GemStone User Guide

Table of Contents
GemStone User Guide ........................................................................................................ 1
License and Warranty ......................................................................................................... 3
Quick Start ........................................................................................................................ 5
Latest Versions .................................................................................................................. 7
New Features in GemStone ................................................................................................. 9
System Requirements ........................................................................................................13
Installation and Setup of GemStone Client...........................................................................15
Installation and Setup of Battery Server for GemStone .........................................................17
Registering your new software ...........................................................................................19
Overviews ........................................................................................................................25
Overview Section ...........................................................................................................25
Overview: Workspace Names and User Interface ..............................................................26
Overview: What is a Probability State Model? ...................................................................27
Overview: Building a Model ............................................................................................31
Overview: Using a Model ................................................................................................32
Overview: Software Batteries ..........................................................................................33
Tutorials...........................................................................................................................35
Tutorials .......................................................................................................................35
Model Building 101 ........................................................................................................36
Strategy ....................................................................................................................36
Getting Started...........................................................................................................36
Choosing CD19 Parameter ...........................................................................................37
Modeling CD19 ...........................................................................................................38
Modeling the SSC Parameter .......................................................................................40
Modeling the TdT Parameter .......................................................................................41
Using Estimate X Positions ..........................................................................................42
Adding a 1P Plot .........................................................................................................43
Getting Statistics ........................................................................................................43
Saving our Model........................................................................................................45
Conclusions ................................................................................................................45
See also: ...................................................................................................................45
Model Building 102 ........................................................................................................46
Strategy ....................................................................................................................46
Open Model101.gs ......................................................................................................46
Read the File..............................................................................................................46

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Adding a Parameter Overlay Plot..................................................................................47

Modeling CD20 ...........................................................................................................47
Modeling CD10 ...........................................................................................................50
Redefine Zones ..........................................................................................................53
Create a 2P Plot .........................................................................................................54
See also: ...................................................................................................................55
Model Building 103 ........................................................................................................56
Open the Model..........................................................................................................56
Disable the Profiles .....................................................................................................56
Open the Data File .....................................................................................................57
Adjust and Enable CD19..............................................................................................57
Turn Off Means and Confidence Limits for SSC..............................................................58
Adjust and Enable SSC ................................................................................................58
Enrich........................................................................................................................59
Adjust and Enable TdT ................................................................................................60
Adjust and Enable CD10..............................................................................................61
Using the Frequency Plot ............................................................................................62
Adjust and Enable CD20..............................................................................................63
Add the Remaining Parameters ....................................................................................64
Saving our Model........................................................................................................65
Other Enhancements ..................................................................................................65
See also: ...................................................................................................................65
Model Building 104 ........................................................................................................67
Open the Model..........................................................................................................67
Cell Type 1: The Kappa Cell Type.................................................................................67
Duplicate Cell Type .....................................................................................................68
Cell Type 2: The Lambda Cell Type ..............................................................................69
Saving the B-Cell Kappa Lambda Model ........................................................................70
Auto Analyze the Data ................................................................................................70
Summary ...................................................................................................................72
See also: ...................................................................................................................73
Creating a Model for a Multi-Tube Panel ..........................................................................74
Concept and Strategy .................................................................................................74
Choosing Good Common Parameters............................................................................74
Tube 1 ......................................................................................................................75
Select the Data Files ...................................................................................................75
Setting up Selection Parameters ..................................................................................75

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GemStone User Guide

Verifying with a 2P Plot ...............................................................................................77

Defining the Progression .............................................................................................78
Adding the Remaining Parameters from Tube 1 ............................................................82
Creating a Parameter Overlay Plot ...............................................................................82
Analyzing Tube 2 ........................................................................................................83
Adding Tube 3 ...........................................................................................................85
Summary ...................................................................................................................86
See also: ...................................................................................................................86
Model Building 201 ........................................................................................................88
Getting Started...........................................................................................................88
Relations ...................................................................................................................91
Modifying a Relation ...................................................................................................93
Adding a Relation .......................................................................................................93
Reading a Relation Expression .....................................................................................94
Relations with Other Parameter Profiles ........................................................................94
Saving Relations .........................................................................................................95
Conclusions ................................................................................................................95
See also: ...................................................................................................................95
Heat Map and Animated Regions.....................................................................................96
Animated Regions.......................................................................................................98
How to Remap Parameters in a Model ........................................................................... 101
The Problem ............................................................................................................ 101
Two Approaches ....................................................................................................... 101
Option 1: Remapping the Model Parameters ............................................................... 102
Saving the Changes to a New Model .......................................................................... 103
Option 2: Using the Parameter Database .................................................................... 103
Open the Parameter Database ................................................................................... 103
Matching the Parameters .......................................................................................... 104
Finishing Edits .......................................................................................................... 106
Summary ................................................................................................................. 106
See also: ................................................................................................................. 106
Batch Processing ......................................................................................................... 107
Background of the Example ....................................................................................... 107
First Time Processing ................................................................................................ 107
Open the GemStone Model Document ........................................................................ 107
Select the Files to Analyze ......................................................................................... 107
Edit the Fields for the Database ................................................................................. 108

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Set the Batch Options ............................................................................................... 110

Process the Files....................................................................................................... 111
After the First Time................................................................................................... 112
Using Levels............................................................................................................. 112
Summary ................................................................................................................. 114
Batteries ........................................................................................................................ 115
How to Work with Software Batteries ............................................................................ 115
Why Software Batteries? ........................................................................................... 115
How do the Batteries Work? ...................................................................................... 115
Recommendations for Battery Use ............................................................................. 115
What is a Web Battery Account? ................................................................................ 115
Free Trial Battery ..................................................................................................... 116
Working with Battery Charge ..................................................................................... 116
Purchasing Battery Charge ........................................................................................ 117
Charging the Software Battery ................................................................................... 117
Battery Charge Method 1: Redeem a Battery Certificate via the Wizard ......................... 117
Battery Charge Method 2: Redeem a Battery Certificate using a web browser ................ 118
Charge Battery Method 3: Download charges directly from your Web Battery Account ... 119
Charge Battery Method 4: Battery Charge Codes from a Web Battery Account ............... 120
Charge Battery Method 5: Request charges from a Battery Server ................................ 120
Charge Battery Method 6: Battery Charge Codes from a Battery Server......................... 121
Uploading Battery Charge to Your Web Account .......................................................... 122
Checking the Balance of Your Web Battery Account ..................................................... 122
Summary ................................................................................................................. 122
See also: ................................................................................................................. 122
How to Work with Battery Server for GemStone ............................................................. 124
Setting up the Battery Server .................................................................................... 124
Registering the Battery Server ................................................................................... 124
Configure the Battery Server ..................................................................................... 125
Charging the Battery................................................................................................. 126
Configuring the System to Automatically Run the Battery Server................................... 126
Common Steps to Automatically Run the Battery Server .............................................. 126
Automatically Run the Battery Server on Windows....................................................... 127
Automatically Run the Battery Server on Os X ............................................................. 128
Using the Battery Server ........................................................................................... 130
Summary ................................................................................................................. 130
See also: ................................................................................................................. 130

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 GemStone User Guide

Reference....................................................................................................................... 131
Reference Section ........................................................................................................ 131
Control Panels ............................................................................................................. 132
File Batch and Database panel ................................................................................... 133
Batch Levels panel .................................................................................................... 139
Batch Options panel.................................................................................................. 141
Global Properties panel ............................................................................................. 146
Cell Type Properties panel ......................................................................................... 147
Parameter Profile Properties panel ............................................................................. 159
Graphic Objects ........................................................................................................... 178
Cell Type Canvas ...................................................................................................... 178
Cell Type Canvas Context Menu ................................................................................. 179
Edit Canvas Properties Dialog .................................................................................... 180
Edit Background Page Properties Dialog ..................................................................... 181
Cell Type Widget ...................................................................................................... 182
Properties of Cell Type Widget ................................................................................... 184
Graphic Options for Parameter Profile: Surface............................................................ 185
Graphic Options for Parameter Profile: Dots ................................................................ 186
Graphic Options for Parameter Profile: Means ............................................................. 187
Graphic Options for Parameter Profile: Confidence Limits ............................................. 188
Graphic Options for Parameter Profile: Model .............................................................. 189
Graphic Options for Parameter Profile: Animate........................................................... 190
Other Graphic Options .............................................................................................. 191
Parameter Overlay Plot ............................................................................................. 193
Graphic Options for Parameter Overlay Plots ............................................................... 195
Other Options for Parameter Overlays ........................................................................ 196
1P Histogram ........................................................................................................... 200
Graphic Options for 1P Histograms ............................................................................. 202
1P Animation Options ............................................................................................... 204
Other Graphic Options .............................................................................................. 205
2P Histograms .......................................................................................................... 207
Properties for 2P Surface........................................................................................... 209
Graphic Options for 2P Dots ...................................................................................... 210
Graphic Options for 2P State Vectors .......................................................................... 213
Animation for 2P Histograms ..................................................................................... 214
Region Label Properties ............................................................................................ 216
Heat Map ................................................................................................................. 218

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Edit Graphics for Heat Map: Surface ........................................................................... 219

Edit Graphics for Heat Map: Dots ............................................................................... 220
Edit Graphics for Heat Map: Zones and P-Values ......................................................... 221
Edit Graphics for Heat Map: Animation ....................................................................... 222
Region Label Properties ............................................................................................ 223
Parameter Graph ...................................................................................................... 225
Zone Statistics Table ................................................................................................. 226
Legend .................................................................................................................... 227
Table....................................................................................................................... 228
Table Context Menu.................................................................................................. 230
Toolbars and Shortcuts ................................................................................................ 231
Main toolbar............................................................................................................. 231
Batch Controls toolbar .............................................................................................. 232
Workspace toolbar .................................................................................................... 233
Parameter Profile toolbar .......................................................................................... 235
1P Graphics toolbar .................................................................................................. 236
2P Graphics toolbar .................................................................................................. 237
Overlay Graphics toolbar ........................................................................................... 239
Shortcuts ................................................................................................................. 241
File Menu .................................................................................................................... 243
New Document ........................................................................................................ 243
Open Document ....................................................................................................... 244
Save Document ........................................................................................................ 245
Save Document As ................................................................................................... 246
Select FCS Files ........................................................................................................ 247
Edit FCS Information................................................................................................. 249
Close FCS File .......................................................................................................... 250
Synthesize Data ....................................................................................................... 251
Export Model Data .................................................................................................... 252
Manage Settings....................................................................................................... 253
Print All Cell Types .................................................................................................... 255
Exit (Quit) ................................................................................................................ 256
Edit Menu ................................................................................................................... 257
Edit Parameter Database........................................................................................... 257
Edit Properties for Parameter..................................................................................... 259
Properties for Linear Transformation .......................................................................... 262
Edit Parameter Conditions ......................................................................................... 264

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GemStone User Guide

Edit Properties of Parameter Condition ....................................................................... 265

Edit Color Palette...................................................................................................... 266
Properties for Color Palette........................................................................................ 267
Edit Logging System ................................................................................................. 268
View All Application Information ................................................................................ 269
Edit Information Equations ........................................................................................ 270
Edit Equation ........................................................................................................... 273
Equation Functions ................................................................................................... 274
Show Tool Button Labels ........................................................................................... 280
Edit Preferences ....................................................................................................... 281
Preferences .............................................................................................................. 282
Analysis Menu ............................................................................................................. 299
Classify Data ............................................................................................................ 299
Optimize Model ........................................................................................................ 300
Auto Analyze ............................................................................................................ 301
Enrich Data .............................................................................................................. 302
Classify File .............................................................................................................. 303
Fidelity Analysis ........................................................................................................ 304
Battery Menu............................................................................................................... 308
Battery Menu ........................................................................................................... 308
Battery Status .......................................................................................................... 309
Buy Now .................................................................................................................. 310
Charge Battery ......................................................................................................... 311
Charge Battery Wizard .............................................................................................. 312
Check Balance .......................................................................................................... 313
Upload Credits.......................................................................................................... 314
Server Menu................................................................................................................ 315
Server Menu ............................................................................................................ 315
Properties for Battery Server ..................................................................................... 316
Transfer Charge ....................................................................................................... 317
Help Menu .................................................................................................................. 318
Help Index ............................................................................................................... 318
Ask Tech Support ..................................................................................................... 319
Make a Suggestion ................................................................................................... 320
Report a Problem ..................................................................................................... 321
Application Info ........................................................................................................ 322
About ...................................................................................................................... 323

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 Table of Contents

Index ............................................................................................................................. 325

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GemStone User Guide

Copyright 2008-2013 by Verity Software House

All Rights Reserved.
Updated 22 Feb 2013

This guide is intended for GemStone version 1.0 and minor revisions.

Quick Jumps:
Quick Start
Overview Section
Tutorials
Reference Section

Verity Software House

Phone: (207) 729 6767
Fax: (207) 729 5443
Email: [email protected]
Web: www.vsh.com

1
License and Warranty

License Agreement
Copyright laws of the United States and international treaty protect this software. Verity
Software House provides this software under the following conditions:
Software is intended to be installed and used on one computer. An additional user license is
required for each additional installation.
Some software is licensed for metered-usage with a software "battery". A software battery is
licensed to one machine for one user account.
Backups of the software must be used only for archival purposes.
Duplication of the manual is strictly prohibited without the expressed written permission of
the copyright holder.
If you transfer the use of this software to another party, that party must accept the terms of
this agreement, and you must transfer all copies of the software or destroy them.
Your license to use this software will terminate if you are found to be in violation of this
agreement.
The laws of the State of Maine and the United States of America govern this license.

Limited Warranty
Verity Software House (VSH) warrants the physical media and the physical documentation, if
included, to be free of defects in materials and workmanship for a period of 30 days from
date of purchase. If VSH receives notice of defects in materials or workmanship within the
warranty period, VSH will replace the defective media or documentation.
VSH makes no other warranties, either expressed or implied, with respect to this manual or
with respect to the software described in this manual, its quality, performance,
merchantability, or fitness for any particular purpose. VSH software is licensed "as is". The
entire risk as to its quality and performance is with the buyer. Should the software prove
defective following their purchase, the buyer (and not VSH, its distributor, or its retailer)
assumes the entire cost of all necessary servicing, repair, or correction and any incidental or
consequential damages. In no event will VSH be liable for direct, indirect, incidental, or
consequential damages resulting from any defect in the software, even if VSH has been
advised of the possibility of such damages. Some states do not allow the exclusion or
limitation of implied warranties or liability for incidental or consequential damages, so some
of the above limitations or exclusions may not apply to you.

3
Quick Start

Install GemStone.
On a Macintosh:
If you received an electronic download, extract the GemStone Client Mac Installer into a
temporary folder on the Macintosh computer for installation. Then, launch the installer
program and follow directions on the screen.
If you received an installation CD, insert the CD and double click on the installer package.
Then follow the directions on the screen.

On a PC:
If you received an electronic download, extract the GemStone Client Windows Installer into a
temporary folder on the Windows computer for installation. Then, launch the installer
program and follow directions on the screen.
If you received an installation CD, insert the CD. If a browser does not automatically open,
double click on the CD and then double click on default.htm. Dismiss any warning message
from the browser. Click on the Install GemStone 1.0 link and follow the directions on the
screen.

Redeem the Battery Certificate. 1

Run GemStone.
Click OK to close the banner dialog.
Choose the Charge Battery command in the Battery menu.
Select the Redeem Battery Certificate via the Wizard option2.
Click Next.
Enter the Battery Certificate Code.
Click Next.

Get Busy!
The complete GemStone User Guide, as well as the latest videos and tutorials can be
accessed online at www.vsh.com/documentation.
We recommend starting with the Tutorials.
For assistance, please contact technical support at 207.729.6767 or [email protected].

1
If your version included a Battery Certificate, it is included as part of the installation CD
packaging or as part of an email received after an online purchase has been made.
2
This option requires that your computer be connected to the Internet.

5
Latest Versions

To find the latest versions for GemStone and its documentation, visit the Verity Software
House web site at www.vsh.com. This site provides a complete version of the latest revision
of the online documentation, patches, and technical support information for the program.
You can also email us at [email protected] to determine whether or not you have the latest
version. Please include the version that appears on the start-up dialog box, and the file date
for the program with your email.

7
New Features in GemStone

This section contains major changes in GemStone.

Build 1.0.35

Build 1.0.34
+ Created a region animation system that enables the identification of any population in all
GemStone graphic systems. (cbb, 01Jan10).
+ Created a heat map system capable of revealing very small populations that do not belong
to a model. (cbb, 01Jan10).
+ Increased the speed of drawing dots by 10%. (cbb, 01Jan10).
+ Fixed occasional problem with 1P scaling. (cbb, 21Dec09).
+ Statistics have been added to the properties of region labels. (cbb, 20Dec09).

Build 1.0.33
+ The accuracy of fitting multiple cell types has also been improved. (cbb, 08Dec09).
+ The accuracy of fitting has been improved substantially, especially with higher exclusion
probabilities. (cbb, 08Dec09).
+ AutoAnalysis did not work correctly if there wasn't at least one Constant parameter profile.
Fixed. (cbb, 20Nov09)
+ A bug was fixed with synthesizing data that was created in version .32. (cbb, 19Nov09).

Build 1.0.32
+ Documents now store the active cell type and return to that cell type after reading in the
document. (cbb, 12Nov09).
+ All tabs for parameters and cell types are now at the bottom of the application. This makes
selecting the page of interest more efficient. (cbb, 12Nov09).
+ GemStone stored documents are now more compact. (cbb, 12Nov09).
+ GemStone will no longer decrement the battery for files that are exported via the Export
Model Data command. This will prevent users from being charged for generated data files
they have created, for example when following the GemStone tutorials. (cmb, es,tc,bb
10Nov09)
+ When loading a document, you can now gate it without the file being loaded in. (cbb,
08Nov09).
+ The program now has a Model Heatmap Graph cell type graphical object. This graph allows
you to see how every event fits the model. A powerful way to use this new object is to use
Enrich to fill the packet with the events you want to investigate. Once the packet is enriched,
type in 0 for the Exclusion Probability cell type property. This forces the system to classify
just about every event in the packet. You can then use the heatmap to find small populations
that don't fit the model well. (cbb, 08Nov09).

9
 New Features in GemStone

+ The global ColorByGateCombination now works on classified and unclassified data. Regions
now have a property, Color By Region Combination, that allows you control what regions are
creating the color combinations. This enhancement makes it much easier to find outlier
populations in your modeling. (cbb, 08Nov09).
+ The vector states now have the ability to trace medians and modes. The medians are so
much better than means that they are now the default method for drawing the vectors. (cbb,
07Nov09).
+ When editing properties in a table pressing Enter or Return will move down one row and
begin an edit. (cmb, es, 3Nov09)
+ Prevented tool tips from blocking user input when shown on Mac systems. (cmb, tc,
3Nov09)
+ Program now gives you an opportunity to save your document before quitting. (cbb,
31Oct09)
+ <Control>S now saves the document to the last model with no dialog shown. (cbb,
02Nov09).
+ Program now gives you an opportunity to save your document before quitting. (cbb,
31Oct09).
+ Fixed a subtle bug on the mac that caused it to crash if the cursor was on a cell type
parameter profile shrink tool when the system was analyzing data (ES). (cbb, 29Oct09).
+ New surface graphics have been added to two-parameter and parameter profile graphs.
(cbb, 24Oct09).

Build 1.0.31
+ A new surface graphics option has been added to parameter state graphs. (cbb, 22Oct09).
+ A new program banner file has been created that expires in Feb, 2010. (cmb, 21Oct09).
+ When a GS document is read in, it always updates the matched database parameter's
transform so that when the associated data file is read in, it looks exactly the same as on the
model's author's system. (cbb, 07Oct09).
+ Added a preference for the fidelity plot in the cell type's frequency plot (Preffer). (cbb,
06Oct09).
+ Improved the ability of old versions of the program to read new versions of GemStone
documents. (cbb, cmb 05Oct09).

Build 1.0.30
+ GemStone now optimally smoothes mean and standard deviation values. (cbb, 25Sep09).
+ When a CDP relation was deleted, it wasn't properly deleting the back relation. (cbb,
22Sep09).
+ Fixed a bug that would cause the definition points of a model to be incorrectly positioned
when the model was saved and later opened. The problem was setup by changing the name
of a database parameter with a model loaded that included that parameter. (cbb, cmb,
18Sep09)

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GemStone User Guide

+ GemStone no longer remembers the pathway to a document if it is executed from an email

attachment. (cbb, 18Sep09).
+ Increased the maximum size of the information table from 20 to 200. (cbb, 18Sep09).
+ Add a "Paste from clipboard" option to context menus for parameter overlay. (cbb,
06Sep09).
+ Add a "Set all match status flags" option for the cell type actions. (cbb, 06Sep09)
+ Allow multiple parameter resolutions to be merged (Amnis files). (cbb, 05Sep09)

11
System Requirements

Minimum System Configurations

PC: Windows XP, Vista or greater with 1GB memory and 50MB free disk space for program
files.
Macintosh: OSX 10.3.9 or greater with 1GB memory and 50MB free disk space for program
files.

13
Installation and Setup of GemStone Client

GemStone Client software is easy to install on a Mac or Windows machine. In order to open
and analyze your own data files, it needs a "software battery". The software battery allows
GemStone to open and analyze a pre-defined number of files.
If you purchased GemStone Client with a Battery Certificate, you can use the certificate to
create a software battery after you install the software. You can also obtain charges for a
software battery from the Verity Software House web site or from a Battery Server for
GemStone if your institution has purchased and installed one on your network.

Windows Version

GemStone for Windows requires Windows XP, Vista or greater.

To install GemStone Client for Windows

● Log on to your computer with an account that has Administrator privileges. Most operating
systems require administrative rights during installation.
● If you have an installation CD, insert the program CD into the CD-ROM drive. If you
downloaded the software, extract the installation files from the download into a temporary
folder.
● Run the program named Setup or Setup.exe. Follow the directions on the screen to
complete the installation.

To run GemStone Client for Windows

● Log on to your computer with your own user account. This should be the account that you
normally use on the computer.
● Open the Start menu, and choose All Programs. Then navigate to the Verity folder, and
choose GemStone Client.

To install a software battery or battery certificate

● See the section How to Work with Software Batteries .

Macintosh Version

GemStone for Mac requires OSX 10.3.9 or greater. You will need to have administrative rights
to install the software.

To install GemStone Client for Macintosh

● Log on to your computer with an account that has Administrator privileges. Most operating
systems require administrative rights during installation.

15
 Installation and Setup of GemStone Client

● If you have an installation CD, insert the program CD into the CD-ROM drive. If you
downloaded the software, extract the installation files from the download into a temporary
folder.
● Run the GemStone Installer program. Follow the directions on the screen to complete
the installation.

To run GemStone Client for Macintosh

● Log on to your computer with your own user account. This should be the account that you
normally use on the computer.
● Start the program by double clicking on the GemStone Client for Mac application in the
Applications/GemStone folder.

To install a software battery or battery certificate

● See the section How to Work with Software Batteries .

16
Installation and Setup of Battery Server for GemStone

You will need to register Battery Server for GemStone with Verity Software House in order to
unlock the program on the computer. This can be done using the registration wizard, the
online registration at vsh.com, or by contacting technical support. The Serial Number and CD
Key provided with your software are required for registration, along with some information
that identifies the computer on which the software is installed.

Windows Version

GemStone for Windows requires Windows XP, Vista or greater.

To install Battery Server for GemStone (Win)

● Log on to your computer with an account that has Administrator privileges. Most operating
systems require administrative rights during installation.
● If you have an installation CD, insert the program CD into the CD-ROM drive. If you
downloaded the software, extract the installation files from the download into a temporary
folder.
● Run the program named Setup or Setup.exe. Follow the directions on the screen to
complete the installation.
● See the section on registering your software for information on how to register your
software.

To run Battery Server for GemStone (Win)

● See the section How to Work with Battery Server for GemStone .

To install a software battery or battery certificate

● See the section How to Work with Software Batteries .

Macintosh Version

GemStone for Mac requires OSX 10.3.9 or greater. You will need to have administrative rights
to install the software.

To install Battery Server for GemStone (Mac)

● Log on to your computer with an account that has Administrator privileges. Most operating
systems require administrative rights during installation.
● If you have an installation CD, insert the program CD into the CD-ROM drive. If you
downloaded the software, extract the installation files from the download into a temporary
folder.
● Run the Battery Server Installer program. Follow the directions on the screen to
complete the installation.

