X
Enter Std Curve Adv Settings
X
X
Enter Std Concentration Units
X
X
Enter Recovery Range
X
X
Select External Standards
X
X
X
Functions that Apply to Results File, Enter Sample Info View
Enter Sample Info (Sample
Description)
X
X
Enter Sample Info (Sample
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 Raw Data 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 Regression Type for
All Analytes
X
X
Logistic Weighting Options
X
X
Labels
X
X
X
X
X
Error Bars
X
X
X
X
X
184
Appendix: Security Edition: User Access by Function
Function
Admin Supervisor Service Clinician 2 Clinician 1 Reviewer
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 Assign Users To Groups
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Bio-Plex Manager User Guide
186
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.
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 freeze/thaw manipulations.
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, R-phycoerythrin. 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, while the emission wavelength is 575 + 12nm. Specific
information on R-phycoerythrin is shown in the following table:
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Bio-Plex Manager User Guide
R-Phycoerythrin
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
Fluidics
There are two fluidic paths in the array reader. The first path includes a syringedriven 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, called 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,
called 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.
188
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 that 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 User Guide
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.
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 × 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.
190
Appendix: 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 to 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.
191
Bio-Plex Manager User Guide
192
Reporting Problems
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.
For critical problems, contact Bio-Rad technical support at the numbers listed on
page 12.
To use Solobug, select Solobug from the Start menu, enter your information and a
description of the request in the Solobug window, and click on Save. This will create
a report that you can attach to an e-mail and send to Bio-Rad.
Please send your Solobug files to Bio-Rad at:
[email protected] (in the U.S.)
[email protected] (International)
193
Bio-Plex Manager User Guide
194
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
195
Bio-Plex Manager User Guide
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.
196
Index
Aggregated beads...............................92
Air bubbles, removing .......................33
Alcohol
Flush..............................................46
Wash .............................................45
Analytes ...............................................1
Custom, creating ...........................55
Editing...........................................57
Panels ............................................54
Selecting in protocol .....................53
Array Reader........................................1
Calibration.....................................25
Canceling an operation..................46
Connecting ....................................13
Controls.........................................21
Disconnecting ...............................15
Ejecting plate ................................44
General cautions............................21
Miscellaneous functions................45
Optics .......................................... 188
Principles of operation ................ 187
Reconnecting.................................15
Removing bubbles.........................33
Sample needle ...............................17
Shutting down ...............................46
Start up..........................................23
Status.............................................44
Unclogging....................................34
Warm up .......................................24
Washing ........................................33
Array Reader Validation .See Validation
Assay reading..................... See Reading
Audit trail......................................... 159
Auto save after run.............................91
Autofill
Settings..........................................62
Turning off ....................................63
Back flush ..........................................46
Bead map......................................... 104
Analytes selected .......................... 99
Changing axes ............................ 105
Display options........................... 107
Displaying .................................... 98
Level multiplier .......................... 107
Log/linear scale view.................. 106
Magnifying ................................. 106
Resizing...................................... 106
Beads ................. See Also Microspheres
Aggregated ................................... 92
Low number ................................. 92
Photobleached .............................. 92
Beads per region counted .................. 89
Bio-Plex Array Reader ...........See Array
Reader
Bio-Plex Manager
Installation .................................... 10
Interface........................................ 15
Licenses.......................................... 5
Security Edition.......................... 147
Starting ......................................... 13
Bio-Plex Suspension Array System..... 1
Components.................................... 2
Concepts ..................................... 187
Controls ........................................ 21
BioPlexdata.mdb file ......................... 11
Blank wells, defining......................... 67
Calibration......................................... 25
Adding new control numbers ....... 27
Log file ......................................... 31
Log viewer.................................... 31
Monitoring.................................... 30
Preparing plate.............................. 29
Target values ................................ 27
Canceling an instrument operation.... 46
Cautions............................................. 21
CFR, Title 21, Part 11 ..................... 147
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Bio-Plex Manager User Guide
COM port settings ............................. 13
Combining panels of analytes ........... 59
Compressed mode ......................50, 116
Computer
Requirements.................................. 9
Screen resolution ............................ 9
Concentration in range .................... 131
Connecting to the reader.................... 13
Control wells, defining ...................... 66
Controls
Dilution series............................... 84
Entering information .................... 82
General system ............................. 21
Recovery rate.............................. 130
Wells............................................. 66
Copying
Raw data ..................................... 113
Report table ................................ 132
Standard curve............................ 142
Custom analytes and panels .............. 55
DD gate range.................................... 93
Changing in Results file ............. 120
Displayed in histogram............... 101
Density plot ....................................... 98
Describe protocol .............................. 53
Desktop license ................................... 5
Disconnecting from the reader .......... 15
Doublet discriminator channel .......... 93
Displaying .................................. 101
Draining the fluidics system.............. 45
Editions ............................................... 5
Eject/retract plate............................... 44
Errors in sampling ............................. 92
Excitation ........................................ 188
Exporting
Raw data ..................................... 122
Report table ................................ 132
XML file..................................... 143
External standards ............................. 73
File types ............................................. 8
Files
Protocol ........................................ 49
Results ........................................ 115
198
Files, secure ..................................... 153
Fluidics ............................................ 188
Draining ........................................ 45
Priming ......................................... 45
Removing bubbles from................ 33
Sanitizing ...................................... 45
Unclogging ................................... 34
Washing ........................................ 33
Format plate ........See Microplate format
Gate range................ See DD gate range
Glossary ........................................... 171
Grouping............See Microplate format,
grouping
Hardware Protection Key................... 12
HASP key .......................................... 12
Heater................................................. 42
Histogram ........................................ 100
Analytes selected .......................... 99
Channel selection........................ 101
DD gate lines .............................. 101
Displaying..................................... 98
Magnifying ................................. 103
Resizing ...................................... 103
Scaling data................................. 103
Installation
Software........................................ 10
Instrument information ...................... 44
Instrument Operations Log ................ 47
Level multiplier ............................... 107
Licenses ............................................... 5
Loading plate ..................................... 95
Luminex IS 2.3, compatibility ............. 6
MCV plate ................................... 23, 24
MCV Plate III .................................... 21
Menus ................................................ 15
Microplate
Loading ......................................... 95
Microplate format .............................. 59
Deleting/changing......................... 68
Formatting wells ........................... 61
Group reference ............................ 70
Grouping ....................................... 68
Printing ......................................... 71
Index
Setting the number of unknown
samples................................... 157
Microplate wells
Autofill..........................................62
Blanks ...........................................67
Controls.........................................66
Deleting formatting .......................68
Grouping .......................................68
Numbering ....................................62
Replicate groups............................62
Resequencing ................................65
Standards.......................................65
Unknowns .....................................61
Microsoft.net......................................11
Microspheres.................................... 187
Calibration.....................................27
Handling...................................... 189
Per region counted ........................89
Network license ...................................5
Opening
Protocols .......................................49
Results......................................... 115
Optics
Shut down .....................................24
Warm up .......................................24
Outliers ............................................ 129
Panels of analytes
Combining.....................................59
Custom, creating ...........................55
Custom, deleting ...........................58
Custom, renaming .........................58
Editing...........................................57
Selecting........................................54
Phycoerythrin................................... 188
Plate ID number ............................... 122
Plate loading ......................................95
Platform heater...................................42
Temperature error flag ..................92
PMT voltage ......................................94
Priming the fluidics system................45
Printing
Raw data ..................................... 123
Report table................................. 136
Standard curve............................ 142
Protocol
Analytes selection......................... 53
Creating ........................................ 49
Describing .................................... 53
Entering controls info ................... 82
Entering unknown samples info ... 85
Format plate... See Microplate format
Generating results......................... 97
New protocol from results ............ 50
Opening ........................................ 49
Rerunning ................................... 113
Running ........................................ 95
Saving........................................... 50
Standards info............................... 71
Window ...................................51, 52
Protocol files ..................................... 49
Quick Guide ...................................... 16
Ratio of member value to reference .. 68
Raw data.......................................... 121
Copying ...................................... 113
Exporting.................................... 122
Printing ....................................... 123
Sampling error codes.................. 110
Raw data table ................................. 108
Display Options.......................... 111
Reading ............................ See Also Run
Parameters .................................... 49
Raw data ..................................... 108
Rerun/recovery ........................... 113
Results ........................................ 115
Sampling errors ............................ 92
Status ............................................ 97
Stopping manually........................ 97
Recovery percentage ......................... 81
Reference........................................... 68
Region
Beads counted per ...................... 109
Number......................................... 54