17
 Installation and Setup of Battery Server for GemStone

● See the section on registering your software for information on how to register your
software.

To run Battery Server for GemStone (Mac)

● See the section How to Work with Battery Server for GemStone .

To install a software battery or battery certificate

● See the section How to Work with Software Batteries .

18
Registering your new software

Why You Should Register

With this version of GemStone, registration is more important than ever before. In fact, your
software will not be fully functional until you register. Here's why.
This version of the program uses software security that allows it to be used without a
hardware lock. Software security requires you to register your software in order to receive an
Unlock Code to make the program fully functional. You can run the program up to 10 times
prior to registering the software.
Of course, registration is useful for other reasons, too. We can inform you of bug fixes and
updates, and provide you with better technical support.

How to Register
When you start the program for the first time, it will automatically start the Registration
Wizard. You can access this dialog at any time by clicking the Registration button on the
dialog box that is displayed when the program starts up.

Click the Next button to begin the registration process.

19
 Registering your new software

The wizard will ask how you want to register the program. Select the appropriate option and
click Next.

Then next step in the wizard shows some information about the program. There are 3 fields
that you need to fill in for this step: Serial #, CD Key, and Email. You can also enter or edit
the Computer Name field to identify your computer.
The Serial number and CD Key can be found on the outside of the box the program came in,
or in email if the program was provided to you by download. Enter these exactly as they

20
GemStone User Guide

appear; they are case-sensitive. If you have lost or misplaced the Serial number or CD Key,
contact Verity technical support at [email protected] or (207) 729 6767.
For the Email field, make sure to enter a valid email address. Otherwise, you may not receive
the Unlock Code and the program will not be fully functional.
Click Next to continue the registration process.

If you have more than one user account at vsh.com, you will be presented with another step.
This step only appears if you have more than one account.

Choose the web account that you want to use for registration, and then click Next.

At this point, the program will request an unlock code from the registration server at
vsh.com. If successful, you will see the confirmation step and the Unlock Code for your
computer.

21
 Registering your new software

You can print the registration information by clicking the Print button. Click Finish to close
the wizard and continue working with the program

If there was a problem with the registration, an informative message will explain the problem
and steps to take to correct it.

Once you have completed the registration successfully, the program will be fully functional.

If the registration wizard cannot connect to vsh.com

Sometimes firewalls can prevent the registration wizard from connecting to vsh.com. In this
case, you can try registering with your browser. Choose the second option in the list and click
Next to launch your browser with the correct registration information.

22
GemStone User Guide

Follow instructions on screen to complete the process.

To register your software using email

The registration wizard can help you send email to Verity Software House with your
registration information. Choose the third option in the list and click Next to create an email
message.

23
 Registering your new software

Follow instructions on screen to complete the process.

If you do not have Internet access

If you do not have access to the Internet, you can send the registration information below to
Verity Software House by fax, mail, or telephone. Our technical support staff can register the
software for you with the information you provide, and supply you with the Unlock Code to
enter into the Registration Wizard.
Here is the information we need. You can print this page and fill it in.
These fields are found in the Registration dialog box.
Program Name:
Program Version:
Computer ID:
Serial number:
CD Key:
User Information
Name:
Institution:
Address:
City:
State/Province:
Zip/Postal Code:
Country:
Phone:
Fax:
Email:
Our technicians will use the information below to create a web user account for you on our
web site.
Name to use on VSH web:
Password to use on VSH web:
A question we can ask to verify who you are:
The answer to the question to verify who you are:
If you are updating from a previous version, you will need to supply the serial number of the
previous version.
Serial Number of previous version:

Contact Verity Software House

Verity Software House
PO Box 247, Topsham, ME 04086
USA

Email: [email protected]
Web: www.vsh.com
Phone: (207) 729 6767
Fax: (207) 729 5443

24
Overviews
Overview Section

The following sections provide overviews of some of GemStone's key concepts.

Overview: Workspace Names and User Interface

Overview: What is a Probability State Model?
Overview: Building a Model
Overview: Using a Model

25
 Overviews

Overview: Workspace Names and User Interface

The annotated graphic below shows the GemStone workspace, along with the names for the
various elements that it is comprised of.

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GemStone User Guide

Overview: What is a Probability State Model?

Flow cytometry data analysis focuses on identification of populations in a sample and

providing statistical results about those populations. Typically, regions are drawn on
univariate or bivariate plots, and then used to create a hierarchy of subsets by "gating" the
events. The resulting subsets are analyzed for frequency, intensity, and other common
statistics to characterize the cells that were analyzed.
Cytometrists are trained to identify subsets of interest. We learn the characteristics of the
subsets and which parameters are used to select them out of the many cell types that our
samples contain. The learning curve can be long, and we often find it difficult to explain our
plots and strategies with other scientists unfamiliar with cytometry.
GemStone approaches data analysis quite differently by using a Probability State Model
(PSM) to identify and quantify subsets. So what is a Probability State Model, and why is it a
better approach?
Let's examine the words that make up the name to get an idea of what a PSM is.

Probability: the statistical likelihood of a particular event occurring. A PSM classifies events
using probabilities rather than user-defined gates. The gating approach is subjective, and
relatively small errors made in drawing gates are compounded with each subsequent gate in
the hierarchy. By using probability, a PSM actually reduces the error with each additional
parameter that is added to the model. The likelihood that you are actually identifying the
cells you are interested in increases as you build a PSM.

State: a set of conditions that discretely characterize something. A PSM probabilistically

classifies events into a set of states, based on the characteristics defined for each parameter.
The state for each event can be thought of as an additional parameter, one that identifies the
particular set of characteristics that define that state. GemStone uses the state as the
common, X-axis for its parameter plots. This allows all parameters to be compared on a
common axis, making it possible to correlate and compare all of the parameters in a single
plot.

Model: a mathematical representation of a process. A PSM uses fitting routines to classify

cells into the most probable states, or to adjust the model to match the data. Using this
approach, GemStone can provide objective measurements of how well the model represents
the data. There are no hard-edges in a PSM. The modeling process allows populations to
overlap based on probabilities.

27
 Overviews

A PSM allows us to classify events into populations probabilistically based on a model that we
define. The model is made up of one or more Cell Types. Each Cell Type defines the
characteristics of a different subset that we want to analyze. We use what we know about
the markers in our experiments to create a set of Parameter Profiles for each Cell Type
subset. A Parameter Profile uses a set of control points to define how the subset transitions
over the state index axis.
The state index axis is typically labeled "Progression", but it does not have to be a progress
at all. In fact, simple models can be designed to identify and quantify cell populations where
the parameter profiles are simply set for constant intensities.

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GemStone User Guide

The PSM approach allows cytometrists to evaluate their analysis more objectively and more
scientifically. By using a model, we can measure and report confidence limits. We can
evaluate the goodness-of-fit with reduced chi-square (rcs). And we can co-plot our
parameters in a single graphic that allows us to examine the coordinated transitions of
markers in the cells we analyze.

29
 Overviews

See also:
Overview: Building a Model
Model Building 101

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GemStone User Guide

Overview: Building a Model

The model-building process is much like the scientific process. It requires careful observation
and attention to detail. It requires you to separate what you know from what you do not
know. It requires you to move methodically through a process, not moving on to the next
step until you fully understand the step you are on.

Start Simple
There are just a couple basic rules for creating models. The first is to start with the
parameters that are the simplest and move toward those that are complex. The second rule
is closely related: start with the parameters that are best defined (those that have the most
obvious transitions) and move on to those that have more subtle characteristics.
For example, let's say that you are only interested in analyzing a cell subset that shows high
expression of some marker, and no other cells have high expression for that marker. This is
an easy subset to identify using a constant parameter profile to select the bright cells for the
marker. We refer to this as a selection parameter. In general, model building starts with the
selection parameters.

Dramatic Transitions
Once the selection parameters are defined, you move on to the markers that show the most
obvious, dramatic transitions. For example, if you have one marker that drops 2-decades in
intensity in a simple step-down manner and another marker that changes only 1/2 decade in
the progression, you would almost certainly choose to model the dramatic, 2-decade shift
first. It is easier to position control points on dramatic transitions; control points are used to
provide the initial estimates to the modeling system.

Save As You Go
When designing a model, save the model often. Create a system for naming models so that
you have a history of revisions. For example, append a letter or number to the name each
time you resave the model. When you need to roll-back to an earlier version, your history of
models will be greatly appreciated.

Test As You Go
As you build a model, test each change that you make. If you add a new parameter and set
up a profile for it, try the model out with a few data files to be certain it classifies properly. If
you see something you don’t expect to see, take the time to decide whether this indicates a
problem with the model or something new you didn’t know about your data. Research the
question to see if there is support in the literature for your observations. Most importantly,
don’t move on until you have the best explanation or you have modified the model
appropriately.

See also:
Overview: What is a Probability State Model?
Model Building 101

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 Overviews

Overview: Using a Model

Simple Process
Using a GemStone model can be as simple as opening a GemStone document, selecting the
data files you want to analyze with it, and reading each file into the model for analysis. In
most cases, you will use the Auto Analysis command after opening a data file into your
GemStone model document. This tells GemStone to adjust the model to match the data as
best it can and then analyze all events in the file. If you have a model that is designed for
the markers in your data files, you can use this simple process.
After the analysis is complete, you may want to save the finished analysis in another
GemStone document file so that it can be recalled at a later time. The Save Document
command is used for this purpose. You can also save and print graphics for individual objects
or the whole workspace.

Matching Parameters
If your data files contain markers that have different names in the GemStone document, you
have a little more work to do. In this case, you would need to tell GemStone how to match
the parameters in your files with the parameters in the model. You only need to do this one
time if you save your model to reflect the changes. For details on how to do this, see How to
Remap Parameters in a Model . Once a model is properly set up for the markers in your files,
you can use the simple process of opening the model, opening the files, and analyzing them.

Batch Processing
Analysis of sets of data files can be automated by using batch processing options described
in the Batch Processing tutorial.

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GemStone User Guide

Overview: Software Batteries

The primary licensing model for GemStone is a per-use system that uses "software battery"
technology. A software battery provides a certain amount of "charge" to GemStone. Each
charge can be used to open one data file. The program keeps track of files it has already
opened, and only uses a charge the first time it reads a data file. So you can reopen the
same file as many times as you like without drawing down the software battery.
There are a number of ways that you can "charge" GemStone's software battery on your
computer. This provides flexibility to support single users as well as large institutions with
many users. The flow chart below provides an illustration of the different sources for battery
charges and how you use each one.

In order to charge your software battery, determine which of the three sources you will draw
from. Then determine whether your computer is able to connect directly to either
www.vsh.com or to a Battery Server. Using the flow chart, you can determine the best
method for charging you software battery.
You can purchase additional battery charges at anytime by using the online store at the
Verity Software House web site, www.vsh.com.

See also:
How to Work with Software Batteries
How to Work with Battery Server for GemStone

33
Tutorials
Tutorials

We are constantly working on new tutorials for GemStone. You'll find them on our web site,
www.vsh.com, along with many other resources for learning and using GemStone.

Model Building 101

Model Building 102
Model Building 103
Model Building 104

Creating a Model for a Multi-Tube Panel

Model Building 201
Heat Map and Animated Regions
How to Remap Parameters in a Model
Batch Processing

How to Work with Software Batteries

How to Work with Battery Server for GemStone

35
 Tutorials

Model Building 101

This tutorial introduces some basic principles behind model building. You will learn about how
to build models that represent a single process - a "Cell Type" in GemStone. We will start
with the simplest pieces and add a little complexity with each step. A follow-up tutorial,
Model Building 102, will continue the model development process where this tutorial leaves
off.
Strategy
Here is a cartoon of how we are going to approach model building.

We start by reading some synthesized listmode data. We create a very simple model for it,
and analyze the data with our model. Then, we will add another parameter, enhance the
model, and analyze the data. We will repeat the cycle until we have fully developed a model
for the data.
We start with a synthesized data file because we want to define what "truth" is for our data.
This file was generated by GemStone, and so we expect an almost-perfect analysis result.
"Real" data is easier to understand after we have designed this first model with "toy" data.
We will be building the skills and understanding to analyze an actual normal bone marrow
file. Our model will help us understand the B-cell lineage and prepare us for "real" data files
later on.

Getting Started
To get started, launch GemStone and pass through the start-up dialogs.
Click the Select FCS Files button on the main toolbar and navigate to the Sample Files folder.
Select the file 101_CD19_SSC_TDT_CD20_CD10.fcs.
Click Open. The program will add the file to the File Database panel and read its data.

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GemStone User Guide

Choosing CD19 Parameter

Click the Cell Type shrink tool to expose some of the important editors we will be working
with. We'l learn a lot more about these editors as we proceed through this tutorial.

Click the last button in the Cell Type toolbar to display a list of parameters in this file. Find
CD19 in the list and select it. (The name might be CD19_CD19.)

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 Tutorials

The parameter is added to the Cell Type widget and displayed as a band of dots on a plot.

Model Building Tip: Start with Selection Parameters

Start building a model with the simplest parameters first, and work toward
parameters that have more complicated transitions. Parameters that are constant
throughout the progression and that select the populations of interest should be
defined first. We refer to these as selection parameters.

Modeling CD19
The CD19 plot is now the "active" plot. Notice that its label is highlighted. We also see that
CD19 appears in the Parameter Profile properties panel. In that panel, click the dropdown
control next to Parameter Profile in the property list to display a list of choices. Choose
Constant from the list.

Model Building Tip: Another Way to Select a Profile

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GemStone User Guide

You can also select a parameter profile from the context menu of the parameter
plot. To do so, right-click the mouse on the parameter plot, select the Choose
Parameter Profile option, and choose a profile from the list.

You should now see two Control Points in the CD19 plot. The vertical placement of the
Control Points should be in the center of the band of dots. Click and drag either point to
identify center of the CD19+ events at around 10^3.

Model Building Tip: Control Points

Control Points are the objects that allow you to manipulate a Parameter Profile.
They are also referred to as "Control Definition Points" because they define the
important transitions in the profile.

At this point, we have set up a very simple GemStone model that will select CD19 positive
events. Let’s tell GemStone to analyze the data with this model. Our expectation is that we
will "classify" most of the events as matching our CellType1 model definition.
Click the Classify Data button the main toolbar.

We now see some dots are darker than others, and there are some additional decorations on
the plot. The darker dots are those that have been classified by our model as being CD19
positive. Light gray dots are considered "unclassified" – not belonging to a cell type at this
point. The graphics show the 95% confidence limits, as well as the mean of the data and the
mean of the model.

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 Tutorials

Good so far. We're ready to add SSC to our model.

Modeling the SSC Parameter

Click the parameter selection tool button and choose SSC. A new plot is added to the Cell
Type widget, and SSC appears in the Parameter Profile properties panel.

B-cells have a constant, low side scatter measurement, so we will again select a Constant
profile. Right-click on the dots in the SSC plot and select the Choose Parameter Profile,
then choose Constant from the list. Position the Control Points in the vertical center of the
SSC population at around 10^2. Then click the Classify Data button on the main toolbar.

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GemStone User Guide

Model Building Tip: Start with Simple, Dramatic Transitions

Once the selection parameters are defined in the model, you are ready to move
on to parameters that transition during the progression. Start with parameters
that have the simplest and most obvious changes. These will help create a
backbone for the model. They will provide clues about how the more complex
parameters behave.

Modeling the TdT Parameter

Click the parameter selection tool button and choose TDT. A new plot is added to the Cell
Type widget, and TDT appears in the Parameter Profile properties panel.

TdT expression in B-cells is a little more complicated. It starts with elevated expression and
then transitions to a low intensity as a function of B-cell progression. We see two bands of
dots for TdT, one at about 10^3 and another heavier band at about 10^1. We need to use a
stair-step profile for this.
Choose a Step Down profile from the dropdown control next to Parameter Profile.

41
 Tutorials

With this profile, the first two points move together in the Y direction, and last two points
move together. We need to position the first level on the bright TdT events and the second
level on the dims.
Move one of the first two Control Points so that it is located at approximately 10^3 on the
Y-axis – the center of the band of bright events. Since there are fewer events in the upper
band, we can position this point near the left end of the X axis.
Now move the third Control Point to approximately 10^1 on the Y-axis. It should look
approximately as shown here.

Click the Classify Data button on the toolbar to analyze the data. You can also use Ctrl-A on
the PC keyboard, or Apple-A on the Mac keyboard to initiate the Classify Data command. The
plot should look similar to this:

GemStone has reordered the events along the progression axis (the X axis), because our
model now tells it that the bright TdT events are early in the progression and dim TdT events
are later in the progression. This new ordering of events is applied to all of the plots,
including CD19 and SSC.

Using Estimate X Positions

The initial placement of control points for TdT is probably close, but not perfect. GemStone
can help us optimize the X positions with the Estimate X Positions tool in the toolbar on
the right of the TdT profile plot.

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GemStone User Guide

Locate the Estimate X Positions button and click it. GemStone will move the definition
points on the TDT profile to create a better match for the model. You can click this button
more than once and improve the model each time.

Adding a 1P Plot
We can create some conventional histograms to help us visualize the data. Click the 1P
histogram tool in the Workspace toolbar.

In the empty space to the right of the Parameter Plots, click and draw a box for the
histogram. You can also click and hold down the Shift key to create a fixed size histogram.
When you release the mouse button, the histogram is displayed.
Click the X-Axis label on the 1P histogram and choose TdT.

Getting Statistics
Let's create three "zones" to measure TdT positive, TdT in transition, and TdT negative
populations. To do this, we will use the Control Points on the TdT profile plot.

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 Tutorials

Double-click the second Control Point on the TdT plot. The Edit BeginDown dialog
appears. "BeginDown" is the name of this point, and this dialog is used to edit properties of
it.
Click the <--- Create Zone button, and notice that events to the left of the BeginDown
point are now colored differently. These events fall into the new zone we created for TdT
positives. Click OK to close the dialog.
Double-click the third Control Point. The Edit EndDown dialog appears. Click <--- Create
Zone to create the intermediate TdT zone.
Finally, click Create Zone ---> to create the TdT negative zone to the right of the definition
point. Click OK to close the dialog.

The zone coloring is applied to dots in all of the Parameter Profiles. Because of the way
Probability State Models work, you will notice that the system accounts for the overlap
between adjacent populations. This is especially evident in the 1P histogram of TdT, where
we easily see overlap of the 3 zones.
Now that our statistical zones are created, let's name them. Locate the Zone Statistics
Table on the canvas and drag it to a convenient location.
To rename a zone, click the zone label and edit the text. Label the zones "TdT+", "TdT
trans", and "TdT-", respectively. Notice that the percentages are approximately 10%, 10%,
and 80%.

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GemStone User Guide

Saving our Model

As you build models, it is always a good idea to save them as you add complexity. Keep a
history of the model's revisions by adding a number or letter to the end of the name each
time you save a new version. Let's save our CD19 TdT model now.
Click the Save Document button on the main toolbar. The Save GemStone Document
dialog is displayed. Type "Model101" for the file name and click Save.

Conclusions
In this tutorial, we introduced many of the principles of model building with GemStone. Our
strategy was to create a simple model, and then to analyze synthesized data with the model.
We gradually added complexity and enhanced the model.
This approach is a sort of "self-fulfilling prophecy". We would be very surprised if our model
was not able to analyze the generated data properly. However, there are a number of real
benefits to this technique. It forces us to describe the characteristics that our real data files
will contain. As we add more parameters to our model and synthesize new data, the
distributions in our generated data should also become more "realistic". We can take a very
complex analysis and break it into incremental steps that can be easily tested.
In our follow-up tutorial, we will add complexity to build a model that can be used with a real
B-cell sample.

See also:
Model Building 102
Cell Types
Cell Type widget
Parameter Profile Descriptions

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 Tutorials

Model Building 102

This tutorial continues where Model Building 101 leaves off. We will add detail to that model
to provide a more complete analysis. The Parameter Overlay plot will be used to help
visualize the B-cell progression in 5-parameter space.

Strategy
Remember the cartoon from the first tutorial? We analyzed synthesized data, analyzing the
simplest parameters first. We then add to our model and cycle through the testing process
again, until we have developed it with enough detail to handle data that has been acquired
on a flow cytometer.

Open Model101.gs
To get started, let's open the model we created in Model Building 101.
Click the Open Document button on the main toolbar. In the Open Document dialog,
navigate to and open "Model101.GS". Click the Open button.
The program will open the model and the simulated data that we generated with it. At this
point, the model defines CD19, SSC, and TdT for our B-cell lineage.

Read the File

When GemStone opens a document, it also closes the current FCS file. This is because the
document itself contains some of the data for the file it last analyzed. So, we need to tell
GemStone to read our test file again so that we can add more parameters to the model.
The quickest way to read a file again is to double-click the file in the File Database panel. Do
that now.

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GemStone User Guide

Adding a Parameter Overlay Plot

The Parameter Overlay plot is one of the most useful graphical tools in GemStone. It
presents all of the parameters in our model in one, correlated plot. Each parameter appears
as a ribbon of color on the plot. Let's create one now.
Click the Overlay button on the Workspace toolbar to select the overlay drawing tool.

In an empty portion of the Cell Type canvas, click and drag to define a rectangle for the
overlay plot. When you release the mouse, the plot will be displayed. The progression for
CD19, SSC, and TdT is shown.

We will refer to this plot as we develop the model with additional parameters.

Modeling CD20
Let's add CD20 to the model.

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 Tutorials

Click the parameter selection tool button and choose CD20 from the list. A new plot is added
to the Cell Type widget, and CD20 appears in the Parameter Profile properties panel.

CD20 is not expressed until part way through the progression, and we can see that without
even modeling the parameter.

We will use a step-up profile for CD20. Right-click the CD20 plot and select Choose
Parameter Profile in the context menu. From the list, choose Step Up.
Position the Control Points approximately as shown in the illustration below:

Now we can tell GemStone to Estimate X Positions and it will automatically adjust the
positions and analyze the data. Click the Estimate X Positions button.

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GemStone User Guide

Now if we look closely at the result, we can see that the CD20 fit needs improvement.

Notice that the little "whiskers" on the Control Points do not match the 95% confidence lines
on the data. This is because the data has a different "line spread" than our parameter profile.
Think of line spread as a measurement of broadness, or standard deviation. The parameter
profile uses the whiskers on the Control Points as an estimate of line spread. In the low-
intensity region, our model's line spread is not as wide as it should be for this data. If we
look at the bright CD20 events, we see that the Control Point whiskers are actually a little
wider than the 95% confidence lines. The model's line spread is wider than it should be. So
we need to learn how to adjust the line spread for Control Points. We start by selecting one
of the Control Points that we want to adjust.
Click the second Control Point to select it.
Next, in the Parameter Profile panel change the Variance Source to Manual Point Entry.
Now, move the Line-spread Value slider to adjust the line spread to about 7.

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 Tutorials

Notice now that the whiskers for the first two Control Points are wider than they were, and
that they more closely match the 95% confidence limits of the data. In most cases, it is good
to set the line spread to be a little wider than the observed data; the automatic adjustment
routines in GemStone give better results with slightly wider settings.
Click the third Control Point to select it.
Move the Line-spread Value slider to adjust the line spread to about 3.5. You may also
need to adjust the vertical position of the definition point.

Model Building Tip: Use the Scroll Wheel for Line Spread Adjustments
If your mouse has a scroll wheel, there’s a quick and easy way to adjust the line
spread for a Control Point. Position the mouse over the center of the Control
Point you want to adjust, and then roll the scroll wheel. Scrolling up increases
the line spread; scrolling down decrease the line spread.

Click the Estimate X Positions button to allow GemStone to adjust the model and
reanalyze the data.

Our CD20 profile now matches the data more accurately. The low-intensity events are
modeled with a broader line-spread, and the high-intensity events have a narrower spread.

Modeling CD10
Click the parameter selection tool button and choose CD10 from the list.

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GemStone User Guide

Now we see one of the most interesting aspects of a GemStone model. We have not yet
attempted to model CD10, and yet the structure of the CD10 progression is plainly visible.
Three distinct levels of expression are shown, and the levels match our expectations for
CD10.

For CD10, we know that it is highly-expressed early, followed by a moderate expression and
finally a low expression. How did GemStone figure this out? In each of the previous
parameters, we had to select a parameter profile and position Control Points before the
events were properly distributed along the progression axis.
What has happened is that our model now has enough structure to reveal structure in
parameters that we have not modeled. Our TdT profile defined a transition early in the
progression. CD20 defined a transition later in progression. These pieces of the model create
enough of a scaffold to order the progression of events. We can use this to explore markers
that are completely unknown.
Let's go ahead and model CD10 so that our model has an even stronger scaffold.
Right-click the CD10 plot and select Choose Parameter Profile in the context menu. From
the list, choose Three Levels. Position the Control Points so that they match the transitions
in the data as shown in the illustration below:

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 Tutorials

Now click the Estimate X Positions tool to allow GemStone to fine-tune the Control Point
locations and analyze the data.

Our fit now looks like this:

Just as we saw with CD20, the line spread for the CD10 profile could use a little fine-tuning
too. The whiskers on the control points are a little narrow. If you would like to adjust these,
go ahead and do so using the same process we used for CD20.

If we look at our Overlay Plot, we now see the coordinated expression of CD19, TdT, CD20,
and CD10. The light scatter parameter is also displayed.

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Redefine Zones
The zones we initially created are not showing us all that we know at this point. Let's
redefine the zones to cover the progression in more detail.
In the Cell Type Properties panel, fine the Edit Zones entry and click the Edit button. A
dialog appears showing the zones we created.
Select the last zone in the list, TdT-, and then click the Delete button. Click OK to close the
dialog.
In the CD10 plot, double-click the 4th control point.

Click the <--Create Zone button and then click OK.

Double-click the 5th control point.
Click the <--Create Zone button. Next, click the Create Zone--> button. Finally, click OK.
We now see dot coloring that reflects 5 zones. Let's edit the names of these zones.
To rename a zone, click the zone label in the Zone Statistics table and edit its label. Label
the zones Total, I, II, III, IV, and V respectively

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 Tutorials

Create a 2P Plot
We can create a standard dot plot to see whether our model is producing data that looks like
what we expect.
From the Workspace toolbar, select the 2P histogram tool and draw a rectangle for the
histogram in an empty space on the Cell Type canvas.

Select CD20 for the X-axis and CD10 for the Y-axis by clicking on the axis label and
choosing the parameter from the list.

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GemStone User Guide

Our model has created events that form a familiar pattern of CD10 vs. CD20 expression for
B-cells. Because we have defined these in terms of a progression, GemStone can overlay
arrows that show us how B-cells develop, even in the dot plot.
Let' save our new, improved model.
From the main toolbar, click the Save Document button. The Save GemStone Document
dialog is displayed. Type "Model102" for the file name and click Save.

We are ready to try this model out on a real B-cell sample, which is the subject for Model
Building 103.

See also:
Model Building 103
Cell Types
Cell Type widget
Parameter Profile Descriptions

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 Tutorials

Model Building 103

In this tutorial, we will put our B-cell model to the test and use it to analyze a real, B-cell
sample file. We will start with the model we created in Model Building 101 and 102, and we
will work through the process of adjusting the model to a real sample. At that point, we
should be able to select the additional parameters that are in our sample file and see how
they behave in the B-cell progression – without having to model them ourselves.
At this stage, we are jumping out of our model development cycle and into the real world of
analyzing real samples.

Open the Model

On the main toolbar, click the Open Document button. Select "Model102.gs" and click the
Open button to load our working model.

Disable the Profiles

Normally, when GemStone opens an FCS data file, it automatically analyzes the file with the
model it currently has open. In making the transition from synthesized data to real data, we
do not want this to happen because we want to see the unanalyzed data from our real
sample. So we are going to want to turn off the pieces of the model. We can do this with one
action.
Click the Utility Action button on the Workspace toolbar, and select Disable Profiles
from the menu.

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All of our plots will redraw without color, showing all events in an unanalyzed state.

Model Building Tip: Use Disable Profiles

Disable Profiles is used to turn off the profiles for all parameters in the cell type.
This can be useful if you want to restart the model building process but don't
want to reconstruct the model from scratch.

Open the Data File

On the main toolbar, click the Select FCS Files button. Navigate to GemStone's Sample
Files folder, select BCellLineage.fcs, and click Open. The file is added to the File Database
and read into our model.
Now we will enable the pieces of our model, one at a time, starting with the CD19 and SSC
selection parameters. Selection parameters, as we learned in Model Building 101, are
those that have constant parameter profiles and are used to select populations of interest.
In this case, CD19 and SSC will be used to select B-cells from our sample file.

Adjust and Enable CD19

Click the shrink tool for the CD19 Parameter Profile plot to expand the plot.

The B-cells are those with high expression of CD19. We need to move the CD19 Control
Points down to identify the correct cells. Click and drag one of the control points to identify
the band of CD19 positive cells.
We also need to adjust the line spread to encompass the bright CD19 events. Use the Line-
spread slider or mouse scroll wheel to adjust the slider to select a broad band of bright
CD19 events.

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You might worry that we are being too generous in selecting with CD19 here. That is
intentional at this point: we don't want to exclude dim CD19 events yet. As we adjust the
model, those dim events will start to drop out.
Click the Enable tool on the CD19 parameter profile to turn it on , and then click the
Classify Data button on the main toolbar .

Turn Off Means and Confidence Limits for SSC

We're ready to move on to SSC. Click the shrink tool for the SSC Parameter Profile plot to
expand the plot.
GemStone is trying to be helpful by showing 95% confidence limits and the mean of the
analyzed events. In this case, however, the extra graphics are making it difficult to see what
we need to see. Let's turn off the extra graphics.
Right-click in the SSC plot area and choose Edit Graphics from the context menu.

In the Edit Graphic Options dialog, select the Means tab. Uncheck the Enable Means
option.
Select the Conf Limits tab and change Line Spread Mode to Model SD Bars.
Select the Model tab and uncheck Enable Model.
Click OK. The SSC plot should now display only the dots and the control points.

Adjust and Enable SSC

Slide the Control Points for SSC up, centering the points on the low-intensity band of
events. Adjust the line-spread for the profile. The result should look something like this:

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Click the Enable tool on the SSC parameter profile to turn it on , and then click the
Classify Data button on the main toolbar .

Enrich
B-cells are a relatively small percentage of the cells in this sample, less than 3%. That can
make things difficult to visualize. So we'll use a feature called Enrich Data to search out all
of the B-cells (those that have high CD19 and low SSC) and select them exclusively. We do
this after adjusting our selection parameters and before we move on to the parameters
that transition during the progression.
On the main toolbar, click the Enrich Data button. Now we've got plenty of B-cells to
analyze.

Model Building Tip: Use Enrich Data to See More of the Cells of Interest

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Using Enrich Data allows you to include for consideration only those events that
match the current model. This allows concentration of events that represent a
small fraction of the entire sample.

Adjust and Enable TdT

Click the shrink tool for the TdT Parameter Profile plot to expand the plot.
Here we can see two bands of events: a sparse band of high-intensity and a dense band of
low-intensity TdT. Let’s move our Control Points down to the approximate centers of the
bands.
Adjust the control points down as shown:

Model Building Tip: Place Definition Points at the Right Intensity

When determining the initial position for the definition points, it is most
important to get close to the right intensity level. The definition point should be
placed near the center of the band of data it represents. The line spread should
be set to approximate the variance of the data. The state index position of the
definition points can be estimated based on the density of the dots at the given
intensity.

Use the Estimate X Positions adjuster tool for TdT to fine-tune the control point positions.

The profile now shows the step-down progression we designed into our model.

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At this point, we have selected B-cells and created the first structure in the progression
backbone using the TdT profile. GemStone has re-ordered the events so that the high-
intensity TdT events are at the start of the progression, the low-intensity TdT events are at
the end of the progression, and intermediate TdT events are in the transition between high
and low. This order is reflected in all of the parameter profile plots, and we start to see the
hidden structure appearing in the other parameters.

Adjust and Enable CD10

The CD10 plot shows 3 levels of intensity, just what our model is designed for. The first level
of high-intensity events is clustered at the beginning of the progression. Then we see a
slightly lower intensity band of dense dots, and a diffuse band of low intensity events.

Once again, we'll adjust the positions of the Control Points to get a good starting estimate.
Move the controls approximately as shown, paying attention primarily to the Y-axis
positioning to place the points in the middle of the bands.

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Now here is a new trick. Notice that the width of the CD10 negative band of dots is
significantly wider than the intermediate and high CD10 bands. We can help GemStone out
here by adjusting the line spread for that low-intensity band to be wider than the other
bands. Here is how we do that.
Click the 5th Control Definition point in the plot. This will select the point.

Now position the mouse over the 5th point and use the mouse scroll wheel to adjust the line-
spread wider. If you do not have a scroll wheel on your mouse, use the Line-spread slider in
the Parameter Profile panel.

This adjustment will make it easier for the auto-adjusting functions. Use the Estimate X
Positions adjuster tool for TdT to fine-tune the control point positions.

The finished result has matched our data well.

Using the Frequency Plot

How do we know that the model is really matching the data well? We use the Frequency Plot
to help make that assessment. This is the bottom element in the Cell Type widget. There are
3 things we examine at on this plot to determine how well the model is working.
At the top, RCS shows us the reduced chi square value, which is a statistical measure of how
well the model matches the data. A value approaching 1.0 is just about perfect. Typical
values can be between 3 and 7 with more complex models.

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Across the plot is a jagged black like showing us the frequency distribution for each state
in our model. There is also a horizontal red line showing the ideal distribution of events.
The ideal distribution simply takes the number of events we are working with and divides
that by the number of states in the progression. A Probability State Model attempts to
distribute events in this manner, so this is ideally what our model would do. The frequency
distribution line shows how our model is actually distributing events, and it should bounce
above and below the ideal line as we see in our model so far. The smoothed frequency
shows a weighted average frequency distribution. See the discussion on what is a Probability
State Model for more on this.
So, our model is looking pretty good at this point.

Adjust and Enable CD20

The CD20 plot shows two levels of intensity.
Move the Control Points to the vertical center of the dense bands of dots. Adjust the line-
spread for the control points. Then click the adjuster button.

This gives us a pretty good fit with the data. We can see an up regulation that then drops off
again, which is what our Three Levels profile is designed for. However, this Step Up profile is
also providing a very good fit, so we’ll keep it. We can always come back and change it to a
Three Level profile later.

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Add the Remaining Parameters

This data file has many more parameters than we are currently modeling. What do you
suppose will happen if we display the remaining parameters without defining any
expectations in our model? Will there be any structure in them, or will they be evenly
distributed bands of dots on the profile plots? Let's find out.
In the Cell Type Properties panel, click the Edit button for Select Parameters. Select all
of the "Live" parameters in the list on the left side of the dialog (even the ones we have
already added), and then click the Add button.

Click OK to close the dialog.

Now with 11 parameters loaded, the best way to see the other parameters is to look at the
Parameter Overlay plot.

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Even without creating a model for CD45, CD43, CD34, CD23, and others, we can see how
these parameters behave with B-cells in our progression. Our model defined the progression
well enough that the other parameters reveal information without any effort on our part. For
example, we can see that CD45 is lowest at the start of the progression, it increases in zones
I and II, and then levels off in zone III. It elevates again at the end of III and levels off in IV
and V.

Saving our Model

We have made several enhancements to our B-cell model. This model is now capable of
analyzing real data, so we should save a new copy of it.
From the main toolbar, click Save Document. Type "Model103.gs" for the file name and
click Save.

Other Enhancements
If you are familiar with B-cell maturation, you know that at some point along the way a B-cell
will either express Kappa or Lambda, not both. When you have a branch like this, you
generally need more than one Cell Type to properly characterize the process. This will be
the focus of Model Building 104, the last in our series.

See also:
Model Building 104

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Cell Types
Cell Type widget
Parameter Profile Descriptions

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Model Building 104

This tutorial introduces the use of more than one Cell Type in a model. It continues the
model-building process from Model Building 101, 102, and 103.
When we finished Model Building 103, we had developed a fairly detailed model of B-cell
maturation. We used a 12-parameter FCS file to test the model, and we were able to observe
correlated transitions in marker expression even for parameters that we did not model.
Our model does not yet account for a branch that occurs in B-cell maturation. As the cells
mature, they will either express Kappa or Lambda, but not both. We want to capture this
with our model to quantify the relative fractions of Kappa and Lambda cells, and to see if
other markers have different characteristics for the two subsets.
To do this, we will create a second Cell Type. In general, whenever you need to analyze
more than one population or subset, you will need to create a separate Cell Type for each.
We will handle the mechanics of doing that here.

Open the Model

We will start by opening the model that we have developed.
Click Open Document on the main toolbar. Select "Model103.gs" and click Open.
The model will be read and displayed in its last state.

Cell Type 1: The Kappa Cell Type

We are going to make the first Cell Type for Kappa B-cells. We will select Kappa cells using a
Constant profile on the Lambda parameter to identify Lambda-negative cells. The advantage
to this approach is that our model will be able to show clonal excess for Kappa cells. Saying
that another way, the model will not filter out events that are very positive for Kappa.

Click the Cell Type shrink tool to expose the editors we will be working with.

In the Cell Type Properties panel, change the Cell Type Name to "Kappa", and edit the
Abbreviation to "K".

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Locate the Lambda parameter in the Cell Type widget. It should be the last parameter. Click
the Lambda label to activate the parameter.
In the Parameter Profile property panel, choose Constant for the Parameter Profile. Also,
enable the Lock Fit checkbox. This tells GemStone not to move this profile, which is very
important when identifying "negatives" in this manner.

Position the Control Points for the profile to identify the low-intensity Lambda events as
shown.

We will not analyze the data yet. First, let's create the Lambda Cell Type.

Duplicate Cell Type

The only difference between the Kappa and Lambda Cell Types will be in the use of the
Kappa and Lambda parameters. All of the other parts of the model for these two Cell Types
will be the same. That means that we can duplicate the work we have done already and the
make a few changes in the second Cell Type to model the Lambda cells.
Click the Utility Action button on the Workspace toolbar, and select Duplicate Cell Type
from the menu.
GemStone will make a copy of all of the elements in our Kappa Cell Type, and name the copy
"CellType2". It also switches to the tab for CellType2.

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Model Building Tip: Duplicate Cell Type

Use the Duplicate Cell Type command to make a copy of an existing Cell Type.
The command will copy the model definition, as well as the layout and graphics
for the Cell Type.

Cell Type 2: The Lambda Cell Type

Click the Cell Type shrink tool to expose the editors for CellType2.

In the Cell Type Properties panel, change the Cell Type Name to "Lambda", and edit the
Abbreviation to "L".

Now we need to make two more changes. We have to turn off the parameter profile for
Lambda in this Cell Type. Then, we need to model the Kappa parameter to select Kappa
negative events.
Locate the Lambda parameter in the Lambda Cell Type widget. Click the Lambda label to
activate the parameter.
In the Parameter Profile property panel, choose None for the Parameter Profile.

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Next, locate the Kappa parameter in the Lambda Cell Type widget and click the Kappa label
to activate the parameter.
In the Parameter Profile property panel, choose Constant for the Parameter Profile.
Enable the Lock Fit checkbox as we did for the Lambda profile in the Kappa Cell Type.
Position the Control Points for the profile to identify the low-intensity Kappa events as
shown.

Saving the B-Cell Kappa Lambda Model

This is a very useful B-cell model at this point. We should save a new copy of it.
From the main toolbar, click Save Document. Type "Model104.gs" for the file name and
click Save.

Auto Analyze the Data

We are ready to analyze our full Kappa-Lambda B-cell model. Let's use Auto Analysis to do
this. Auto Analysis does many of the steps we have been doing manually: it adjusts the
constant selection parameters, it enriches, and it then adjusts the parameter profiles for each
of the remaining parameters (except those with the Lock Fit option enabled).
Click the Auto Analysis button on the main toolbar.
Like the Classify Data command, Auto Analysis acts on all of the Cell Types that are
defined in the model document. An event can be classified as belonging to one of the cell
types or it can be considered unclassified. An event can never be classified into more than
one of the cell types at the same time. In the model we have created, the Kappa and
Lambda Cell Types are assigned events probabilistically.

Model Building Tip: Stopping Auto Analysis

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There may be a point at which the RCS and frequency distribution are "good
enough" and the optimization is no longer making real improvement to the fit of
the model. You can stop the current estimation or the auto analysis process by
clicking the appropriate check box in the status bar.

Let's take a look at the Parameter Overlay plot for each of our two Cell Types. In the Lambda
Cell Type overlay, we can see the elevated expression of the Lambda marker starting late in
zone III. The Kappa expression for Lambda cells remains low.

Conversely, in the Kappa Cell Type, the Kappa expression increases in zone IV, and Lambda
expression remains low throughout.

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Summary
In this series of tutorials, we have developed a GemStone model from scratch to a point
where it is a very detailed analysis of B-cells with Kappa and Lambda differentiation.
We outlined a model-building strategy where we design part of a model, synthesize data with
GemStone, increase the complexity of the model, and continue this cycle to a point where we
are ready to test the model on real samples. This approach allows us to test our assumptions
about what we "know" about the cells we are going to analyze. The use of synthesized data
makes it much easier to increase complexity gradually in model development, and as we saw,
the synthesized events take on very "real" characteristics as we build the model.
The model-building tips we learned along the way are worth reviewing here. Models should
start out by defining the Constant parameters, which we referred to as selection
parameters. These are the simplest parameters for us to set up and for GemStone to
analyze. Then we set up simple parameters that have the most dynamic transitions over the
progression. The dramatic transitions are easier for us to visualize, and we can make
confident decisions about where the control point should be positioned. Finally, we modeled
some of the more complicated parameters. GemStone helped us with these because of the
simple parameters we modeled first.
We created zones for analysis and used the Parameter Overlay plot to view the correlated
expression of all of the parameters in our files. And finally, we learned how to handle
subpopulations (Kappa and Lambda) with multiple Cell Types.

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Use these principals to develop models for the cells you want to analyze, and you should
produce robust models that can be used with GemStone for a great variety of applications.

See also:
Tutorials
Cell Types
Cell Type widget
Parameter Profile Descriptions

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Creating a Model for a Multi-Tube Panel

In the previous Model Building tutorials, we learned how to build a model for a single-tube B-
cell assay. Those tutorials are pre-requisites for this one.
Much of the time we use panels with several tubes - and therefore several listmode files.
That presents a different set of problems for analysis. This tutorial will focus on the
differences in how you approach model building for panels with more than one tube.

Concept and Strategy

The model that we build needs to do everything that the single-tube model does. It needs to
help us understand the coordinated expression and transitions of the markers in the panel.
However, GemStone is going to read the files in our panel one at a time. So, how can we
build a model that correlates all markers if they are spread across several tubes?
GemStone helps us with this task by retaining the results for the markers it has analyzed.
Here is a simple example. Let's say that tube 1 has CD10 and some other markers, and tube
2 has CD34 among its markers. GemStone will store the means and standard deviations for
every state of the CD10 in tube 1 and allow us to correlate it with CD34 in tube 2. After
GemStone has analyzed all of the tubes in the panel, the results for the entire panel are
available to us. We can display Parameter Overlay plots and view statistics for any of the
markers in the panel. We can even look at dot plots for markers that are actually in separate
tubes.
Our model-building strategy is similar to the one we used to build our single-tube model. The
difference is that we are more dependent on a good set of common parameters. The term
"common parameters" refers to the set of markers that are common to all tubes in the panel.
Using these parameters, we will build the skeleton of a model that will span the several tubes
in our panel. The common parameters provide the backbone that allows us to make
correlations among all of the markers.

Model Building Tip: Common Parameters

Common parameters are the parameters that are included in all tubes of a panel.
Good common parameters make it possible to create a framework for comparing
markers that are not in the same tube.

Choosing Good Common Parameters

A good set of common parameters is the key to success. For most cytometers, the light
scatter parameters are available in every file. Unfortunately, they do not help us correlate
markers in most cases. Ideally, the markers we choose to be common will have simple, well-
defined transitions in the progression.
For our tutorial, we will set up a model for a 3-tube bone marrow panel, each tube having 7
colors, for a total of 11 fluorescent markers. The tubes have 5 common parameters: CD19 is
required in all tubes so that we can identify the B-cells; CD34 will help us identify early B-
cells and is a simple, step-down parameter; CD20 is up-regulated later in B-cell maturation;

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CD10 starts high and is lost in two later stages; and CD45 will also help identify transitions in
the B-cell progression.
Along with these common parameters, tube 1 has CD58 and CD38. Tube 2 contains CD33
and CD13. And finally, tube 3 includes CD9 and CD22, rounding out this panel.
Let's get started.

Tube 1
Select the Data Files
Click the Select FCS Files button on the main toolbar. Navigate to the GemStone Sample
Files folder, and select 3 files in the list: BCell_BM_Tube_001.fcs,
BCell_BM_Tube_002.fcs, and BCell_BM_Tube_003.fcs. Click Open. The files are
added to the File Database and the first file is read into GemStone.

Model Building Tip: Renaming Parameters

The parameter names for the files in this tutorial may not be especially helpful
the first time you read them into GemStone. This can be easily corrected by
selecting the Parameter Database command in the Edit menu. In this dialog, you
can rename parameters (and do lots of other things, too). For additional details,
see Edit Parameter Database.

Setting up Selection Parameters

We will build the model by first setting up the selection parameters: CD19 and SSC. Click the
Choose Parameter button in the Workspace toolbar, and select CD19 in the list.

Let's use the context menu in the CD19 parameter plot to select a Constant profile. Right-
click inside the CD19 plot and from the menu, select Choose Parameter Profile. In the
popup menu, select Constant.

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With the markers in this panel, we will not be using the three auto-adjustment buttons as we
have in other tutorials. Those routines are helpful in many cases, but they are not smart
enough to handle the data in these files. We will size and position the definition points
manually, and then use the Estimate X-Position button to fine-tune the placement.
Position the profile on the band of high-intensity CD19 cells. Use the Line Spread slider in
the Parameter Profile panel to increase the line spread to about 7.0, and then click the
Classify Data button on the main toolbar to analyze the events.

Next, we will repeat this process for SSC and identify cells with low SSC values.

Click the Choose Parameter button in the Workspace toolbar, and select SSC in the list.
Right-click inside the SSC plot and from the menu, select Choose Parameter Profile. In
the popup menu, select Constant. Position the profile on the band of low-intensity SSC
cells. Click the Classify Data button to analyze the events.

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Verifying with a 2P Plot

Now before we go on, let's see if we have really captured the cells of interest by using a
conventional 2P dot plot display of SSC vs CD19. Click the Create 2P button in the
Workspace toolbar.

Find an empty space on the Cell Type Canvas, click and drag to define a 2P plot. Select SSC
on the X-axis and CD19 on the Y-axis.

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If we look carefully at the plot, we can see that we have cut off some of the B-cells with low
CD19 and some with higher SSC. At this stage in the model-building process, it is very
important not to eliminate cells that may be of interest. Since we are about to enrich for the
cells we have selected, it is better to be a little generous on the edges at this step. So, we
need to increase the line-spread settings for both CD19 and SSC before going on.
Click the CD19 parameter profile plot to activate it, and then increase the line-spread setting
to approximately 8.40. Increase the line-spread for SSC slightly. Click the Classify Data
button (or use the short-cut, Ctrl-A on the PC or Command-A on the Mac). You should now
see more dark gray events in the 2P plot.
Let's enrich for B-cells. Click the Enrich button on the main toolbar.

Defining the Progression

Now we are ready to really define the progression. We will analyze the common parameters
from the simplest to the hardest: CD34, CD20, CD10, and CD45.
Click the Choose Parameter button in the Workspace toolbar, and select CD34 in the list.
The CD34-negative population is very broad and dim, and the positives at approximately 150
on the Y-axis. We will use a Step-Down profile for it. Right-click inside the CD34 plot and
from the menu, select Choose Parameter Profile. Choose the Step Down profile in the
list.
Position the upper step at ~ 150 and the lower step at ~0 on the Y-axis. Next, change the
Variance Source in the Parameter Profile panel to Manual Point Entry. This allows us to
adjust the line-spread for the upper and lower steps separately.
Click the first point in the lower step and adjust the Line-spread Value to 10 or 11. Then,
click Estimate X-Positions to activate the profile and allow GemStone to determine the
best location horizontally.

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Model Building Tip: Tweaking the Profile

When you are manually adjusting definition points and not using the 3 adjuster
buttons, you can tweak the positions of the points and re-analyze to find optimal
positions and line-spread settings. Try moving points up, down, and sideways
just a little and reanalyzing. Then, use the Estimate X-Positions button to
fine-tune it.