Regression methods......................... 138
Selecting in protocol..................... 79
Remove bubbles ................................ 33
Replicate groups................................ 62
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Bio-Plex Manager User Guide
Changing ...................................... 65
Defining........................................ 62
In report table ............................. 128
Report table ..................................... 123
Columns ..................................... 124
Conc in Range column ............... 131
Copying ...................................... 132
Error conditions.......................... 179
Exporting.................................... 132
Formatting .................................. 131
Obs/Exp*100 column ................. 130
Options ....................................... 126
Outliers ....................................... 129
Printing ....................................... 136
Replicate groups ......................... 128
Reporter fluorochromes................... 187
Rerun/recovery mode ...................... 113
Resequencing microplate wells ......... 65
Results
Analytes selection....................... 119
Calculated values........................ 123
Controls info............................... 119
Exporting raw data ..................... 122
Exporting report table................. 132
Generating from protocol ............. 97
Opening ...................................... 115
Plate format ................................ 119
Printing raw data ........................ 123
Protocol description.................... 119
Raw data ..............................108, 121
Report table ................................ 123
Reviewing/changing protocol
settings................................... 118
Samples info ............................... 119
Saving......................................... 116
Standard curve............................ 136
Standards info............................. 119
XML export................................ 143
Results files ..................................... 115
Run ............................ See Also Reading
Protocol ........................................ 87
Settings ......................................... 89
Run conditions, documenting.......... 143
200
Sample groupingSee Microplate format,
grouping
Sample needle, adjusting ................... 17
Sample size ........................................ 91
Sampling errors
Raw data table............................. 110
Sanitizing the fluidics system ............ 45
Saving
Automatically after reading .......... 91
Compressed mode................. 50, 116
Protocols ....................................... 50
Results......................................... 116
Screen resolution ................................. 9
Security Edition ........................... 5, 147
Audit trail.................................... 159
Audit trail, viewing ..................... 161
CFR, Title 21, Part 11 ................. 147
Document ID............................... 156
Electronic records ....................... 152
File security................................. 152
Locking software ........................ 163
Log files ...................................... 153
Logging off ................................. 163
Passwords ................................... 149
Protocol files ............................... 153
Protocol files, adjusting samples. 157
Results files................................. 153
Results files, generating .............. 159
Secure Mode ............................... 148
Secure mode, enabling and disabling
............................................... 150
Signed files ................................. 154
Standard Mode............................ 148
Starting........................................ 148
System Requirements.................. 148
Unsigned files ............................. 153
User access by function .............. 181
User authentication ..................... 151
User Levels ................................. 149
Users ........................................... 149
Select analytes ................................... 53
Set Number of Unknown Samples... 157
Shut down .......................................... 46
Index
Signed files ...................................... 153
Solobug ............................................ 193
Standard curve ................................. 136
Copying....................................... 142
Equations .................................... 139
Log versus linear......................... 141
Logistic weighting.........................80
Printing........................................ 142
Regression method, selecting in
protocol ....................................79
Regression methods .................... 138
Standard Edition ..................................5
Standard wells, defining.....................65
Standards............................................71
Back-calculation ......................... 130
Concentration units .......................78
Current plate..................................74
Curve........................................... 136
Entering concentrations.................76
External.........................................73
Recovery percentage range ...........81
Recovery rate .............................. 130
Wells .............................................65
Start up...............................................23
Starting Bio-Plex Manager ................13
Status bar ..................................... 15, 97
Stopping a reading .............................97
System Requirements...........................9
Technical support ................................ 4
Toolbars............................................. 15
Total beads counted........................... 89
Triggers ............. See Errors in sampling
Troubleshooting .............................. 165
Unclog ............................................... 34
Unknown samples
Defining wells .............................. 61
Entering information .................... 85
Validation.......................................... 35
Control numbers ........................... 36
Kit................................................. 35
Loading Plate................................ 38
Log ............................................... 40
Results .......................................... 39
Set Up........................................... 36
Warm up............................................ 24
Warranty.......................................... 195
Wash
Alcohol ......................................... 45
Basic ............................................. 45
Between plates.............................. 33
Wells .................... See Microplate wells
Workflow ............................................ 7
Workstation license ............................. 5
xMAP Technology .............................. 1
XML export..................................... 143
Automatic export after reading..... 91
201
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