Let's move on to CD20. Click the Choose Parameter button in the Workspace toolbar, and
select CD20 in the list. We want to consider everything less than ~150 as negative, and the
events centered at ~800 as positive. Right-click inside the CD20 plot and from the menu,
select Choose Parameter Profile. Choose the Step Up profile in the list.
Position and size the definition points approximately as shown in the picture below.

Trigger the Classify Data command from the menu, from the toolbar, or using the shortcut
keys. Then, click the Estimate X-Positions button to allow GemStone to tweak the
horizontal placement.

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Click the Choose Parameter button in the Workspace toolbar, and select CD10 in the list.
With CD34 and CD20 defined, the expression characteristics of CD10 become more obvious.
We can easily see 3 levels of expression that we will model.

Right-click inside the CD10 plot and from the menu, select Choose Parameter Profile.
Choose the Three Levels profile in the list. Position the levels to match the transitions that
appear in the distribution.

Model Building Tip: 3-Levels or 4-Levels?

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We know that when B-cells become mature, they lose CD10. In this example,
there are actually a few cells that have become fully mature and show virtually
no CD10 expression. We could model this with a Four Levels profile, and set the
last level very low at the end of the progression to capture those mature B-cells.
For this tutorial, we’ll keep it simple with Three Levels.

Click the Estimate X-Positions button to allow GemStone to tweak the CD10 definition
points.

Now let's add CD45 to the model. This marker starts low on early B-cells and elevates twice
in the progression. Click the Choose Parameter button in the Workspace toolbar, and
select CD45 in the list. Right-click inside the CD45 plot and from the menu, select Choose
Parameter Profile. Choose the Three Levels profile in the list. For the initial placement of
the definition points, use the graphic below as a guide.

Model Building Tip: Positioning Definition Points

The horizontal spacing of Definition Points is based on the number of events that
the points relate to. If there are many events that relate to a pair of points, there
is more space between them on the X axis. If there are few events between the
pair of points, the points are close together on the X axis.

Now use the Estimate X-Positions tool to fine-tune the positions and classify the data.

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The adjusted profile will look similar to the image shown below.

At this point, our model is defined. We have modeled 6 parameters common to all tubes.
Two parameters (CD19 and SSC) were used to select the B-cells. The other four parameters
(CD34, CD20, CD10, and CD45) were used to define a scaffold for the progression.
We can now use Auto Analysis to optimize the fit. Click the Auto Analysis button.

Adding the Remaining Parameters from Tube 1

We can now add the remaining parameters in tube 1. However, we will not need to do any
modeling for these markers because the model is already well-defined. This is one of the
benefits to using GemStone for the analysis: with a well-defined model, you can explore how
other markers - even those with unknown expression characteristics.
Add CD58 and CD38 to the model.

Creating a Parameter Overlay Plot

Let's add a Parameter Overlay plot to the model, and then save the document.
Click the Parameter Overlay tool on the Workspace toolbar, hold the Shift key down and
click the mouse on the canvas. The plot will be displayed.
Because the CD34 negatives are so dim, much of the CD34 ribbon in the plot is off scale. To
adjust for this, we need to change the Y-axis scaling for the plot.
Double-click the axis tics for the Y-axis to display the Edit Properties for Y-Axis Area
dialog. Change the Axis Scaling Mode to Fixed Scale, and set the Axis Min Scale to -10.
Click OK.

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On the main toolbar, click the Save Document button, and save the model document as
"BCell-Multi-Tube.gs".

Analyzing Tube 2
We are ready to open the next file in the panel. Click the Next File button on the batch
toolbar to read it into the model.

The first things you will notice when the second file is loaded are that some of the plots now
show no dots and that the Parameter Overlay plot did not appear to change. Here is why.
The parameters that are common between tube 1 and 2 have been reanalyzed. The
parameters that were in tube 1 and not in tube 2 still show the means and confidence limits
for the analysis - but no dots. Those parameters can still be plotted on the Parameter Overlay
plot based on the analysis from tube 1.

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Let's add the parameters from tube 2 all at once. Click the Choose Parameter button in the
Workspace toolbar, and select Choose in the list. The Edit Parameters dialog is displayed.
Add CD13 and CD33 by double-clicking each one in the list on the left. Click OK to close the
dialog.

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We do not need to add Parameter Profiles for these markers because the model is already
well defined. We can see the expected transitions for the markers, which come from the
parts of the model that we did define. If the markers had characteristics that were not shown
at this point, we probably would try to model them here.

Model Building Tip: Watch the RCS when opening a new data file.
The RCS (Reduced Chi-Square) is a measurement of how well the model
matches the data. With a multi-tube panel, it can also be used as a quality
control tool. If the RCS changes much when a new tube is read into the model,
there is a good chance that one of the common parameters has changed from
the previous tube. If this occurs, you should stop and determine what has
changed before moving on. GemStone has tools that can normalize parameter
intensities from one file to the next.

Let's move on to tube 3.

Adding Tube 3
Click the Next File button on the batch toolbar to read it into the model. Using any
technique you prefer, add CD22, CD9, and FSC-A to the model.
Here again, if we needed to define any of these additional parameters, we could. It is not
necessary in this case.

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Our parameter overlay now shows us the correlated expression for all of the markers in the
panel.
We would typically want to create some analysis zones for the model. That is left to the
reader as an additional exercise, since it has been covered in earlier tutorials.
Let's save our model, now that it represents the entire panel of tubes. Click the Save
Document button on the main toolbar. Type a name for the model and click Save.

Summary
One of the most important aspects about multi-tube panels is the selection of common
parameters that will be included in all tubes. These parameters are used by GemStone to
create a backbone for the analysis, allowing correlations of markers in separate tubes to be
explored as if they were in the same tube.
When creating a model for a panel of files, the common parameters are heavily relied upon.
Markers that are found in only one tube are modeled when they provide key information to
the progression. Once the model is fully defined with common parameters, GemStone can
display and correlate parameters from different tubes.

See also:

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Tutorials and How To Section

Parameter Profile Descriptions

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Model Building 201

This tutorial introduces several advanced topics in model building. It assumes that you have
worked through the Model Building 101, 102, 103, and 104 tutorials.

Getting Started
In this section, you will learn some of the subtleties of parameter profiles. The most
important controls are the relations that can exist between control points. These relations
create dependencies to our models so that they can operate reliably with Auto Analysis.
To get started, just launch GemStone. We do not need to load a GemStone document or data
file for this tutorial.
Click the Choose Parameter button in the Workspace toolbar, and then click Choose to
display the Edit Parameters dialog.

Find and select TDT and CD10 in the list. If these parameters are not listed, just choose two
log parameters. Click Add and then OK.

Control Points
Right click the TDT plot, and select Choose Parameter Profile from the context menu.
Choose the Step Down profile for TDT.

Let's look carefully at the first Control Point on the left. There are arrow tips pointing up and
down. This indicates that this control point can fit data up or down. Now, look at the second
Control Point. It has left and right arrow tips that indicate that it can fit data to the right or
left. The third Control Point can fit data in any direction. Finally, notice that the last Control

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Point has no arrow tips, indicating that it does not fit data in any direction. Move each of
these points to get a feeling for how they interact with each other.

Model Building Tip: Arrows Indicate the Direction of Movement

The arrow tips on a Control Point indicate the directions that the point can be
moved by the program when it is fitting the model to the data. In many cases,
you can manually move a point in other directions because it has a relation with
another point.

These attributes of the Control Point are editable, and for some modeling situations you have
to make changes to achieve the behavior your model requires. Let's take a look at some of
these important properties.
Double-click the first Control Point to display the editor.

We can edit characteristics related to the parameter intensity, state position, and standard
deviation. Param controls properties related to the way the Control Point adjust on the
parameter intensity axis. State controls the position on the progression, or state axis. SD
relates to the line spread on the parameter intensity axis. Let's drill deeper.
With Param selected in the list, click the Properties button in the Param group box at the
bottom of the dialog. This will display the Param property editor.

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The Value property shows the current position of the point on the parameter intensity axis.
GemStone's dynamic range for parameter values is a normalized scale of 0.0 to 100.0. We
can directly edit the value here, but in most cases its easier to drag the point with the mouse
or use one of the left or right arrows on the keyboard.
The next property is the Fit Status property. If it is checked, the point's parameter value
can be adjusted by GemStone's fitting routines. This property determines whether or not up
and down arrow tips are displayed on the graphical control point.
If the Can Change property is unchecked, you will not be able to move this CDP at all. Let's
see how this affects this and the other points in the parameter profile.
Uncheck the Can Change property and click on OK twice.
Now, try to move the first Control Point. You can't.
Try to move the second point up or down. You can't move it either.
Now try moving the third Control Point up to the top of the plot. When it is almost as high as
the second point, you can't move it any farther.

Why? The reason is that there is a relation between the third and second point. A relation
defines a rule between two Control Points that restricts the movement in some way. In this
case, there is a Less Than relation between the third point and the second. The third point
must have a parameter intensity value that is less than the second point, and the system will
not allow you to violate that relation. When you move the third point up, the system tries to

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move the second point up. However, when we changed the Can Change property, we told it
that it could not move. Relations can be quite intricate and ultimately allow us to create
models with some amazing properties.

Model Building Tip: Relations Can Restrict Movement

A relation is a rule defined between two Control Points that restricts the
movement in some way. Relations can be based on parameter intensity or
position along the progression (state) axis. They can affect points in the same
profile as well as points in other profiles.

Let's change our property back to what it was.

Double click on the first Control Point, select Param in the list, and click the Properties
button at the bottom of the dialog.
Enable the Can Change property, and then click OK twice. The points can once again move
as they had before.

Let's review what we have discovered about these Control Points. Each Control Point is
composed of three parts: Param, State, and SD. Param controls the up and down
movement of the Control Point, State controls the left and right movement, and the SD
controls the line spread. The Fit Status property for Param and State allow or disallow the
estimation and minimization routines to change these values.
If you want a Control Point to be set to a value and not change, uncheck the Can Change
property.
These properties give you some sophisticated control over how Auto Analysis does its job.
You can tell it what values to manipulate and which ones to leave alone.

Model Building Tip: Use "Fit Status" and "Can Change"

When the Fit Status property is checked, the automatic adjustment routines in
GemStone have permission to adjust the value of the Control Point. Uncheck this
property when you only want the user to manipulate the value. Uncheck the Can
Change property when you do not want the user or the adjustment routines to
change a value.

Let's now look at the relations in more detail.

Relations
Slowly move the second Control Point down and watch what happens with the other points.
Notice that the second point always moves vertically with the first. When the second point
gets to the third point's vertical position, it pushes the third point down. As the third point

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moves down, the fourth moves vertically with it. Let's see why the behavior of the Step Down
profile works this way.
Double-click the second Control Point to display its editor. Now double click the Param entry
to show its two, defined relations.

Double click Relation1 to see its details.

Relation1 is enabled and related to CellType1: TDT: First with an Equal to relationship.
This is why the first Control Point always accompanies the second one as we move it up and
down.
Click OK to close the dialog, and then double click Relation2 to see its properties.

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The parameter intensity value for the second definition point has a Greater than relation
with the third point, CellType1: TDT: EndDown. This relation is what forces the second
point to be greater than the third point.

Modifying a Relation
Suppose you wanted this point to remain greater than the second point by more than 20
units. Is it possible to create a relation with an offset of 20? Yes.
Type a value of 20 into the Offset property, and then click OK three times.
Now, move the second CDP down and watch what happens. As it approaches the third point,
the third point starts moving down, too. If you move the third point up, you will see that the
second point starts to move up. The two Control Points maintain a separation of more than
20 units (on a scale of 100).
The way to "read" the relation expression is represented by this equation:
BeginDown > (EndDown + 20)

Adding a Relation
Just to make sure you appreciate how sophisticated you can make these relations, let's
augment what we have done so far. We have changed the Greater than relation between
the second and third Control Point so that the second is always 20 units or more greater than
the third. Suppose we also want the second point to be no more than 40 units away from the
third. In order to do this, we will need to add an additional relation. Here is the expression:
BeginDown <= (EndDown + 40)

Double click on the second point again to bring up the editor. Double click Param and click
New to create a new relation and view its editor. Choose the Less than or equal relation.
Then, choose CellType1: TDT: EndDown as the Link Name. Enter 40 for its offset.

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Click OK three times to close all the editors. Now, move the second Control Point up and
down to appreciate what we have done. We have established kind of window between these
two points. The points can be no closer than 20 units, but no farther than 40 units from one
another.
When we changed the second point's relations to the third, the system automatically made
the reverse entries for the third point's relations. GemStone will always try to make these
relations work bilaterally, which is normally what you want. If you need to have a
unidirectional relationship, you can delete the relationships that are automatically added to
the target point.

Reading a Relation Expression

We can generalize the relation expression. Given:
CDP_a is the Control Point we are editing
relationship is the relation to the other point, e.g. Greater than, Less than.
CDP_b is the target Control Point in the Link Name field
m is the Multiplier value
offset is the Offset value
Then the general expression for the relation is:
CDP_a relationship (m * CDP_b) + offset

Relations with Other Parameter Profiles

A Parameter profile can have sophisticated relations that constrain its shape and movement.
You can also create relations with Control Points in any other Parameter Profile, even in other
Cell Types. For example, suppose we wanted to create a model that coordinated the down
regulation of TDT with the down regulation of CD10. Here is how you would do that.
Click on the CD10 parameter plot to activate it. Then right click and select Choose
Parameter Profile. Pick the Step Down parameter profile.
In the TDT plot, double click the second Control Point again. This time, double click the
State item in the list and click New.

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In the editor, choose Equal to for Relation to Link. Now click the Link Name and choose
Celltype1: CD10: BeginDown. We are telling the GemStone that TDT and CD10 will begin
the down transition at the same location on the progression (state) axis.

Click OK three times to close the editors.

Now move the second Control Point in the TDT profile back and forth and notice that the
second point in CD10 moves back and forth as well. The two profiles are independent of one
another in the vertical direction (the Param value).

Saving Relations
How do you save all this information? The relations are stored in the model document when
you use the Save Document command. However, you can also save your customized
relations in your own Parameter Profile file. This allows you to reuse your work in another
model document. To do this, right click the TDT parameter plot and choose Edit Parameter
from the context menu. In the Edit Properties dialog, use the Save and Open buttons to
store and read a customized Parameter Profile.
If you save these parameters to disk and then retrieve them, the system will automatically
make all the hookups you have specified when they are both read into a model.

Conclusions
Editing and creating relations is an advanced feature for GemStone. For some applications, it
is important to add relations to ensure the system works reliably for complex analyses.

See also:
Tutorials

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Heat Map and Animated Regions

The Heat Map graphic allows the visualization and analysis of populations that statistically do
not belong to the model. These aberrant populations appear in the heat map with
unprecedented clarity and sensitivity. To use this new feature effectively, we need to
examine how it works.
Normally when a GemStone Cell Type is created, the Exclusion Probability property is set
to 0.05.

The property causes GemStone to exclude some fraction of outlier events from being
classified by a Cell Type. If we did not have this option, unwanted events from nearby
populations would become part of the Cell Type. For example, if we are modeling bone-
marrow B-cells, we do not want myelo-monocytic events to contaminate our B-cell Cell Type.
This property ultimately defines the extent of our Cell Type in any number of measurement
dimensions.
Let's suppose for this example that you have a progression similar to that shown below.

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This model defines the B-cell progression probabilistically in all dimensions. If we had
originally enriched our analysis packet with CD19 SSC B-cells, there could be events that for
one reason or another do not belong to this normal progression. The Heat Map graphic is the
best way to examine these events. Just click the Heat Map tool button and define the area
where you want the graphic using your mouse.

If you want to see all the events in the analysis packet, set your Exclusion Probability
property to 0.00. This will cause every event to be classified by your Cell Type. Here is what
the heat map looks like for this bone marrow B-cell example.

The X-axis represents our states in the B-cell progression. The Y-axis is the difference
between the events and our model. The horizontal gray line is currently set to 0.01. Anything
above this line has less than 1 out of 100 chance of being part of our cell-type. The higher
the difference (higher on the Y-axis), the more likely the population is statistically different.
We see quite a number of populations in this heat map. The long horizontal bands below the
p=0.01 line are presumably due to imperfections in the normal distributions of one or more
parameters due to events piling up at the axis origin. The more interesting populations are
further up on the Y-axis. If we zoom in on all the populations that are above the 0.01 p-value
line, we can clearly see a number of small, outlier populations

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What we really want to do here is to see other characteristics of these populations. For
example, we might like to see where the cells fall on a CD38 vs. CD20 plot. We can do that
very easily. Let's put a region around one of the populations.

We will label this region as "Unknown 1" or "UK1". We can see that it comprises about 0.12%
of our B-cells, or about 0.001% of the total events. To find out where these events are in all
the other GemStone plots, we need to use another GemStone feature: Animated Regions.

Animated Regions
You can animate any region in GemStone. Simply right click the region and choose Toggle
Region Animation. You can also double-click the region and check the Animate Region
property.
What happens when you animate a region? Every graphic in GemStone shows where the
region events are located by blinking those events with the region color. In dot plots, the
animated dots are drawn larger than the other dots so it is easy to see the events.

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The arrows in the figure above show where the UK1 events are in our example. These events
seem to be plasma cells that have low CD45.
You can also use animated regions in reverse, to investigate where known populations fall on
the heat map. For example, if we turn off animation for UK1 and turn on animation for R2:
Plasma Cells region shown above, we can see the blinking dots in the heat map graphic:

In this example, we can see quite a few plasma cells in the heat map graphic, and they are
not all in the same cluster on the heat map. One of the parameters that differentiates these
cells is CD45. The plasma cells near B3 have higher CD45 than the ones near B1.

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The Heat Map is a great exploratory tool. It can be used to find rare, malignant events in the
marrow or to find new normal populations. If tumors have subtly different clones, the Heat
Map will show them as separate spots. By using the Heat Map with animated regions
together, we can understand characteristics in abnormal cells that were never possible
before.

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How to Remap Parameters in a Model

The Problem
Suppose that you have created a detailed model for one of your analysis applications, and
you want to analyze a data file that was run on a different instrument. The new data file has
the same markers that your model was built upon, but the parameter names are a little
different. What happens when you read the data file into your model?
Nothing!
Some of the Parameter Profile plots show that the parameters are inactive. The label for the
parameter is dimmed when the parameter is not found in the data file, and no events appear
in the plot.

Does this mean that you can't analyze this data file with the model you worked so hard to
create? Absolutely not. You just have some remapping to do, to tell GemStone which
parameters in the file match up with the parameters in the model.

Two Approaches
There are two ways to approach this: one that modifies the model to match the new
parameters, and one that modifies GemStone's parameter database so that it can
automatically remap the parameters for you. Modifying the model is quick and easy. The
limitation with that approach is that you need to maintain a separate model for each of the
naming variants that your data files use. Modifying the parameter database requires a little
more effort, but it has the advantage of not requiring any changes to the model. We will look
at both techniques in this how to section.

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Option 1: Remapping the Model Parameters

The first approach is to remap the parameters in the model to the appropriate parameters in
the new data file. Here is how that is done.
First, open the GemStone Model Document that you want to use. Then open the new data
file that has different parameter names than those used in the model.
Now, show the Cell Type editors by clicking the Cell Type shrink tool.

Next, click the label of the first Parameter Profile plot that you want to remap. For example,
in the illustration above we would choose the CD19_CD19 plot label. This activates that plot
and shows its properties in the Parameter Profile Properties panel.
On the right edge of the Parameter Profile Properties editor is a toolbar. Click the button at
the top of the toolbar, with the tooltip "Match to Database Parameter".

A list of parameters found in the data file appears. Choose the parameter in the list that you
want to map to the model parameter. Using our example, we would choose PE-Cy55-
A_CD19.

As soon as we make the selection, GemStone fills the CD19 Parameter Profile with events
from the data file and reactivates the parameter.

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This process is repeated for each inactive parameter in the model. If the model has more
than one Cell Type, you will need to repeat the process for each.
Saving the Changes to a New Model
Once the model has been remapped for the new parameter names, it should be saved as a
new model so that it can be used again with data files with this format for file names. The
original model remains unchanged so that it works with the parameter names in the original
data files.

Option 2: Using the Parameter Database

The second approach is to make use of the Parameter Database to map the new file's
parameters to existing parameters in the database that the model was designed for. Here is
how that is done.
Once again, start by opening the GemStone Model Document that you want to use. Then
open the new data file that has different parameter names than those used in the model.
As before, you will see some of the labels on the Parameter Profiles are dimmed, indicating
that the parameters do not exist in the new data file.

Open the Parameter Database

Click the Parameter Database button on the main toolbar. The Edit Parameter
Database dialog is displayed.

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The "Live" column shows a value of "On" for the parameters that are found in the current
FCS file. A value of "Off" means that the parameter is in the database, but not found in the
current FCS file. Our job is to tell the database how to match the parameters in this new file
with parameters that are already in the database.

Matching the Parameters

This technique requires us to edit the names of the "Live" parameters to match existing
parameters. For example, in the dialog shown above, we see a parameter named "PE-Cy55-
A_CD19". Our model and the parameter database already know about a parameter named
"CD19_CD19". So, we need to edit the name of the Live parameter to match "CD19_CD19"
Click on the label of a Live parameter that you want to edit. In our example, we will start
with "PE-Cy55-A_CD19". Edit the name to match the existing parameter, in this case
"CD19_CD19". Then, press the Tab key to finish the edit.

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As soon as you press Tab, GemStone realizes that the name you typed matches a name it
already has in the Parameter Database. It presents a "Merge" confirmation dialog, with a
message similar to the one shown here:

Choosing "Yes" means that the program will merge the two parameter names into one entry
in the database. It will treat the names as aliases for the same parameter, and our model will
be able to work with either data file. Choosing "No" will allow us to choose a different name,
in case we made the change by accident.
In this case, we want to choose "Yes". After we click "Yes", the entry we just edited is
flagged to be deleted from the parameter database.

So if this entry is going to be deleted, how will the program keep track of the alias we just
created? Let's take a look.
Scroll down in the list and you will eventually find another entry labeled "CD19_CD19" that
has "Live" set to "Off". Double-click this entry to display the Edit Properties dialog.
Click the Edit All Conditions button to display the Edit All Parameter Conditions dialog.

We see there are two entries in the condition list: one for "CD19_CD19" as a short name, and
another that has "PE-Cy55-A" for the short name and "CD19" for the long name. GemStone
will consider parameters with these keyword values as equivalent. Note that you can
manually create new conditions or remove existing conditions with this dialog. See Edit
Parameter Conditions for details.

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Click OK to close the dialog, and once again to close the Edit Properties dialog.

Finishing Edits
Repeat this editing process for each of the parameters in the new data file until you have
mapped each one to an existing entry in the parameter database. Take care to match the
names with the names used by your model exactly.
When you finish your edits, click OK to close the Edit Parameter Database dialog. As soon as
you close the dialog, the parameters in the model that had been inactive should activate and
display events.

Summary
We have looked at two methods for telling GemStone how to map parameters in a new data
file to the parameters in an existing model. In the first case, we modified the model to use
the parameters in the new file. This was very quick, but required us to create a second copy
of the model to handle both of the file naming styles.
In the second approach, we used the Parameter Database to let GemStone map the
parameters to the same database entry. We edited the new parameter names in the
database to match existing parameters, and merged the parameters together. This created
aliases for the parameter by defining "conditions". The advantage to this approach is that
one model can now work with files that have different parameter names.

See also:
Edit Parameter Database

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Batch Processing

In this tutorial, we will learn the basics of batch processing. Any time that you need to
process a number of files using a repetitive pattern for the analysis, batch processing
features in GemStone can save you time and provide consistency. The system is very flexible,
allowing you to do different things to files in different groups. You can store your command
scripts so that they can be reused the next time you need to repeat the process.

Background of the Example

The example we will use in this tutorial is a model for a research project to monitor fetal red
blood cells in cases of fetomaternal hemorrhage (FMH). The model was developed in
conjunction with Dr. Bruce H. Davis of Trillium Diagnostics, LLC. See "Multicenter Clinical
Experience With Flow Cytometric Method for Fetomaternal Hemorrhage Detection",
Cytometry 50:285-290, 2002, published by Wiley-Liss, Inc for additional background.
The model uses three Cell Types, one for Adult RBCs, one for Adult F-Cells, and one for Fetal
RBCs. We will set up batch processing for a set of files from different patients to quantify the
relative percentages of each cell type.

First Time Processing

Open the GemStone Model Document
In most cases, you will use one GemStone model document for a given batch process, but it
is certainly possible to use a set of model documents, each applied to a different group of
files. We will keep things simple for this tutorial.

Click the Open Document button on the main toolbar. Navigate to the folder in which
GemStone was installed and find the Model Examples folder. Select "FetalRBC.gs" and
click Open.

Activate the Adult RBC Cell Type by selecting its tab at the bottom of the Cell Type
workspace.

Batch Processing Tip: Open the GemStone Model Document First

It is a good idea to open the GemStone document that you are going to use for
batch processing as your first step. This allows you to inspect the model before
you begin batch processing with it.

Select the Files to Analyze

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Next, we select the FCS files that we want to analyze.

Click the Select FCS Files button on the main toolbar. Navigate to the folder in which
GemStone was installed and find the Sample Files folder. Select the five "QQ" files in the
list and click Open.

GemStone will add the files to the File Database panel and read the first file into our model.

Edit the Fields for the Database

Since this is the first time we have done this analysis, we will need to set up our database
fields so that we store the information we want. Once we select the fields, we will save them
to a disk file so that we do not have to repeat the process next time.

Right click the File Database panel and choose Edit Fields from the menu.

We want to define a new set of fields from scratch. In the Edit Columns dialog, click the
Delete All button to remove any fields that might have been defined for another application.
A few fields will be left in the list; these are fields that cannot be deleted.
Now we are ready to select the new fields.

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In the tree control on the left side of the dialog, you will see a section named Application.
Within this section, find two fields: Application_Document Name and
Application_Opened Document Name. Select these fields and click the Add button.
These fields will store the name of the GemStone model document that we are using
(FetalRBC.gs), as well as a GemStone document that we will save for each data file that we
analyze.

Next, scroll down in the tree to the CellType1 section. In this group, select
CellType1_Zone0_Adult RBC_NumberOfEvents and CellType1_Zone0_Adult
RBC_PcntOfTotalEvents. Click the Add button to add these fields to our database.

Scroll to CellType2, and select two fields: CellType2_Zone0_Adult

RBC_NumberOfEvents and CellType2_Zone0_Adult RBC_PcntOfTotalEvents. Click
the Add button.

Finally, scroll to CellType3, and select two fields: CellType3_Zone0_Adult

RBC_NumberOfEvents and CellType3_Zone0_Adult RBC_PcntOfTotalEvents. Click
the Add button.

Our database is set up to store the number of events and percentages for each of the cell
types in the model. Let’s save this database structure so that we can reuse it without having
to find the results again in the tree.
Click the Save button on the right of the dialog. Navigate to a convenient location to store
this file, type FetalRBCFields for the filename, and click Save.

Batch Processing Tip: Save your Database Structure

Since you will probably do more than one kind of analysis with GemStone, the
information you want to store in a database will change. Use the Save and Open
buttons in the Edit Columns dialog to store and retrieve the appropriate database
structure for the analysis you are doing.

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Click OK to close the Edit Columns dialog.

Set the Batch Options

Now that we have selected the files and set up our database, we are ready to define what
happens as each file is processed. For this, we use the Batch Options panel, found below the
File Database panel on the left side of the GemStone workspace.
Here is what we want the batch to do. After it loads one of our data files, we want it to re-
open the FetalRBC model document. We do this so that the model is in the same state when
we begin our analysis. Then, we want GemStone to trigger the Auto Analyisis command. And
finally, we want the program to save the finished analysis in a separate GemStone model
document.
In the Batch Options panel, enable the Open Document command by checking it. Then,
click the Edit button for Open Document Options.

In the dialog that is displayed, we will specify the GemStone document that we want to open.
Set the Location Option to Use the specified Target File.
Click the Target File property, and in the Choose File dialog, select the FetalRBC.gs
document and click Open. Click OK to close the document options dialog.

In the Analysis section of the Batch Options panel, enable the Auto Analyze Data option,
and uncheck any other options.
In the Finish Up section, enable the Save Document option and uncheck other options.
Then, click the Edit button for Save Document Options.

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In the dialog that is displayed, we will specify where we want GemStone to save our finished
analysis and how to name it.
Set the Location Option to Use the data file name and the Target Path. This will name
the GemStone document using the data file name that was analyzed.
Click the Target Path property, and in the Choose Folder dialog, choose a folder that you
have write-access to and click OK. Click OK to close the document options dialog.
Let's save this set of batch options so that we can use the same commands again.
Just below the Batch Options panel, there are Open and Save buttons. Click Save, navigate
to a convenient location, and type FetalRBCBatchCommands for the file name. Click Save
to store the commands.

Batch Processing Tip: Save your Batch Options

You can save time setting up for batch processing by saving your batch options
to disk. The next time you perform the same analysis, open your saved batch
options and you are ready to go.

Process the Files

We are all set up and ready to go. The best approach for batch processing is to do the first
file, make sure everything is working as planned, and then to let the batch process run
automatically through the rest of the files.
Click the Redo button on the batch toolbar at the bottom of the GemStone workspace.

GemStone will open the first data file in the batch and then run the commands we selected.
It opens the FetalRBC.gs model document, performs an Auto Analysis, and then saves a new
GemStone model document based on the name of the data file we analyzed. We also see the
analysis results in the File Database.
To automatically advance through the rest of the files, click the Auto Advance button on the
batch toolbar.

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As GemStone processes each file, it repeats the batch options that we selected and adds the
analysis results to the File Database. Notice that the Application_Document Name field
shows the name of the document that was saved with the finished analysis, and the
Application_Opened Document Name shows the name of the original model document.
These fields are very useful for keeping track of how files were analyzed and where the final
analysis is stored.

After the First Time

If we wanted to run this batch process again tomorrow, our task would be easier because we
have saved the database fields and the batch commands. Our process for setting up would
be simplified:
1. Open the GemStone model document.
2. Select the FCS files to analyze.
3. If the file database fields need to be set up, we use Edit Fields, and then Open the file we
saved (FetalRBCFields.xml) with the database structure.
4. Use the Open command at the bottom of the Batch Options panel to read in our
FetalRBCBatchCommands.xml file.
5. Process the first file by clicking the Redo button. Inspect to make sure everything is
working OK.
6. Process the remaining files by clicking the Auto Advance button.

Using Levels
There are situations in batch processing where you need to treat some files differently than
others in the batch. For example, suppose you have a GemStone model document that is
designed to analyze 8 markers for a 4-tube panel. When the first file in the panel is read, you
need GemStone to open the appropriate GemStone model document from disk and do an
analysis. When the second and third files are read, you just want the program to analyze the
data. And when the last file in the panel is read, you need to analyze the data, save the
document, and print a report. Your analysis is not complete until you have read and analyzed
all four tubes.
In a situation like this, you can use Batch Levels to perform different actions on certain files
in the panel. Here is how you would handle the example described above.
After selecting the files for the cases you are going to analyze, expand the Batch Levels
panel. This displays some additional controls that we will use to create the levels in our
batch.

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Next, you would select the second and third file in the list, and click the Indent button to
indent the files to Level 2.

Similarly, you would select the fourth file in the list, and click the Indent button twice to
indent the file to Level 3. If you have more than one case, you would repeat the process for
the additional cases.
Now that you have 3 "levels" of files in the file database, you can set the batch options for
each level separately. Notice that the Batch Options panel now shows a tab for each of the
three levels.

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To set the options for the Level 1 files, click the Level 1 tab. In our example, we would set
the options on this tab to Open Document and Auto Analyze Data.
On the Level 2 tab, we would set the options to simply Auto Analyze Data.
And on the Level 3 tab, we would choose Auto Analyze Data, Print Workspaces, and Save
Document.
As the files are processed, GemStone looks to see which level in the outline the files are on
and selects the appropriate commands for that level.
This feature provides great flexibility for processing files automatically.

Summary
Batch processing with GemStone simplifies the task of processing more than one file at a
time. A batch process requires some initial setup to configure the database and the
processing options you want to perform. Once the process is defined, files can be
automatically analyzed and results stored.
The batch system allows more advanced processing through the use of outline "levels" in the
file database. Files on different levels can be processed in different ways. Levels are useful
for processing panels of files and other processes that require more than one analysis
strategy for the files in the batch.

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How to Work with Software Batteries

This tutorial introduces the software battery concept and describes how to work with
software batteries in GemStone.

Why Software Batteries?

Software batteries provide a different from of software licensing than the typical commercial
software license. The software battery is a per use style of license, which allows for much
greater flexibility than other software license types. Since the battery charge can easily be
transferred or shared the use of the software is highly portable. This allows you to install
GemStone for use on any number of computers, allowing many individuals to utilize the
application as needed. Software batteries eliminate the need to purchase licenses for each
user who may use the application.

How do the Batteries Work?

Each time a new FCS file is loaded into GemStone, the battery charge is decremented by one
unit. When the battery charge is depleted GemStone will prevent you from opening additional
FCS files. You will still be able to open any files that have previously been loaded.
When the battery is depleted, additional Battery charge can be obtained either via a purchase
from Verity Software House or a request to a Battery Server for GemStone at your institution.
In addition to remembering the files that have been previously loaded, GemStone associates
a set of files with each stored analysis or GemStone Document. The GemStone Document
can be shared with others who will be able to load the associated files into that analysis
without requiring any battery charge. This means that an analysis can be shared, adjusted,
or modified by many users, or the same user on different computers, with only one charge
per associated file.
Each user account on a computer will have its own software battery. This allows many users
to use GemStone on the same computer without using up battery charges of another user.

Recommendations for Battery Use

In most cases it is recommended to leave most of your battery charge in your Web Battery
Account. And, that you only transfer enough charge to your computer to accomplish the task
at hand. Doing this will prevent loss of battery charge in the event that you computer's hard
drive needs to be reformatted, replaced, or the OS needs to be reinstalled. Having the
majority of your battery charge in your Web Battery Account also allows you to transfer
charge easily to another computer or to another user. This enables you to quickly "activate"
GemStone on another computer or for another user.

What is a Web Battery Account?

A Web Battery Account is an account that can contain a number of battery charges for a
specific application. Your Web Battery Account is associated with a Member Account on
www.vsh.com. Battery charges can be put into your account via a purchase, by uploading

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remaining charge from an installation of GemStone, or via a transfer from another user's
Web Battery Account. Battery charges are removed from your Web Battery Account when
you download them to an installation of GemStone, or by transferring them to another user's
Web Battery Account. When you login to your Member Account, you will see a number of
actions that can be preformed on the Web Battery Account.

Free Trial Battery

If you are new to GemStone you can request a free trial battery which will allow opening of
your own data for evaluation. To request a free trial battery from GemStone you can click on
the Free Trail button on the banner dialog that is displayed when GemStone first launches.
This button will take you to Verity's website where you can fill in information needed to
supply you with the trial battery.

Working with Battery Charge

There are a number of actions that act upon GemStone's battery. If your computer is
connected to the Internet, you can buy additional battery charge from www.vsh.com,
redeem a battery certificate, check the balance of your web account, download charges from
your web account, or upload charges to your web account. If your computer is connected to
an internal network that hosts a Battery Server for GemStone, you can request battery
charge from the Battery Server. If you computer is not connected to a network you can use
the offline transfer mechanism to obtain battery charge.
Some of the actions listed above have more than one way to access them. For our
discussion, we will consider the use of the Battery Menu. Let's first introduce the other two
locations of battery interaction.
The first is the Battery Meter, which is located in the lower right corner of the GemStone
main window, and shown below. It will indicate the current percent of charge remaining in
the battery. The tool tip displayed by holding the mouse over the Battery Meter will give the
actual remaining charge amounts. The context menu, shown by right clicking the Battery
Meter, contains a few shortcuts to actions also contained in the Battery Menu.

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The second is the Battery Status dialog. The Battery Status dialog, shown below, can be
accessed via the Battery Menu, the Battery button on the Banner dialog, or the context menu
of the Battery Meter. The Battery Status dialog contains a few buttons that allow you to
perform actions also available under the Battery Menu.

Purchasing Battery Charge

If you need to purchase battery charge the process can be initiated by selecting the Buy Now
option in the Battery Menu. You will be directed to the Verity Software House online store
where the purchase will be completed.

Charging the Software Battery

Selecting the Charge Battery command in the Battery Menu initiates the Charge Battery
Wizard. The Charge Battery Wizard will guide you through the process of charging the
battery.
There are three sources of battery charge that can add charge to GemStone's software
battery: a Battery Certificate, a Web Battery account on Verity's web site, or a Battery Server
on your local area network. For each of these sources, there are several methods for actually
charging the software battery.

Battery Charge Method 1: Redeem a Battery Certificate via the Wizard

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If your computer is connected to the Internet you will be able to redeem a Battery Certificate
directly using the wizard. Note that firewalls can sometimes interfere with this action. To
redeem the certificate, select method 1 and click Next. The following page will be displayed.

Enter the Battery Certificate Code in the field. When you click Next the wizard will attempt to
redeem the certificate. The wizard will either move to a success or failure page when the
request is completed. If this method fails you can redeem the certificate using method 2.

Battery Charge Method 2: Redeem a Battery Certificate using a web browser

If your computer is not connected to the Internet you will need to use this method to redeem
your Battery Certificate. Choose method 2 in the wizard and click Next. A page is displayed
with a Transaction Code and your Computer ID. Take these codes and your Battery
Certificate to a computer with internet access and visit www.vsh.com. Click Redeem a
Battery Certificate in the Quick Links section, enter the information and follow the
instructions given.
The web site will provide you with several codes to complete the process. When you have the
new codes, click Next in the wizard to move to the next page. On this page you will need to
enter the three Battery Charge Codes generated by the website.

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After you enter the codes, click Next. The codes will be evaluated and the wizard will either
move to a success or failure page. If this method fails please contact technical support at
[email protected] for assistance.

Charge Battery Method 3: Download charges directly from your Web Battery
Account
If your computer is connected to the Internet, you can download charges directly from your
Web Battery Account on the Verity web site. Note that firewalls can sometimes interfere with
this action. To begin this method, select option 3 in the wizard and click Next. The following
page will be displayed.

Enter your Verity Software House web account information and specify a number of charges
to download. Click Next to initiate the download request. The wizard will either move to a
success or failure page when the request is completed. The actual charge downloaded may
be reduced if your web account has fewer charges than you have requested. If this method
fails, you can download charges indirectly using method 4. The method will fail if the wizard

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cannot connect with the Verity web site, the user account is not valid, or the account has no
charges in it.

Charge Battery Method 4: Battery Charge Codes from a Web Battery Account
If your computer is not connected to the Internet, you can use this method to obtain battery
charges from a Web Account on Verity's web site. Choose method 4 in the wizard and click
Next. A page is displayed with a Transaction Code and your Computer ID. Take these
codes to a computer with internet access and visit www.vsh.com. Login to your Web Account
and choose the Get Battery Charge Codes link. Enter the information and follow the
instructions given.
The web site will provide you with several codes to complete the process. When you have the
new codes, click Next in the wizard to move to the next page. On this page you will need to
enter the three Battery Charge Codes generated by the website.

After you enter the codes, click Next. The codes will be evaluated and the wizard will either
move to a success or failure page. If this method fails please contact technical support at
[email protected] for assistance.

Charge Battery Method 5: Request charges from a Battery Server

If your computer is connected to a local network that hosts a Battery Server for GemStone,
you can request charges from the Battery Server. Note firewalls can sometimes interfere with
this action. To begin the process, choose method 5 and click Next. The following page will
be displayed.

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'
Enter the network name or the IP address of the Battery server, the password required by
the Battery Server, and enter the number of charges requested. When you click Next, the
request is sent to the Battery Server. The wizard will move either to a success or failure page
depending on the result of the request. The actual charge downloaded may be reduced the
Battery Server has fewer charges than you have requested or if there is a limit on how many
charges you can requested at one time. If this method fails you can obtain charges form a
Battery Server using method 6.

Charge Battery Method 6: Battery Charge Codes from a Battery Server

If your computer cannot connect directly to your Battery Server, you can use this method to
obtain battery charges. Choose method 6 in the wizard and click Next.
A page is displayed with a Transaction Code and your Computer ID. Take these codes to
the Battery Server administrator. The administrator can enter the codes into the Battery
Server to generate the Battery Charge codes you need to complete the process.
When you have the charge codes, click Next in the wizard to move to the next page. On this
page you will need to enter the three Battery Charge Codes generated by the Battery Server.

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After you enter the codes, click Next. The codes will be evaluated and the wizard will either
move to a success or failure page. If this method fails please contact the Battery Server
Administrator for assistance.

Uploading Battery Charge to Your Web Account

If you computer is connected to the internet, you have the option of uploading all remaining
battery charge to your Web Battery Account. Note, firewalls can sometimes interfere with this
action. Uploading your remaining battery charges allows you to redistribute your remaining
battery charge to other computers or users. You can also prevent the loss of your battery
charge by uploading your battery charge before reformatting or replacing your hard drive or
reloading your operating. To upload the battery charge, choose the Upload Credits option
from the Battery Menu. A dialog like the one shown below will be displayed. You will need to
enter your web account login and password, and optionally choose to have the password
remembered on this system. When you click OK the Upload will proceed and alert you as to
the result, either success or a reason for failure.

Checking the Balance of Your Web Battery Account

If you computer is connected to the Internet, you will be able to check the balance of your
Web Battery Account directly from the application. Note, firewalls can sometimes interfere
with this action. To check your Web Account Balance, choose the Check Balance option from
the Battery Menu. Enter your web login and password and optionally choose to have the
password remembered, in the dialog, shown below, that is displayed. When you click on OK,
GemStone will attempt to check the balance of your Web Battery Account, and will either
display the current balance or a reason the action failed.

Summary
In this tutorial we have considered the why and how of software batteries. We have learned
the benefits over other software license structures. We covered recommendations for using
the battery system. We learned about the Web Battery Account. We then covered use of the
battery system, and how to obtain the battery charge needed to complete analysis on a
computer.

See also:

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Battery Menu
Server Menu
How to Work with Battery Server for GemStone

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How to Work with Battery Server for GemStone

This tutorial covers the setup and use of the Battery Server for GemStone application. The
Battery Server is used as a central hub on a network to distribute battery charges to client
versions of GemStone. For more information on the use of software batteries see the tutorial
How to Work with Software Batteries.

Setting up the Battery Server

Install the Battery Server on a computer that can be accessed by all of the GemStone Client
computers. The client computers do not need to be in constant contact with the server; they
periodically communicate with the server to get battery charges which allow the client
software to read data files.
After installation of the Battery Server application is completed, there are a few steps
necessary to prepare it to distribute charges:
1. Register the Battery Server.
2. Configure the Battery Server to distribute charges.
3. Charge the "software battery" for the server so that it can deliver charges to clients.
4. Optionally, configure the computer to automatically launch the Battery Server
application.

Registering the Battery Server

When the application is first launched it will need to be registered. Read Registering your
new software for instructions on how to register you software. After a successful registration,
you are ready to configure the Battery Server.

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Configure the Battery Server

You will be then presented with a couple dialogs that will assist in configuring the Battery
server to distribute charges. The first dialog will ask you to enter a password. The Battery
Server can be protected from unauthorized access by use of a password. Users will need to
know this password when making battery requests.

The second dialog, will allow you to specify the port that the Battery Server will monitor for
battery requests. In most cases, the default port selection should be used. If there is a
conflict, a different port may be specified. If you do not use the default port, users making
battery requests will need to know the port number you have chosen. They must modify the
Battery Server Port specified in the Miscellaneous section of the Preferences. See Edit
Preferences for additional details.

Firewall Alerts!
When the Battery Server attempts to monitor the IP port selected, firewall
software on the computer may display an alert. This is because the Battery
Server is attempting to listen for incoming requests on an IP port - something
that firewall software is designed to prevent.
The Battery Server needs to monitor a port in order to service requests from
GemStone Client users. Firewall software settings need to be adjusted to allow
this.

Once the port is selected, the Battery Server will begin monitoring the port for battery
requests. You should see a message indicating success.

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Charging the Battery

The Battery Server's battery can be charged using any of the options covered in the tutorial
How to Work with Software Batteries. These options are found in the application's Battery
menu. Choose the appropriate method and charge the battery. For new installations, use the
"Battery Certificate" that was included with the software. The Battery Certificate encodes a
set of charges for the Battery Server. When the battery has been successfully charged you
should see a success message. The battery meter in the bottom right of the application
window will show that it is fully charged.
At this point, the Battery Server is ready to fulfill battery requests from GemStone Client
software on other computers. This can be tested by requesting a battery from the Battery
Server itself. To do this "self-request", use the Request from Server option in the Battery
Server's Battery Menu, and use localhost in the Battery Server Name field. You should again
see a success message and have a full battery.

Configuring the System to Automatically Run the Battery Server

The Battery Server should be available to GemStone Client installations at all times. The best
way to ensure this is to set up the Battery Server to automatically launch when the computer
is restarted. This allows the Battery Server to distribute charges without interaction on the
part of the user. There are a few steps to set this up, depending on the operating system you
are running.

Common Steps to Automatically Run the Battery Server

In order for the Battery Server to run automatically, the banner dialog and the user login
need to be disabled. This can be done using the Preferences dialog. On the Mac, choose
Preferences from the application menu. On Windows, choose Preferences from the Edit
menu.
In the Edit Preferences dialog, choose the Miscellaneous section. Uncheck the Show
Banner checkbox and choose Don't Show for the User Login Option.

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Automatically Run the Battery Server on Windows

If you are running Battery Server for GemStone on Windows, you will need to setup a
scheduled task to launch GemStone each time the computer reboots. From the Control Panel,
choose Scheduled Task, Add Scheduled Task.

This will start the Scheduled Task Wizard. Click Next.

Use the Browse button to navigate to the location where the Battery Server for GemStone
was installed.
Choose the Battery Server for GemStone.exe application file. Note, you may or may not see
the ".exe" extension. If you are unsure which entry is the application you can right click and
choose Properties. The "Type of file" field for the application will specify Application.
Select to run the application "When my computer starts" and click Next.

In the next pane of the wizard, you will need to enter a user account and password under
which the Battery Server task will be run. Use the Windows OS user and password that was
used to register Battery Server for GemStone. This is the user name and password that you
use to login to your computer.

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Click Next.
There are a few additional settings to configure, so enable the "Open the advanced
properties for this task when I click Finish".

Select the Settings tab. Uncheck the Stop the task checkbox. Then click OK to finish the
task.

At this point, the Battery Server for GemStone should run in the background each time your
computer starts up. It will monitor the IP port and be able to fulfill battery requests from
GemStone Client users even if no user is logged in to the computer. To ensure that the
Battery Server will always be available when needed, the system should also be configured
not to sleep after a certain period of time, unless network activity can wake it up (Power
Options).

Automatically Run the Battery Server on Os X

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If you are running Battery Server for GemStone on Macintosh OSX, you will need to add the
Battery Server to a list of applications that start when a user logs in, and set that user to be
automatically logged in when the system restarts. From the System menu choose System
Preferences.

In the System Preferences, dialog click on the Accounts icon. From the list of users, select
the user who preformed the Battery Server registration. Click the "Login Items" or "Startup
Items" tab. Click the "+" button in the Login Items section, browse to and select the Battery
Server for GemStone application file. If you want the Battery Server to run minimized check
the Hide checkbox.

Now click on Login Options in the lower left. If the control is disabled you will need to click
on the lock button in the lower left and authenticate to make the needed changes. In the
Login Options section, check the Automatically Login As checkbox. Then choose the user
that preformed the Battery Server registration. You may need to re-authenticate for this step
as well.

At this point, the Battery Server for GemStone should run in the background each time your
computer starts up. It will monitor the IP port and be able to fulfill battery requests from

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GemStone Client users even if no user is logged in to the computer. To ensure that the
Battery Server will always be available when needed, the system should also be configured
not to sleep after a certain period of time, unless network activity can wake it up (Energy
Saver Settings). Also the user should not be logged out after a period of inactivity (Security
Settings).

Using the Battery Server

When you need to interact directly with the battery server, you will need to log in to the
operating system with the user login that was used to register the Battery Server.
If you are running the Battery Server on Windows and the server is configured to run
automatically as outlined above, you will need to terminate the server background process,
and then re-launch the Battery Server application directly after logging in to the operating
system. To terminate the Battery Server process, you can run the "Stop_Battery_Server.bat"
file that is installed in the same directory as the Battery Server for GemStone application.
You can view and adjust the server configuration using the Properties option in the Server
menu. The server properties will show you the information needed by the user when making
battery requests from the server. If changes need to be made to the Battery Server's
configuration they can be made here. You can also view the Battery Creation Log, which
shows information about the batteries that have been generated and distributed. From the
Server Menu you can satisfy an offline transfer request from a user by choosing the Transfer
Charge option. You will need enter the needed information from the user into the Transfer
Charge dialog and return to the user the battery transfer codes that are generated. For more
information on these options see the Server Menu section.

Summary
In this tutorial we have considered the how to configure and use Battery Server for
GemStone. This included registration, initial configuration, charging the battery, configuring
the system to run the Battery Server automatically, and using the Battery Server directly
after logging in to the computer on Windows.

See also:
Battery Menu
Server Menu
How to Work with Software Batteries
Tutorials

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Reference Section

The following sections document the features and functionality of all menus, dialogs, and
functions of GemStone.

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Control Panels

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File Batch and Database panel

File Database panel

This panel contains a list of FCS files that have been selected for analysis. The Status column
on the left indicates the current file with a pointer icon. A check in the Status column
indicates that the file has been analyzed. The batch level for the file is also shown, e.g. L1 is
level 1.
Columns can be added to the list to display analysis results and keyword information from
the files. This serves as the database of analysis information and can be exported for
integration into other applications.
The File Database context menu can be displayed by right-clicking on the panel.

If you want to...

Add files Use the Select FCS Files command from the File menu or
the main toolbar.
Remove files Use the mouse to select files, and then press the Delete
key on the keyboard.
Set the current file Right-click the file name and choose "Set Current Batch
Item" from the context menu.
Add or remove columns Right-click the list and choose Edit Columns from the
context menu.
Export the database of Right-click the list and choose "Export Batch Database"
information from the context menu.

See also:
File Database Context Menu
Edit Batch Columns

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File Database Context Menu

This context menu appears when the mouse is right-clicked on the File Database panel. On
computers with a one-button mouse, it can be displayed by holding the Control key and
clicking the mouse. The menu provides a number of actions that relate to the files in the
batch.

Set current batch item

Make the selected item the current batch item.
Reset analysis flags
Set all files in the batch to "unanalyzed".
Delete all items
Clear all files from the batch list and database.
Delete selected items
Remove the selected file(s) from the batch list and database.
Refresh
This command forces the file batch list to update.

Edit Fields
Display the Edit Batch Columns dialog to choose columns for the batch and database.
Export Database
Export the database to an ASCII text file. See Export Database for additional information.

Delete maximum group level

Delete the deepest batch level in the in the batch, if there are no files on that level.

Save batch list

Save the current state of the batch list. When GemStone starts up it restores the last state of
the batch. When it exits, it saves the state of the batch automatically.
Save batch list as
Displays the File Save dialog to save a batch list to disk. See Save Batch List for details.
Open batch list
Displays the File Open dialog to opens a previously saved batch list. See Open Batch List for
details.

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Edit Batch Columns

This dialog is used to select the columns that are displayed in the File Database panel, as
well as the fields that are included in the database of analysis results.

The tree on the left of the dialog contains all of the information available, including analysis
results, FCS keywords, and other useful fields. The list on the right of the dialog shows the
fields that have been selected either for display in the batch list, inclusion in the database, or
both.

If you want to...

Add columns or Select one or more items in the information tree on the left
fields and then click the Insert or Add button.
Remove fields Select one or more items from the list on the right and then
click Delete or Delete All.
Change the order of Select an item in the list and use the Up or Down button.
columns
Include a field in the Add the field to the list and make sure the Enable option is
database checked.
Show or hide a Add the field to the list and check or uncheck the Display
column option.

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Export Database

Use this command to export the analysis information in the batch database to a tab-delimited
ASCII text file. The command will display the standard File Save dialog. Enter a file name for
the database and navigate to the location where you want to store it. Then click the Save
button to complete the export.

ASCII formatted text is one of the most universally accessible file formats available. It can be
easily read and interpreted by database and spreadsheet applications, as well as most lab
information systems.

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Save Batch List

Use this command to save the current files and database to disk. The standard File Save
dialog will be displayed, allowing you to choose a name and location for the database file.

The Open Batch List command can be used to reload the database.

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Open Batch List

Use this command to open a previously saved database from a file on disk. The standard File
Open dialog will be displayed, allowing you to choose a name and location of the database
file.

Use the Save Batch List command to store a database on disk.

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Batch Levels panel

Batch Levels panel

This panel is used to control the "batch level" of files in the File Database panel.

GemStone uses "levels" to differentiate groups of files in the batch, and it allows different
actions to be performed on each level. The level for each batch item is shown in the File
Database panel; it appears in text form and as indentation in the Status column.

When GemStone opens a file in the batch list, it looks to see what level of indentation the file
is on. It then finds the set of actions that relate to that level and executes them. The actions
for each level can be edited in the Batch Options panel. This provides a simple mechanism
for creating sophisticated batch processing. For example, Level 1 could be used to identify
the first file in a panel of related files to open a GemStone document and initialize the model,
and then perform an Auto Analysis. Most other files in the panel could be assigned to Level 2,
which would simply perform Auto Analysis. The last file in the panel could be assigned to
Level 3, which would save the GemStone document and print the final results.

If you want to...

Change the level Select the item and click to increase or to
decrease the batch level.
Set the current batch item Click "Set as Current".
Move an item up or down Select the item and click Up or Down.
Delete one or more items Select the items and click the Delete or Delete All
button.
Clear the analysis flags Select the items and click Clear Flags.
Edit the columns that Click the Columns button.
appear in the batch list and
the fields that are included
in the database

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See also:
File Database panel
Batch Options panel

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Batch Options panel

Batch Options panel

This panel determines the actions that are performed when an FCS file in the batch is read
into the program for analysis. When GemStone reads a file, it determines which "level" the
file is on in the batch. Each batch level has a set of actions associated with it. For more on
batch levels, see Batch Levels panel .

A tab will appear for each level that has been defined in the batch list. Select a tab to view or
edit the actions for the associated batch level.

Enable
Enable the actions defined on this tab.

Custom
Custom Actions
Enable Custom Actions mode and the ability to create custom action scripts.
Advanced Editing
Edit the actions in the script. This is an advanced feature that provides access to additional
scripting commands. See Edit Actions for more information.

Init and Open

New Document
Create an empty model in a new document, clearing any previously opened document and
graphics from memory.
Open Document
Open a specific GemStone document.
Open Document Options
Set options for opening the GemStone document, including the file name and path. See
Properties for File Open and Save for additional details.

Analysis
Normalize Data
Normalize intensity levels from one file to the next for any parameters enabled for
normalization in the Edit Parameter Database dialog.
Normalize
Data Options
Initialize Clears any normalization factors and target mean intensity values.

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This is the default option.

Set targets Stores the linear mean values of parameters flagged for
normalization. The mean values will be used as target mean values
for subsequent files when Use Targets is selected.
Use targets Parameters flagged for normalization are normalized using the
current Target Mean Intensity. Graphics and results for these
parameters will reflect the normalized data.

Classify Data
Initiate the Classify Data command after the file is read.
Auto Analyze Data
Initiate the Auto Analysis command after the file is read.
Enrich Data
Initiate the Enrich Data command after the file is read.
Classify File
Initiate the Classify File command after the file is read.
Merge Options
Choose an option for file merging. File merging allows a set of related files (e.g. a panel of
tubes) to be merged into one file containing the union of all parameters in the set. The
program synthesizes data for missing parameters.
Merge
Options
Don't merge Disables file merging.
Start merge Creates a new file to start merging data.
Add to Adds to a merge file. This option is only valid after a "Start merge"
merge action and before a "Finish merge" action.
Finish Finishes the file merging and closes the output merge file.
merge

Finish Up
Pause and Continue
Enable this option to pause batch processing before printing, saving, and continuing to the
next file. When this command is triggered, a dialog is displayed indicating that processing is
paused. The dialog shows a count-down timer and will automatically close and continue after
the timer runs out. If the user clicks anywhere on the GemStone workspace during the
pause, the timer is shut off and the program will wait until the user clicks the Continue
button before it advances.
Print Workspaces
Print the workspaces for all cell types.

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Save Document
Save the GemStone document after the analysis.
Save Document Options
Set options for saving the GemStone document, including the file name and path.
Save Graphics
Save graphics for objects that have been selected.
Save Graphic Options
Set options for saving graphics, including the file name and path. See Properties for File
Open and Save for additional details.
Stop Auto Advance
Stop the Auto Advance of the batch processor.

Open and Save

These buttons allow you to save and re-open batch scripting setups. This makes it easy to
store scripts for different purposes and reuse them without having to reset all of the options.

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Properties for File Open and Save

These dialogs are displayed from the Batch Options panel to set options for opening and
saving GemStone documents and other file-based actions. The dialogs are very similar; the
Save dialog has one additional property.

Options
Location Option
Choose the naming option to use for the open or save operation.
Options
Use the data file name and path With this option selected, the GemStone
document name and location are based on the
name of the FCS data file that is currently
opened. For example, if the FCS file is
"C:\Data\Experiment123.fcs", the document
that the program attempts to open will be
"C:\Data\Experiment123.gs".
Use the data file name and the With this option selected, the GemStone
Target Path document name is based on the name of the
FCS data file that is currently opened, but the
location of the document is defined by the
Target Path property below.
Use the specified Target File With this option selected, the GemStone
document name and location are defined by the
Target File property below.

Confirm Selection
When this option is enabled, the File Open or Save dialog will be displayed so that the file
name and location can be verified or changed by the user.
Replace Existing File (Save Document dialog only)
Check this option to overwrite a file of the same name without prompting for confirmation.

Names and Paths

Target Path
When "Use the data file name and the Target Path" is selected as the Location Option, this
field sets the path that is used. For other Location Options, this field is ignored.
Target File
When "Use the specified Target File" is selected as the Location Option, this field sets the
path and file name that is used. For other Location Options, this field is ignored.

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Edit Actions

This dialog provides custom editing for batch processing scripts. It is an advanced option and
is not recommended for most users.
The actions listed in this dialog are intentionally left undocumented. If you need to create a
customized action sequence, you will need to contact Verity Software House Technical
Support for assistance.
The dialog displays a list of available actions on the left. The list on the right of the dialog
displays the current script of actions. Additional actions can be added or inserted in the
script, and actions in the script can be edited, reordered, or deleted.
A set of buttons on the right of the dialog allows scripts to be saved to and read from disk.
Additional buttons allow the script to be executed for testing purposes.

If you want to...

Add or insert a new action Select the action(s) in the list on the left of the dialog,
and then click Add or Insert.
Delete an action from the Select the action in the list on the right, and then click
script Delete.
Delete all actions in the Click the Delete All button.
script
Edit the parameters for an Select the action in the list on the right, then click the
action in the script Properties button.
You can also directly edit the Return, Object, and
Parameters columns in the list.
Save a script to a file on Click the Save button.
disk
Open a script from disk Click the Open button.
Run the script Click the Run button.
Pause a script that is Click the Pause button.
running
Run the script one line at a Click the Step button to advance the script one line.
time
Run the script to the Click the Run Until button.
selected line

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Global Properties panel

Global Properties panel

This panel contains properties that apply to all cell types in the current GemStone model.

Specimen keyword
Set the keyword that is used to identify the specimen represented by the current FCS file.
Enclose the keyword with square brackets. FCS file keywords as well as internal keywords
can be used.
Color by Gate Combinations
When enabled, this option applies coloring to unclassified events based on the combination
of gates (i.e. regions) that each event is contained by.

Document Name
The name of the GemStone document currently opened.
Document Path
The path to the FCS file currently opened.

Total Analyzed Events

The number of events in the FCS file analyzed by the model.
Percent Unclassified
The percentage of events that were not classified by the model.

Model Author
This field can be used to enter the name of the person who created the model.
Saved Program Version
This field displays the program version that was used to save the model.

Synthesize Data Using Model

When checked, the model's means and line spreads are used when synthesizing data. If
unchecked, the observed means and SDs of the existing data are used.

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Cell Type Properties panel

Cell Types

A GemStone model can define one or more "cell types", with each cell type describing the
characteristics of one subset of cells. When the model analyzes the events in an FCS file,
each event ("cell") is classified into one cell type or it is considered an "unclassified" event
that does not match any of the cell type definitions.

General characteristics for a cell type are defined in the Cell Type Properties panel. The
model definition for a cell type is defined using the Cell Type widget and Parameter Profile
Properties panel.

If you want to...

Add a new cell type In the Cell Type Properties panel, click the Utility
Actions dropdown and select Add Cell Type.
Duplicate a cell type In the Cell Type Properties panel, click the Utility
Actions dropdown and select Duplicate Cell Type.
Delete a cell type In the Cell Type Properties panel, click the Utility
Actions dropdown and select Delete Cell Type.
Select a cell type At the bottom-left of the Cell Type workspace, click the
tab for the cell type that you want to view or edit.

See also:
Cell Type Properties panel
Cell Type widget
Parameter Profile Properties panel

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Cell Type Properties panel

This panel contains properties and actions that relate to the current Cell Type tab. A
GemStone model can contain any number of Cell Types. Each Cell Type has its own collection
of parameter profiles that define the attributes of events that will be selected into that Cell
Type. For example, it is common practice to create a Kappa Cell Type and a Lambda Cell
Type for a model designed for Leukemia/Lymphoma analysis. Each event (cell) can be
classified into one of the Cell Types or assigned as an "unclassified" event. An event cannot
be assigned to more than one Cell Type.
By default, there are 100 states defined for each Cell Type, but each Cell Type can use a
different number of states. If the progression is a very simple and rapid transition, a lower
value can be used and the classification process will be faster. In some cases, it may be
necessary to increase the number of states for a Cell Type in order to resolve finer, more
gradual transitions in the progression.

Identification
Cell Type Name
Enter a name for the Cell Type.
Enable
Enable or disable classification of events for this Cell Type.
Abbreviation
Enter the short name for this Cell Type.
Description
Enter the description of the Cell Type.
State Index Label
Enter the label that will be used on the X-axis (the state index axis).

Analysis
Exclusion Probability
This value determines the probability at which an event will be excluded from a Cell Type
during the classification process. The value ranges from 0.0 to 1.0. More events are excluded
as the value approaches 1.0. For example, a value of 0.1 will exclude 10% of the events from
the Cell Type. The events excluded will be the ones with the lowest probability of matching
the Cell Type.

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Inclusion Probability
This is an additional filtering option that can restrict the events that are included by a Cell
Type. It sets the minimum probability for events that are to be included in the Cell Type.
Normally it is set to 0.0. Its range is 0.0 to 1.0. As the value approaches 1.0, fewer events
are included in the Cell Type.
Number of States
Enter the number states to define for the progression of events in this Cell Type. The default
value is 100. Smaller values improve analysis speed and can be used when transitions are
distinct and dramatic. Larger values take more time to process, but may be useful for gradual
or complex transitions in the progression.
Gate On
Enable or disable the gating expression by toggling this option. See Gating and Gate
Expressions for additional details.
Gate Expression
Enter a gating expression, if desired, to apply to events in the listmode file prior to the
classification process. The gate is only applied if the Apply Gate option below is enabled. See
Gating and Gate Expressions for additional details.

Statistics
Cell Type Events
The number of events classified by this Cell Type.
Reduced Chi-Square (RCS)
The goodness-of-fit for events classified into this Cell Type. Well-defined models produce RCS
values between 0.0 and 3.0. Values between 3.0 and 5.0 are acceptable for many models.
Complex models with fine distinctions between states may produce even higher RCS values.
% State Index Fidelity

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The field shows the average probability that the classification made by the Probability State
Model is correct, expressed as a percentage. This can be useful when building a model to
determine where the model is well-defined an where it is not well defined.

Editors
Select Parameters
Click the Edit button to edit the parameters that are selected for analysis in the current Cell
Type. The Edit Parameters dialog is displayed.
Edit Zones
Click the Edit button to create and edit Zones defined for this Cell Type. The Edit Zones
dialog is displayed.

Utility Actions
Actions
Update all Updates all parameter profiles in the cell type from the parameter
parameters database.
from database
Deselect Remove the selection of all definition points.
definition points
Link selected Links the set of selected control points so that they maintain the
control points same relative positions on the state index scale. This forces the
control points to maintain the relationship during Auto Analysis and
other fitting operations.
Unlink selected Removes the linkage relationship previously established with the
control points "Link selected control points" action.
Expand Expands all parameter profiles plots in the cell type.
parameter
graphs
Shrink Shrinks all parameter profiles plots in the cell type.
parameter
graphs
Show definition Shows the definition points for all parameter profiles in the cell
points type.
Hide definition Hides the definition points for all parameter profiles in the cell type.
points
Fidelity Analysis Performs a Fidelity Analysis on the cell type. See Fidelity Analysis
for additional details.

Graphics
Show Parameter X-Axes

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Toggle this option to show or hide the state index axis for each Parameter Profile plot.

Animation Controls
Animate
Toggle animation on or off. Animation appears on Parameter Overlay plots as well as 2P
histogram plots.
Animation Speed
Set the speed of the animation in milliseconds.
Animation Pause
Set the number of milliseconds to pause at the end of the progression before the animation
returns to the beginning of the progression.
Marker Separation
Set the separation between the start and end markers for animation. The value is a
percentage of the state resolution scale.

Other
Relative Events to Synthesize
When using the Synthesize Data into Packet action in batch processing (see Batch Options
panel ), this field controls how many events of this cell type are generated into the output.
Each cell type can use a different value for this field, so that the mixture in the output file has
a known composition.
For example, with 2 cell types, if you were to set this field to 200 for Cell Type 1 and 100 for
Cell Type 2, GemStone would attempt to create a 200-to-100 mixture (or 2-to-1) for Cell
Type 1 and 2 respectively in the synthesized file.

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Edit Parameters

This dialog is displayed to select the parameters that are included in a cell type for analysis
or display.
The list on the left side of the dialog displays all of the parameters that exist in the file, as
well as other parameters that GemStone has seen in other files. The parameters in the
current file display "On" in the "Live" column.
The list on the right shows the parameters that have been selected for analysis or display.

If you want to...

Add or insert a new Select the parameter(s) in the list on the left of the
parameter dialog, and then click Add or Insert.
Remove a parameter from Select the parameter in the list on the right, and then
the cell type click Delete.
Remove all parameters in Click the Delete All button.
the cell type

See also:
Parameter Profile Properties panel

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Edit Zones

This dialog allows zones to be added, deleted, and modified. Zones, in GemStone, are
defined on the Progression (State Index) axis and provide statistics and coloring for portions
of the progression.
Zones are most often defined using Definition Points in the Parameter Profiles for a given Cell
Type. This allows the boundaries of the zone to automatically adjust as the Definition Points
are adjusted by the user or by a process in the program. However, it is also possible to
define Zones using percentage values. For example, a zone could be defined so that it
started at 50% of the progression and ended at 75% of the progression.
Each zone has a user-defined name and color. The color is used when drawing dots that fall
within the zone's boundaries and along the top of Parameter Overlay plots when zones are
displayed. If overlapping zones are defined, the color of the last zone containing an event is
used to color dots for that event.
A Zone Statistics Table object appears on each Cell Type canvas to provide statistics for the
zones. The table shows the name, color, active state, and statistics for each zone.
See Edit Properties for Zone for additional details on zone properties.

If you want to...

Add a new zone Click the New button and fill in the Edit Properties for
Zone dialog.
Delete a zone Select the zone in the list, and then click Delete.
Change the order of zones Select a zone, and then use the Up or Down button to
move it in the list of zones.
Save a set of zones Click the Save button. The standard File Save dialog will
be displayed, allowing you to navigate to a location and
name the file.
Open a set of previously Click the Open button. Navigate to the zone file on disk
saved zones and choose Open. The zones in the file on disk will
replace any zones already defined in the list.

See also:
Edit Properties for Zone
Zone Statistics Table

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Edit Properties for Zone

This dialog displays all of the properties for a single zone. Zones are defined on the
Progression (or State Index) axis and provide statistics and coloring for portions of the
progression.

Name
Type a name for the zone. Short names are usually recommended so that they can be read
when displayed on Parameter Overlay plots.
Active
Check this option to enable the zone for coloring and analysis. If this option is unchecked,
GemStone will ignore the zone; dots will not be colored with the zone and statistics will not
be computed.
Show in Graphics
When this option is checked, the zone is displayed in the Frequency plot at the bottom of the
Cell Type widget.
Color
Choose a color for the zone. The color is applied to dots in Parameter Profile plots and 2P
histograms. It is also used as a background for the zone label on Parameter Overlay plots.
Parameter Intensity Stats
When this option is enabled, GemStone computes intensity statistics within the zone for each
parameter. The statistics are available for databasing and display on reports.
% Minimum Purity
This field adjusts a probability filter for including events within the zone. By default, the value
is 0.0, and any events that fall within the zone boundaries are counted as "in" the zone. A
positive value for this field will eliminate events from the zone based on the standard
deviation of events for a given state index location. For example, if "90.0" is entered in this
field, GemStone will only include events that is determines with 90.0% certainty to fall within
the zone boundaries.
Example of Minimum Purity set to 0.0

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Example of Minimum Purity set to 90.0

Link

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Start Link
Select a Control Point to link to for the start of the zone. Each Definition Point in the list is
named based on the parameter and the position in the Parameter Profile that owns the point.
Select "None" if you want to enter a fixed value for the starting location of the zone in the
Start State Value field.
Start State Value
If the Start Link field is set to "None", enter a number between 0.0 and 100.0 to define the
starting location for the zone. The value represents a percentage of the Progression axis for
the starting location of the zone.
If a Definition Point is selected for Start Link, this field is disabled. It shows the location of
the selected point.
End Link
Select a Control Point to link to for the end of the zone. Each Definition Point in the list is
named based on the parameter and the position in the Parameter Profile that owns the point.
Select "None" if you want to enter a fixed value for the ending location of the zone in the
Start State Value field.
End State Value
If the End Link field is set to "None", enter a number between 0.0 and 100.0 to define the
ending location for the zone. The value represents a percentage of the Progression axis for
the ending location of the zone.
If a Definition Point is selected for End Link, this field is disabled. It shows the location of the
selected point.
Label
Vertical Location
This option sets the vertical position of the label in the Frequency plot at the bottom of the
Cell Type widget.
Font
This option sets the font of the label in the Frequency plot at the bottom of the Cell Type
widget.
Statistics
Percent
This read-only field displays the percentage of events in this Cell Type that fall into this zone.
Pcnt Total
This read-only field displays the percentage of all events that fall into this zone.
Events
This read-only field displays the number of events that fall within the boundaries of this zone.

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Gating and Gate Expressions

Each Cell Type can define a gate expression that can be used to filter the events that will be
considered for classification by the Cell Type's model. If gating is enabled for a Cell Type, the
events that satisfy the gate are the only ones that the Cell Type examines for possible
inclusion.
Edit and enable gating using the Cell Type Properties panel. This panel contains a "Gate"
checkbox to enable or disable gating, and a "Gate Expression" edit box to enter the gate
expression.
A gate expression is a Boolean expression that can reference any regions that have been
created. The regions do not need to be in the same Cell Type to be referenced by a gate
expression. If the expression references a region that does not exist, the gate is effectively
disabled. To avoid unexpected gating behavior, make sure that gate expressions only
reference valid regions.
Expressions can contain parentheses for grouping. Regions are referenced by the region
labels, e.g. "R1". The table below shows the valid elements for gating expressions.

Symbol Name Use

Rn Region Identifies a region, where n is the
region ID. For example, R1 identifies
the first region, R2 identifies the
second region, etc.
( Open parenthesis Begin a grouping expression
) Close parenthesis End a grouping expression
& Boolean "AND" is represented by the Events must satisfy the expressions
ampersand. on the left and right sides of the "&"
symbol to pass the gate.
| Boolean "OR" is represented by the Events can satisfy the either the
piping symbol. expression on the left or the
expression on the right of the "|"
symbol to pass the gate.
! Boolean "NOT" is represented by the This operator reverses the result of
exclamation point. the expression to its right. In other
words, of the expression to the right
evaluates to TRUE, the "!" operator
changes the result to FALSE.

Examples
R1 Select events that fall within region R1.
R1&R2 Select events that fall within both R1 and R2
R1&!R2 Select events that are in R1 but not in R2

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R1&!(R2|R3) Select events that are in R1, but not in either R2 or R3.

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Parameter Profile Properties panel

Parameter Profile

Each parameter that is added to a Cell Type for display or modeling creates a Parameter
Profile in the Cell Type widget.

If a Parameter Profile is selected for a parameter, this plot displays a set of Control Points
that are used to define the profile. Also called Control Definition Points, the Control Points
can be moved and have defined relationships with one another, depending on the selected
profile. See Parameter Profile Descriptions for a complete list of available profiles.
When you add a parameter to a Cell Type, the parameter becomes available for display in 1P
and 2P histograms, and in Overlay plots. It also appears in the Cell Type widget, where it can
be simply displayed or it can be modeled.
Properties for each Control Point can be edited by double-clicking the point in a Parameter
Profile plot.

See also:
Parameter Profile Properties panel
Parameter Profile Descriptions
Edit Control Point

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Parameter Profile Properties panel

This panel is used to define the parameter profile for a parameter.

Parameter Name
This field shows the name of the parameter associated with this parameter profile.
Parameter Abbreviation
Enter an abbreviation to use for the parameter. This is used on parameter overlay plots,
among other places in the program.
Description
Enter a description or strategy for this parameter profile. For example, a CD10 profile for a B-
cell model could be described as "Elevated in early stage, moderate in mid stage, and low in
late stage" to indicate the expected progression of CD10 for normal B-cells.
Live
If the parameter associated with this parameter profile exists in the current FCS file, this
option will be checked. If unchecked, the parameter is not found in the current FCS file.
Parameter Profile
Choose the type of profile to use for modeling this parameter. An extensive set of options is
provided. Each option defines a set of Control Points with interdependencies to constrain the
relative positions of the points. When modeling a parameter, choose the simplest option that
describes the progression for the parameter in your experiment.
Enable Profile
Check this option to include this parameter profile in the GemStone modeling process.
Lock Fit
If this option is checked, the Control Points for the parameter profile will be ignored by
Optimize Model, Auto Analysis, and other routines that attempt to adjust definition points to
fit the data. Control Points can still be moved manually by the user.
Lock Estimates
If this option is checked, the estimates for peaks and standard deviations for the parameter
will not be modified by Auto Analysis.
Lock Transform
Check this option to prevent the transformation used by this parameter from changing when
a new file is opened.
Line-spread Mode
This option is only enabled if the parameter is using a linear transformation.
Note: The control is disabled for HyperLog and log transformations. Constant SD mode is
always selected for log-like transformations.

Options

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Constant In this mode, the line spread estimation uses Coefficient of Variation (CV).
CV CV is similar to Standard Deviation (SD), but it is normalized on the
intensity. In other words, while the SD generally increases with the
intensity measurement, the CV does not.
Constant In this mode, the line spread estimation uses the Standard Deviation (SD).
SD

Variance Source
Select the method for estimating the variance for the parameter profile Control Points.
The variance provides a constraint on the intensity axis (Y-axis) of the parameter profile.
Smaller variance values constrain the model and select a more restricted set of matching
events. Larger values allow the definition points more freedom to match events. For the most
accurate modeling, the goal is to match the variance with the uncertainty of the data at each
Control Definition point.
Sequence is important in choosing a Variance Source. Always start with "Estimated Line
Spread" when creating a parameter profile. This is the best way to provide initial estimates to
the modeling system. Once the model has classified data, other options for Variance Source
can be considered.

Variance
Source
Options
Estimated Uses the Line-spread Value as the estimated variance for all Control
Line Spread Points in the parameter profile.
Observed Uses the last calculated variance of the data at each Control Point in
Line Spread the parameter profile. This variance is an average variance of definition
points surrounding each Control Point.
This option should not be selected until the model has analyzed the
data at least once.
Manual Point Allows each definition point's variance to be adjusted independently.
Entry When this option is selected, each definition point must be selected
and adjusted. This can be done one-by-one or by multiple selection of
Control Points.
Observed Uses the last calculated variance of the data at each state index in the
SDs parameter profile.
This option should not be selected until the model has analyzed the
data at least once.

Line-spread Value
When either Estimated Line Spread or Manual Point Entry is selected as the Variance Source,
this control adjusts the variance for the Control Points. The control is disabled if the Variance
Source is either Observed Line Spread or Observed SDs.

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Utility Actions
This list contains useful commands that act on the parameter profile.
Action
Hide All Hides the Control Points for the parameter profile.
Definition Points
Show All Shows the Control Points for the parameter profile.
Definition Points
Update from Takes all properties from the associated parameter in the
Parameter parameter database and updates the parameter profile with them.
Database This includes the transformation used to display the parameter.
Open… Opens a previously saved parameter profile from disk.
Save… Saves a parameter profile to disk.
Export model Exports information about all of the model's Control Points and
point values state indices to a text file. The file includes fields for state index,
mean, SD, observed mean, and observed SD.
Edit Parameter Select this option to display the Properties for Linear
Transformation Transformation dialog, which allows you to edit the transformation
used by the parameter.
Update If the parameter is using the HyperLog transformation, this
Parameter command will re-evaluate the B-coefficient and the negative
Transformation percentage based on the current data. Use this command after
opening a new data file that has different population distributions.
The command has no effect on linear and log transformations.
Distribute [TODO]
Control Points…
Level Control [TODO]
Points…

Custom Properties
Cell Tracking Intensity Shift
For Cell Tracking parameter profiles, this option defines the magnitude of the intensity shift
from one generation to another. The value is normalized to a 0 to 100 scale, so a value of 10
means that the intensity drops 10% from one generation to the next.
Estimation Range
This value restricts the estimation routine that adjusts Control Points on the parameter
intensity (Y-axis) scale. The value is normalized to a 0 to 100 scale, so a value of 10 means
that the estimation routine will consider moving a Control Point as much as 10% up or down
if it finds a peak within that range.
Parameter Color
Choose a color to use when displaying this parameter in Parameter Overlay plots.

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Allow Transitions
Enable this option to force smooth, sigmoidal transitions in the modeled data surrounding the
Control Points. The option applies to Step-Up, Step-Down, Three-Level, Four-Level, Five-
Level, and the Constant Increase parameter profiles. The option requires additional
processing, so analysis will take longer with the option enabled. Also note that each Control
Point has a property that determines whether it supports transition logic or not. This allows
some portions of the parameter profile to show smooth transitions, while other show sharp
transitions.
When this option is unchecked, the transitions at Control Points are sharper and more abrupt.
The default for this option is unchecked, providing faster analysis results.
Allow Interpolation
Normally, the sloped portions of a parameter profile are straight lines. When this option is
enabled for a parameter using a log or HyperLog transform, the sloped portions of the profile
will be linearly interpolated between the control points.
Block Initialization
Enable this option to prevent the Auto Analysis system from initializing this parameter profile
fit information.
Store Definition Points
Enable this option to store the positions of the Control Points for this parameter profile in the
information system. Once stored, the positions become available to the database and tables
for reporting.

Auto Analysis
Pause and Continue
Enable this option to force Auto Analysis to pause after it adjusts the profile for this
parameter. This allows you to review the changes Auto Analysis makes and to make other
adjustments before it continues to the next parameter.
Paused Delay
Enter the number of seconds to pause.

Read-only properties
Average Obs SD
This displays the average of the observed standard deviations.
Lookup Param Name
This displays the name that was used to find the parameter in the Parameter Database.

See also:
Parameter Profile
Parameter Profile Descriptions

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Parameter Profile Descriptions

The Parameter Profiles available in GemStone provide great flexibility for modeling different
intensity characteristics for parameters in the progression being modeled.
The general rule for choosing a profile is to choose the simplest one that describes the
intensity changes you expect to find in your samples. Start with a simple profile, and then as
the model becomes well-defined and additional structure appears, a more appropriate profile
can be selected.

Constant

This profile has 2 Control Points that are linked to the same intensity. It is most often
used to select events for a subset.

Increasing

This profile has 2 Control Points. The first point is forced to have a lower intensity than
the second. It can be used on Time parameters to order events based on acquisition
time.

Decreasing

This profile has 2 Control Points. The first point is forced to have a higher
intensity than the second.

Step Up

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The Step Up profile has 4 Control Points. The first 2 points are linked to have the same
intensity values. The last 2 points are also linked, and are forced to have a higher
intensity than the first two. It is used for parameters that are up-regulated in the
progression being modeled.

Step Down

The Step Up profile has 4 Control Points. The first 2 points are linked to have the same
intensity values. The last 2 points are also linked, and are forced to have a lower
intensity than the first two. It is used for parameters that are down-regulated in the
progression being modeled.

Three, Four, Five Levels

These profiles are very flexible, used to define 3, 4, or 5 levels of intensity. For each
level, there are 2 Control Points that are linked to the same intensity for the
parameter. Each of the levels has no dependencies on the other levels. In other
words, the next level can be higher, lower, or even the same intensity as its
neighboring level.

Cell Tracking 5, 6, 7, 8

These profiles are used for modeling cells stained with cell-tracking dyes, like CFSE

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and PKH-26. With these dyes, there is a well-defined relationship of intensity from one
level to the next. When the cells divide, each daughter cell takes half of the cell-
tracking dye. Therefore, the intensity drops by 50% from one generation to the next.
When choosing between profiles for 5, 6, 7, and 8 generations, choose the one that
will accommodate the divisions in the full course of the experiment.

Increase and Level out

This profile has 3 Control Points. The first point is forced to have a lower intensity than
the others. The second and third points are forced to have the same intensity values.
It is used for parameters that up-regulate at the beginning of the progression, and
then level off.

Decrease and Level out

This profile has 3 Control Points. The first point is forced to have a higher intensity
than the others. The second and third points are forced to have the same intensity
values. It is used for parameters that down-regulate at the beginning of the
progression, and then level off.

Up Pulse

This profile has 5 Control Points that are used to model an up- and down-regulation in
the progression. The first 2 points are forced to be on the same intensity level. The
third point is forced to be higher than the others. The last two points are locked to the
same intensity level, but the level can be different than the first level.

Down Pulse

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This profile has 5 Control Points that are used to model a down- and up-regulation in
the progression. The first 2 points are forced to be on the same intensity level. The
third point is forced to be lower than the others. The last two points are locked to the
same intensity level, but the level can be different than the first level.

Constant Increase Two-fold Constant (DNA)

This profile is similar to the Step Up profile, but is specifically designed to be used with
linear parameters that have a 2-fold increase in the progression. It is most commonly
used for DNA parameters, where the G2-M intensity is twice that of the G0-G1
intensity.

Up Constant Down

This profile uses 4 Control Points to model an up-regulation, leveling off, and
ending with a down-regulation.

Custom 2 through 10 Point

These profiles are extremely flexible. There are no linkages or dependencies on the
points, so the shapes they can model are very open ended. In most cases, it is best to
choose a simpler, more constrained Parameter Profile instead of a Custom profile. In

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general, simpler profiles create more robust models.

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Edit Control Point

This dialog is displayed by double-clicking a Control Point on a Parameter Profile plot. It is

used to view and edit properties of the Control Point.

Each Control Point has 3 editable characteristics: its parameter intensity value (Param), its
state index value (State), and its standard deviation value (SD). Each of these has a value
and a Fit Status flag that indicates whether the automatic adjustment routines like Auto
Analysis can adjust the setting. Additional settings can be edited by selecting the item and
clicking the Properties button below the list. This displays the Properties for Param, State,
and SD dialog box.

This dialog also provides a convenient way to create Zones based on this Control Point. Use
the Create Zone buttons to create a zone between the current Control Point and the end of
the previous zone or to the beginning of the next zone.

See also:
Parameter Profile
Properties for Param, State, and SD
Zone Statistics Table

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Properties of Control Point

This dialog exposes properties for a Control Point. It is accessed through the Edit Control
Point dialog.

Allow To Transition
Enabling this property causes GemStone to use a sigmoidal transition between this Control
Point and the next. If this property is disabled, the transition is linear from one Control Point
to the next.
Linearize the Next Transition
Enabling this option causes GemStone to linearize the transition between this and the next
Control Point.

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Properties for Param, State, and SD

This dialog allows properties for one of the Control Point characteristics to be edited. The
same dialog is used to edit the point's parameter intensity value (Param), its state index
value (State), or its standard deviation value (SD).

Value
This displays the current value of the object being edited. If it is a Param object, it shows the
intensity (y-axis) position of the control point in the parameter. If it is a State object, it shows
the state index (x-axis) position of the control point. If the object is an SD, it shows the
current standard deviation of events in the state index where the control point is located.
Fit Status
If checked, the point's value can be adjusted by GemStone's fitting routines
Can Change
Uncheck this property to prevent the value from changing by any means.
Value Type
This field describes the type of value represented.
Average Value
This field shows the average value for events falling in the same state index. The value will
always be 0.00 when editing State.
Edit Relations
Click this button to display the Edit Relations dialog. This dialog is used to define relationships
between control points.

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Edit Relations

This dialog is used to define relationships between control points.

Note of caution: This is considered an advanced feature. Because any definition point can
have a relationship with any other point, relationship trees can be very deep and even
circular. Only create or edit relations when you have a clear understanding of the existing
relations of control points.

Relations between points are used to provide restrictions on movement of the points. The
relationships can affect a point's frequency, state index, or standard deviation. Typically, the
relationships are relative to other definition points in the same parameter profile. However,
this is not a requirement; relationships can span profiles and even cell types to coordinate
the position and size of control points.

If you want to...

Edit an exiting relation Select the relation in the list, and then click the
Properties button. This will display the Edit Properties
for Relation dialog.
Create a new relation Click the New button. When the new Relation object is
created, click the Properties button to edit the relation's
properties.
Delete a relation Select the relation in the list, and then click the Delete
button.
Save a set of relations Click the Save button. The standard File Save dialog will
be displayed, allowing you to navigate to the desired
location and save the file.
Open a set of relations Click the Open button. The standard File Open dialog is
displayed, allowing you to select the previously saved
relation file.

See also:
Edit Properties for Relation

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Edit Properties for Relation

This dialog allows editing of a relationship between one control point and another. It is
accessed from the Edit Relations dialog.

Note of caution: This is considered an advanced feature. Because any definition point can
have a relationship with any other point, relationship trees can be very deep and even
circular. Only create or edit relations when you have a clear understanding of the existing
relations of control points.

The relationship for the control point is an expression that involves the properties of this
dialog:
This point's value must be [Relation To Link] [Link Name] times [Multiplier] plus
[Offset].
For example:
This point's value must be less than CellType1:SSC:BeginUp times 1.0 plus 0.0.

Name
This is a read-only field containing the name of the relation. It is automatically assigned by
the system when the relation is created.
Enable
The relation can be enabled and disabled using this checkbox. This allows relations to remain
in the list but disabled.
Relation to Link
Choose the relationship from the list of options.
Link Name
Choose the definition point with which to define a relationship.
Multiplier
By default, this value is 1.0. If a value other than 1.0 is entered, the multiplier is applied to
the value of the link prior to making the comparison.
Offset
By default, this value is 0.0. If a value other than 0.0 is entered, the offset is added to the
value of the link after any Multiplier and prior to making the comparison.

See also:
Edit Relations

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Frequency Plot

The Frequency Plot appears at the bottom of the Cell Type widget. It provides information
about how well the model is fitting the data.

At the top of the plot, the RCS value is a measure of quality of fit. Generally, values between
1 and 7 are pretty good, and higher values indicate that the model is not properly
characterizing the data.
The number of events classified into each state of the progression is plotted as the
frequency distribution, which will typically bounce above and below the ideal
distribution shown as a straight, red line. The smoothed frequency distribution is
shows an averaged distribution.
The goal is for the model to evenly distribute events in all states along the progression axis.
Therefore, if the model is fitting properly, the frequency distribution will closely follow the
ideal distribution horizontally. In case where the model does not match as well, the frequency
distributions will show areas where they differ from the ideal significantly.

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Also shown on the Frequency plot are zone boundaries for all active zones in the cell type.

See also:
Properties of Frequency Plot

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Properties of Frequency Plot

This dialog is displayed to edit the graphics for the Frequency Plot.

Frequency
Show
Enable this option to display the frequency line.
Line width
Choose a line width.
Line Style
Choose a line style.

Smooth Frequency
Show
Enable this option to display the smooth frequency line.
Line width
Choose a line width.
Line Style
Choose a line style.

Average Frequency
Show
Show
Enable this option to display the average frequency line.
Line width
Choose a line width.
Line Style
Choose a line style.

State Index Fidelity

Show
Enable this option to display the state index fidelity.
Line width
Choose a line width.
Line Style
Choose a line style.

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Scale Factor
Select a scaling factor for the fidelity line.
Fidelity Color
Select a color for the fidelity line.

State Index Uniqueness

Show
Enable this option to display the uniqueness plot.
Model View
Enable this option to use the model's values for the line. Uncheck to use the data values.
Uniqueness Line Width
Choose a line width.
Uniqueness Line Style
Choose a line style.
Uniqueness Scale Factor
Select a scaling factor for the uniqueness line.
Uniqueness Color
Select a color for the uniqueness line.

Edit Zone
Show
Enable this option to display zone boundaries on the plot.
Edit Zones
Click this button to edit the zones.

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Graphic Objects
Cell Type Canvas

The Cell Type canvas is the workspace that contains GemStone graphical objects, like the Cell
Type Widget, 1P and 2P histograms, and the Zone Statistics table. Objects can be moved and
resized on the canvas, and reports are generated from the objects that reside on it.

See also:
Cell Type Canvas Context Menu

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Cell Type Canvas Context Menu

This menu is displayed when the mouse is right-clicked on the Cell Type Canvas.

Edit Canvas Properties

This command displays a dialog with properties of the canvas. Use the dialog to control grids,
conform mode, zooming, and how the object is copied and saved to disk. See Edit Canvas
Properties Dialog for details.
Edit Background Page Properties
This command displays a dialog with properties of the background page. You can set the
page size, margins, and a number of other background page properties. See Edit Background
Page Properties Dialog for details.
Copy Page to Clipboard
This command copies the entire canvas to the clipboard as a bitmap. Use Edit Canvas
Properties to determine the size and resolution of the bitmap.
Paste
This command pastes an object from the clipboard onto the canvas.
Save Page to Disk
This command pastes saves the canvas to disk in the format specified in Edit Canvas
Properties.
Copy Selected Item to Clipboard
This command copies the selected object on the canvas to the clipboard as a bitmap. Use
Edit Canvas Properties to determine the size and resolution of the bitmap.
Save Selected Item in Standard Format
This command saves the selected object on the canvas to disk in the format specified by Edit
Canvas Properties.
Print Selected Item
This command prints the selected object on the canvas to the printer.
Print
This command prints the entire canvas to the printer.
Save Image Flag
If this option is enabled, the canvas will be saved to disk automatically during batch
processing.

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Edit Canvas Properties Dialog

This dialog edits the properties of the canvas. Use the dialog to control grids, conform mode,
zooming, and how the object is copied and saved to disk.

Grid Snap
When enabled, objects on the canvas move and size based on the grid. This makes it easier
to align objects.
Grid Size
This property determines the size of each grid element, in pixels. It is only used when Grid
Snap is enabled.
Angle Snap
When enabled, objects rotate on fixed increments to assist in alignment.
Angle Snap Size
This property determines the number of degrees of rotation when Angle Snap is enabled.
Zoom Percent
This command adjusts the zooming of the canvas. Choose a size from the list, or enter a
custom zoom value by typing.
Conform Mode
When enabled, some objects on the canvas will conform to the edges of other objects when
they are moved. This is most useful for regions on 2P plots, where it is desirable to have
adjacent analysis areas.
Bitmap Operations
Copy DPI
Type the number of dots per inch (DPI) to use when copying and saving graphic objects.
Higher values produce finer graphics, but also require more memory. The default value is
300.
X and Y Screen DPI
These fields show the DPI for the screen. They are not editable.
Saving Format
Choose a graphic file format to use when saving graphics to disk. All supported formats
appear in the list.
Saved Image Path
Choose a location on disk to use for saving graphics on this canvas.
Save Image
Enable this property to flag this object to be saved to disk during batch processing.

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Edit Background Page Properties Dialog

This dialog edits the properties of the background page. You can set the page size, margins,
and a number of other background page properties.

Measurement Units
Choose English or Metric units.
Percent Zoom
This field displays the current zoom of the canvas.
Page Size
Page Width and Height
Set the overall width and height of the page. These settings do not have to match the paper
size for your printer. The program will scale the page size to the actual paper size when you
print.
Margins
Show Mat
Enable this option to see the mat area surrounding the page. This is useful if you want to
drag some objects off the page so that they are not printed.
Mat Left, Right, Top and Bottom Margin
Set the margins to use for the mat. These values are added to the page size to determine the
actual working area on the screen.
Colors
Page Color
Select a color for the page.
Mat Color
Select a color for the mat.
Shadow Color
Select a color for the shadow.

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Cell Type Widget

The Cell Type widget contains the set of Parameter Profile plots that represent one Cell Type
in the model. In addition, a Frequency Plot appears at the bottom of the widget to provide
information about how well the cell type is being modeled.

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You can expand and collapse parameter profiles within the widget for better visualization of
the data. To collapse a profile that is showing, click the "shrink" icon next to the parameter
name on the left edge. To expand a profile that is hidden, click the "expand" icon next to the
parameter name.
Click to
Shrink

Click to
Expand

When you click on a parameter profile within the collection of profiles, the profile becomes
the active profile and its properties appear in the Parameter Profile Properties panel. See
Parameter Profile and Parameter Profile Properties panel for more about parameter profiles.

After you click on a parameter profile in the Cell Type Widget, you can use the PgUp and
PgDn keys on the keyboard to move to the next or previous parameter profile. This method
automatically opens the next (or previous) profile and activates it.

The Frequency Plot provides valuable information for understanding how well the model for
this cell type is working. The Y-axis shows frequency of events, and the X-axis shows the
Progression axis (normally 100 states). A bold red horizontal line shows the optimal
frequency across the progression axis. The optimal frequency is computed by dividing the
number of events by the number of states on the Progression axis. In other words, the ideal
model would put the same number of events into each state of the progression.
The jagged black line shows the actual number of events that have been assigned to each
state for this cell type. For a model that matches the data well, the black line will follow the
red line, bouncing slightly over and under the red line. In places where the black line is
significantly above the red line, there are too many events being assigned to those states.
Conversely, where the black line falls well below the red line, there are not enough events
assigned. This relationship helps identify which Parameter Profiles need to be adjusted. In
general, when there are too many or too few events in a portion of the Frequency Plot, look
for Parameter Profiles with control points and transitions in those states. Examine those
profiles to be sure the control points are properly positioned and the other characteristics of
the Parameter Profile are appropriate for the model.
The Frequency Plot also shows the Reduced Chi Square (RCS) of the model and the zones
that have been defined.

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Properties of Cell Type Widget

This dialog is displayed when the Cell Type Widget is double-clicked. It displays a number of
properties for the object that can be edited.

Save Image
If this property is enabled, the Cell Type Widget will be saved to disk as an image file when
any Save Graphics command is triggered. Save Graphics is an option in batch processing, and
is also available in the Workspace toolbar.
Position and Size
X Position, Y Position, X Size, Y Size
These fields show the position and size of the object, in inches. The values can be directly
edited. They are also updated when the object is moved or resized using the mouse.
Other Properties
Background
Select a background style for the Cell Type widget.
Title Font
Select a title font for the Cell Type widget

See also:
Cell Type Widget

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Graphic Options for Parameter Profile: Surface

This tab is part of the Edit Graphic Options for Parameter Profiles. It configures the display of
the Surface plot.

Enable Surface
Check this option to display the surface graphics.
Probability Mixing Color
Choose a color that will be used to for the densest portions of the surface plot (the highest
probability). The default color is white.

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Graphic Options for Parameter Profile: Dots

This tab is part of the Edit Graphic Options for Parameter Profiles. It configures the display of
dots.

Enable Dots
Check this option to display dots on the plot. (The 2P histogram can display other kinds of
graphics, which are selected on other tabs in this dialog.)
Auto Dot Size
Enable this option to allow GemStone to determine the best dot size, based on the current
size of the histogram object and other factors.
Dot Size
This field displays the current size of dots in points; a point is 1/72 inches. To set a specific
size for the dots, uncheck the Auto Dot Size option and enter a value between 0.0 and 5.0
for Dot Size.
Show All Dots
Enable this option to display dots for all events. If this option is unchecked, the system will
use the value in Desired Dot Number.
Desired Dot Number
If Show All Dots is unchecked, the system will use the value in Desired Dot Number to
determine the number of dots to display in the plot.
Probability of Inclusion
This setting determines how dots are display when showing Surface plots and Dots
simultaneously. With the default setting of 0.05, only the 5% outlier dots will be drawn. This
setting allows the Surface plot to highlight the dense portions of the plot.

Other Dots
Show Other Types
This option allows dots from one or all other cell types to be display in the plot.
Show Unclassified Events
Enable this option to display dots for unclassified events.

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Graphic Options for Parameter Profile: Means

This tab is part of the Edit Graphic Options for Parameter Profiles. It configures the display of
means of the classified events.

Enable Means
Check this option to display a line plotting the mean of classified events in the Parameter
Profile.
Line Width
This option controls the thickness of the line used to plot the mean. Enter a value from 0 to
10 inclusive.
Line Style
Choose a line style for the mean line.

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Graphic Options for Parameter Profile: Confidence Limits

This tab is part of the Edit Graphic Options for Parameter Profiles. It configures the display of
confidence limits for the classified events.

Enable
Check this option to display confidence limit lines on the Parameter Profile plot.
Line Spread Mode
Choose a method for visualizing line spread.

Line Spread Mode

Options
Model SD Bars Displays the standard deviation bars at each Control Point.

Model SD Envelope Displays the standard deviation of the model as an envelope.

Observed SD Similar to Model SD Envelope, except that the standard

Envelope deviation of the actual data values is used.

SD Bars and Displays the Model SD bars and the Observed Envelope.
Observed Envelope

Number of SDs
This option controls the number of standard deviations (SDs) used for the display. The
default value is 1.96, equivalent to 95% confidence limits.
Line Width
This option controls the thickness of the line. Enter a value from 0 to 10 inclusive.
Line Style
Choose a line style for the line.
Color
Choose a color for the line.

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Graphic Options for Parameter Profile: Model

This tab is part of the Edit Graphic Options for Parameter Profiles. It configures the display of
a line that connects each Control Point in the selected Parameter Profile.
Enable Model
Check this option to display the model line on the Parameter Profile plot.
Line Width
This option controls the thickness of the line. Enter a value from 0 to 10 inclusive.
Line Style
Choose a line style for the line.

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Graphic Options for Parameter Profile: Animate

This tab is part of the Edit Graphic Options for Parameter Profiles. It configures the animation
options for the Parameter Profile.
The Parameter Profile Plot can display additional information when animation is enabled in
the Cell Type Properties panel. When Cell Type animation is enabled, the plot will display two
vertical cursors that move along the progression axis.

Enable Animation
Check this option to enable animation for the Parameter Profile plot.
Line Width
This option controls the thickness of the line. Enter a value from 0 to 10 inclusive.
Line Style
Choose a line style for the line.
Color
Choose a color for the line.
Animation Data Source
When Animation is enabled, it can position its cursors based on either Mean Data or Model
Data. Mean Data uses the mean for the classified events at each state index. Model Data
uses the model’s position based on Control Points at each state index.

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Other Graphic Options

This set of properties appears on the "Other Options" tab in the dialog used for editing a 1P
or 2P histogram. You can view and edit these properties by double-clicking a histogram or by
right-clicking a histogram and choosing "Edit Graphics".
Select the "Other Options" tab to view these properties.

Background Color
Choose a color for the background of the plot.
Show X Grid Lines
Enable this option to show grids for the X-axis. Grid lines will appear for each major tic on
the axis.
Show Y Grid Lines
Enable this option to show grids for the Y-axis. Grid lines will appear for each major tic on
the axis.
Grid Line Width
Enter a value for the width of the grid lines. The default, 0, creates the narrowest possible
line that the screen (or printer) can draw.
Grid Line Style
Choose a line style for grid lines.
Grid Color
Choose a color for grid lines.
Grids On Top
Enable this option to draw grids on top of the other graphics in the plot. When the option is
unchecked, grids are drawn behind the other graphics.

Background Image
A plot can display a background image, on top of which the other graphics are displayed.
This can be useful, for example, to present a comparison of the current analysis with a
previous analysis that was saved as an image to disk.
Image Transparency
Enter a value between 0.0 and 100.0 to define how transparent a background image
appears. Higher values produce more transparency.
Open Image
Click this button to select an image file from disk. This file will be opened and displayed as
the background image for the plot.
Clear Image
If an image has been opened using the Open Image option, the Clear Image option removes
the images from memory.

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See Also:
1P Histogram
2P Histogram

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Parameter Overlay Plot

The Parameter Overlay Plot displays the modeled progression for all parameters
simultaneously.

The cell type, file name, and percentage of events in the cell type are displayed in the area at
the top of the plot. Each cell type can have its own Parameter Overlay plots, but it can also
include parameters from other cell types, too.
The checkbox at the top left of the plot is the "save graphic" checkbox. Check this box if you
want the graphics from the overlay plot to be saved when you click the Save Graphics button
on the Workspace toolbar.
The X-axis of the plot is the Progression axis, or "state index" axis. The Y-axis is a relative
intensity scale from 0 to 100, which allows linear, log, and HyperLog transformed parameters
to be compared on the same plot.
If zones have been defined, they are displayed along the top edge of the ribbon display area.
Each parameter appears as a "ribbon" in the plot, with text labels identifying the parameter
name. The color for each ribbon is determined by the Parameter Color property in the
Parameter Profile Properties panel.

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Tools to manipulate the plot appear on the right edge of the plot window. See Overlay
Graphics toolbar for a description of each tool.

If you want to...

Create a new Parameter Click the Overlay Plot button in the Workspace toolbar,
Overlap Plot and then click and drag to define the area where the
plot should be displayed.
Edit the properties of the Double-click within the ribbon display area, or
Parameter Overlay Plot right-click and choose Edit Graphics.
Delete the Parameter Overlay Select the plot and press the Delete key.
Plot
Create a free-floating window Click the Create Dialog button in the Overlay Graphics
with the Parameter Overlay toolbar.
plot

See also:
Graphic Options for Parameter Overlay Plots
Other Options for Parameter Overlays
Workspace toolbar
Overlay Graphics toolbar

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Graphic Options for Parameter Overlay Plots

Each parameter in a Parameter Overlay Plot has a set of graphic options that determine how
it appears. You can view and edit these properties by double-clicking a Parameter Overlay
plot, or right-clicking a Parameter Overlay plot and choosing "Edit Graphics".

Show Parameter
Enable this option to include the parameter in the Overlay Plot. This does not affect whether
or not the parameter is included in the modeling process.
Line Style
Select a style for the line that borders the parameter ribbon in the Overlay Plot.
Brush Style
Select a style for the fill used for drawing the parameter ribbon in the Overlay Plot.
OK to Smooth
Enable this option to apply smoothing to graphics. Uncheck this option to display the
unsmoothed values from the model.
Show All Cell Types
With this option enabled, this overlay plot will display one ribbon for the selected parameter
for each of the cell types that have been defined. To assist with identification, the cell type
abbreviation is used as a prefix for the ribbon labels. For example, if you wanted to view a
"CD10" parameter ribbon for two cell types, "Kappa" and "Lambda", you would enable this
option. One ribbon would be labeled "Kappa-CD10" and the other "Lambda-CD10".
Parameter Color
This read-only property shows the color used for this parameter. To edit the color, use the
"Parameter Color" property in the Parameter Profile Properties panel.

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Other Options for Parameter Overlays

This set of properties appears on the "Other Options" tab in the dialog used for editing a
Parameter Overlay Plot. You can view and edit these properties by double-clicking a
Parameter Overlay plot, or right-clicking a Parameter Overlay plot and choosing "Edit
Graphics".
Select the "Other Options" tab to view these properties.

Background Color
Choose a color for the background of the Parameter Overlay plot.
Show X Grid Lines
Enable this option to show grids for the X-axis. Grid lines will appear for each major tic on
the axis.
Show Y Grid Lines
Enable this option to show grids for the Y-axis. Grid lines will appear for each major tic on
the axis.
Grid Line Width
Enter a value for the width of the grid lines. The default, 0, creates the narrowest possible
line that the screen (or printer) can draw.
Grid Line Style
Choose a line style for grid lines.
Grid Color
Choose a color for grid lines.
Grids On Top
Enable this option to draw grids on top of the parameter profile ribbons. When the option is
unchecked, grids are drawn behind the ribbons.

Background Image
An overlay plot can display a background image, on top of which the parameter ribbons are
displayed. This can be useful, for example, to present a comparison of the current analysis
with a previous analysis that was saved as an image to disk.
Image Transparency
Enter a value between 0.0 and 100.0 to define how transparent a background image
appears. Higher values produce more transparency.
Open Image
Click this button to select an image file from disk. This file will be opened and displayed as
the background image for the Overlay Plot.
Clear Image

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If an image has been opened using the Open Image option, the Clear Image option removes
the images from memory.

Labeling
Type of Labeling
Choose an option for labeling the parameter ribbons on the overlay plot.
Option Description
Wire Labels follow the ribbon.
Labeling
Zone One label appears for each ribbon
Labeling in each zone.
Number of SDs
This value controls the width of the parameter profile ribbons in the overlay plot. The value
represents the number of standard deviations from center of the ribbon (either the mean of
classified events or the value from the model). The default value is 0.20 SDs.
Label Font
Choose a font to use for the labels on the parameter profile ribbons.
Show Abbreviation
Enable this option to use the abbreviation for the parameter labels on the ribbons. If the
option is not enabled, the full parameter names are used.

Additional Properties
Data Type
Option Description
Model The parameter ribbon is based on the model's Y-axis position at each state
+/- SD in the progression. This value approximately matches the Control Point
locations for the Parameter Profile of each parameter, with interpolation
between control points.
The width of the ribbon is controlled by the Number of SDs property.
Mean The parameter ribbon is based on the mean intensity of the data at each
+/- SD state in the progression.
The width of the ribbon is controlled by the Number of SDs property.
Transparency
Enter a value between 0.0 and 100.0 to control the transparency of the parameter profile
ribbons. Higher values make the ribbons more transparent.
Smooth Data
If "Mean +/- SD" is selected for Data Type, this option will smooth the data displayed in the
overlay plot. The option has no effect if "Model +/- SD" is selected for Data Type.
Smooth Label

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For wire labeling, smooths the angles for the labels.

Drop Shadow
Enable this option to display a drop shadow under each parameter profile ribbon in the
overlay plot.
Show Zones
Enable this option to display zones at the top of the overlay plot.
Show Zone Boundaries
Enable this option to display "grid lines" for the zone boundaries. Note, "Show Zones" must
be enabled for this option to function.

Animation
The Overlay Plot can animate the process of drawing the parameter profile ribbons. The
settings below control that animation.
Seconds
This option controls the number of seconds each parameter profile ribbon is displayed when
animation is enabled in the overlay plot. Animation is turned on and off using tools in the
Overlay Graphics toolbar.
One at a Time
With this option enabled, each parameter ribbon is displayed by itself during animation. If
this option is unchecked, each ribbon remains on the plot until all ribbons have been drawn.
Then the plot is cleared and drawing starts again.

Cell Type Animation

The Overlay Plot can display additional information when animation is enabled in the Cell
Type Properties panel. When Cell Type animation is enabled, the overlay will display two
vertical cursors that move along the progression axis.
Show Animation
Enable this option to display a pair of moving cursors when the cell type is animating. The
cursors define a window on the progression, and many graphical objects make use of these
cursors to show animation for the cell type.
Line Width
Choose a width for the animation cursors.
Line Style
Choose a line style for the animation cursors.
Color
Choose a line color for the animation cursors.

Smoothing

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If the Smooth Data option is enabled, these properties define characteristics of the
smoothing algorithm.
Number of Points
This value controls the degree of smoothing by determining how far to the left and the right
of a given state to include in the smoothing routine. Larger values result in greater
smoothing. The default value is 6.
Polynomial Number
This is the order of the polynomial to use for smoothing. The default is 1, and the range is 1
to 4.

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1P Histogram

The 1P Histogram plot displays a histogram for a selected parameter. Any number of 1P
Histograms can be created.

The name of the cell type is displayed in the area at the top of the plot. Each cell type can
have any number of 1P histograms, but it can also include parameters from other cell types,
too.
The checkbox at the top left of the plot is the "save graphic" checkbox. Check this box if you
want the graphics from the histogram to be saved when you click the Save Graphics button
on the Workspace toolbar.
The X-axis of the plot shows the selected parameter in the transformation selected. The Y-
axis is frequency of events. Click the X-axis label to select a different parameter; a menu of
available parameters will be displayed.
Tools to manipulate the plot appear on the right edge of the plot window. See 1P Graphics
toolbar for a description of each tool.

If you want to...

Create a new 1P Histogram Click the 1P Histogram button in the Workspace toolbar,
and then click and drag to define the area where the
histogram should be displayed.
Edit the properties of the 1P Double-click within the histogram display area, or
Histogram right-click and choose Edit Graphics.

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Delete the 1P Histogram Select the plot and press the Delete key.
Create a free-floating window Click the Create Dialog button in the 1P Graphics
with the 1P Histogram plot toolbar.

See also:
Graphic Options for 1P Histograms
1P Animation Options
Other Graphic Options
Workspace toolbar
1P Graphics toolbar

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Graphic Options for 1P Histograms

This set of properties appears on the "Histograms" tab in the dialog used for editing a 1P
Histogram. You can view and edit these properties by double-clicking a histogram or by right-
clicking a histogram and choosing "Edit Graphics".

Graphics
Resolution
Choose a resolution for the histogram. The default value is 100. Lower values produce
smoother histograms with less detail; higher values reveal more detail in the distribution.
Fill Style
Choose the fill style to use for the histogram.
Transparency
Choose a transparency value between 0.0 and 100.0. A value of 0.0 is opaque; a value of
100 is completely transparent. The transparency setting is most useful when Zones have
been defined and the histogram is displaying a set of overlapping sub-populations.

Cell Types
Show Unclassified Events
Enable this option to display a polygon for the unclassified events, along with polygon(s) for
classified events.
Show All Cell Types
When this option is enabled, additional polygons are displayed representing the selected
parameter in each of the other cell types.

Peaks
Show Peaks
When this option is enabled, a small triangle is displayed on the histogram for each of the
statistically significant peaks. These peaks are used by several of the automatic positioning
routines for Control Points
Peak P-value
This value is a filter used by the peak-finding routine to decide whether or not a peak is real.
The p-value is the probability that the peaks represent statistical noise. The default value of
0.0010 means that there is a 1 out of 1000 change that the peak is due to statistical noise.
Higher values allow more peaks to pass the filter (i.e. it is more likely that the peaks are due
to statistical noise).

See also:
1P Histogram
1P Animation Options

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Other Graphic Options

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1P Animation Options

This set of properties is displayed on the Animation tab of the 1P Histogram Edit Graphics
dialog. It contains properties that relate to animation features of GemStone.

Enable
When this option is enabled and the Animate property is enabled in the Cell Type Properties
panel, this 1P histogram will display animation cursors.
Animation Data Source
When Animation is enabled, it can position its cursors based on either Mean Data or Model
Data. Mean Data uses the mean for the classified events at each state index. Model Data
uses the model position based on Control Points at each state index.
Animation Color
Choose a color for the animation cursors.
Line Width
Choose a line width for the animation cursors.

See also:
1P Histogram
Graphic Options for 1P Histograms
Other Graphic Options

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Other Graphic Options

This set of properties appears on the "Other Options" tab in the dialog used for editing a 1P
or 2P histogram. You can view and edit these properties by double-clicking a histogram or by
right-clicking a histogram and choosing "Edit Graphics".
Select the "Other Options" tab to view these properties.

Background Color
Choose a color for the background of the plot.
Show X Grid Lines
Enable this option to show grids for the X-axis. Grid lines will appear for each major tic on
the axis.
Show Y Grid Lines
Enable this option to show grids for the Y-axis. Grid lines will appear for each major tic on
the axis.
Grid Line Width
Enter a value for the width of the grid lines. The default, 0, creates the narrowest possible
line that the screen (or printer) can draw.
Grid Line Style
Choose a line style for grid lines.
Grid Color
Choose a color for grid lines.
Grids On Top
Enable this option to draw grids on top of the other graphics in the plot. When the option is
unchecked, grids are drawn behind the other graphics.

Background Image
A plot can display a background image, on top of which the other graphics are displayed.
This can be useful, for example, to present a comparison of the current analysis with a
previous analysis that was saved as an image to disk.
Image Transparency
Enter a value between 0.0 and 100.0 to define how transparent a background image
appears. Higher values produce more transparency.
Open Image
Click this button to select an image file from disk. This file will be opened and displayed as
the background image for the plot.
Clear Image
If an image has been opened using the Open Image option, the Clear Image option removes
the images from memory.

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See Also:
1P Histogram
2P Histogram

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2P Histograms

The 2P Histogram plot displays a correlated plot for two parameters. Any number of 2P
Histograms can be created.

The name of the cell type is displayed in the area at the top of the plot. Each cell type can
have any number of 2P histograms, but it can also include parameters from other cell types,
too.
The checkbox at the top left of the plot is the "save graphic" checkbox. Check this box if you
want the graphics from the histogram to be saved when you click the Save Graphics button
on the Workspace toolbar.
The X-axis of the plot shows intensity of a selected parameter in the transformation selected.
Similarly, the Y-axis shows the intensity of another parameter. Click an axis label to select a
different parameter; a menu of available parameters will be displayed.
Tools to manipulate the plot appear on the right edge of the plot window. These include
region drawing tools, which can be used to create gates and to color events. See 2P Graphics
toolbar for a description of each tool.

If you want to...

Create a new 2P Histogram Click the 2P Histogram button in the Workspace toolbar,
and then click and drag to define the area where the
histogram should be displayed.
Edit the properties of the 2P Double-click within the histogram display area or

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Histogram right-click and choose Edit Graphics.

Delete the 2P Histogram Select the plot and press the Delete key.
Create a free-floating window Click the Create Dialog button in the 2P Graphics
with the 2P Histogram plot toolbar.

See also:
Graphic Options for 2P Dots
Animation for 2P Histograms
Other Graphic Options
Workspace toolbar
2P Graphics toolbar

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Properties for 2P Surface

This set of properties appears on the Surface tab in the dialog used for editing a 2P
Histogram. You can view and edit these properties by double-clicking a histogram or by right-
clicking a histogram and choosing Edit Graphics.
Surface graphics offer a smooth, color transition that helps visualize the density of events,
similar to color density or color gradient plots.

Enable Surface
Check to enable surface graphics.
Probability Mixing Color
Choose the color to mix with the zone color to show cumulative probability.

Actions
OK
Click this button to accept the changes and close the dialog box.
Cancel
Click this button to discard any changes and close the dialog box.
Reset
Click this button to reset the object's properties to default values.

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Graphic Options for 2P Dots

This set of properties appears on the "Dots" tab in the dialog used for editing a 2P
Histogram. You can view and edit these properties by double-clicking a histogram or by right-
clicking a histogram and choosing "Edit Graphics".

Enable Dots
Check this option to display dots and other graphics from this tab. (The 2P histogram can
display other kinds of graphics, which are selected on other tabs in this dialog.)
Auto Dot Size
Enable this option to allow GemStone to determine the best dot size, based on the current
size of the histogram object and other factors.
Dot Size
This field displays the current size of dots in points; a point is 1/72 inches. To set a specific
size for the dots, uncheck the Auto Dot Size option and enter a value between 0.0 and 5.0
for Dot Size.
Show All Dots
Enable this option to display dots for all events. If this option is unchecked, the system will
use the value in Desired Dot Number.
Desired Dot Number
If Show All Dots is unchecked, the system will use the value in Desired Dot Number to
determine the number of dots to display in the plot.
Probability of Inclusion
This value controls how dots are included for display in the plot. Enter 1.0 to include all dots.
Enter 0.05 to include only the outermost 5% of dots. This is most useful when plotting
Surface plots along with dots.

Other Dots
Show Other Types
This option allows dots from one or all other cell types to be display in the plot.
Show Unclassified Events
Enable this option to display dots for unclassified events.

Types of Dots
Show Parameter Dots
Enable this option to display dots based on parameter event values as a traditional dot plot.
Show Model Values
When this option is enabled, a rectangular region is displayed for each state index. The
position of each rectangle is based on the model's value for the parameters at each state

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index. The size of each rectangle is based on the Number of SDs option and the standard
deviation of the parameter in the model at that state index.

Show Mean Values

When this option is enabled, an elliptical region is displayed for each state index. The position
of each ellipse is based on the mean value for the parameters at each state index. The size
of each ellipse is based on the Number of SDs option and the standard deviation of the
parameter in the model at that state index.

Show Calculated Dots

This option works in conjunction with the Show Parameter Dots option. When both options
are enabled, dots for classified events are displayed using the calculated mean and standard
deviation from the model to display a Gaussian-like distribution.
The option is useful for comparing modeled vs. actual event data.

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Dots showing actual data values Calculated Dots

Number of SDs
Enter the number of Standard Deviations to use for several of the plotting options (Show
Model Values, Show Mean Values, etc.) The default value is 1.0.

See also:
Graphic Options for 2P Vectors
Animation for 2P Histograms
Other Graphic Options
Workspace toolbar
2P Graphics toolbar

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Graphic Options for 2P State Vectors

This set of properties appears on the "State Vectors" tab in the dialog used for editing a 2P
Histogram. You can view and edit these properties by double-clicking a histogram or by right-
clicking a histogram and choosing "Edit Graphics".
State Vector arrows allow you to track the progression information in the context of two,
correlated parameters. Each arrow represents the same number of states in the progression,
so the length of arrows give an impression of the relative speed of transitions. Longer arrows
indicate more rapid state transitions; shorter arrows suggest that there is not much change
occurring.

Enable
Check this option to display arrows showing the state progression in the 2P histogram.
Number
Enter the number of state vectors to display.
Arrow Data Source
Choose "Mean Data" to position the state vectors based on the mean of the classified events.
Choose "Model Data" to position the vectors based on the model positions.
Perimeter Color
Choose the color for the border of the vectors.
Line Width
Enter the line width to use for the perimeter of the arrow.
Head Height
Enter the height of the arrow head.
Width
Enter the width of the arrow.
Head Proportion
Enter the proportion of the arrow head height to head width. Values greater than 1.0
produce elongated arrow heads.
Angle Indent
This value controls the angle for the base of the arrow head. Enter 0.0 for a square base.

See also:
Graphic Options for 2P Dots
Animation for 2P Histograms
Other Graphic Options
Workspace toolbar
2P Graphics toolbar

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Animation for 2P Histograms

This set of properties appears on the Animation tab in the dialog used for editing 2P
Histograms. You can view and edit these properties by double-clicking a histogram or by
right-clicking a histogram and choosing "Edit Graphics".
When Animation is enabled for the cell type and for this 2P histogram, the State Vector
arrows will also animate.

Enable
Check this option to enable animation for this 2P histogram. When this option is enabled and
the Animate property is enabled in the Cell Type Properties panel, this 2P histogram will
display animation vectors.
Animation Data Source
Choose "Mean Data" to position the state vectors based on the mean of the classified events.
Choose "Model Data" to position the vectors based on the model positions.
Animation Color
Choose the fill color for the vectors.
Perimeter Color
Choose the color for the border of the vectors.
Line Width
Enter the line width to use for the perimeter of the arrow.
Head Height
Enter the height of the arrow head.
Width
Enter the width of the arrow.
Head Proportion
Enter the proportion of the arrow head height to head width. Values greater than 1.0
produce elongated arrow heads.
Angle Indent
This value controls the angle for the base of the arrow head. Enter 0.0 for a square base.

Region/Gate Animations
Dot Size

See also:
Graphic Options for 2P Dots
Graphic Options for 2P Vectors

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Other Graphic Options

Workspace toolbar
2P Graphics toolbar

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Region Label Properties

The properties of a region label can be edited by double-clicking the region label.

Name and Alias

Show Region Name
Show/hide region name
Region Name (Read only)
Name of the region
Show Region Alias
Show/hide region alias
Region Alias
Alias for the region

Statistics
Show Region Counts
Show/hide region counts in the region label
Show Region Percent
Show/hide region percent in the region label
Show Region Percent Total
Show/hide region percent total in the region label
Decimal Digits
Enter the number of decimal digits to display. (0 to 6)

Color and Font

Text Color
Color of the label's text
Label Font
Font of the label's text

Background Properties
Fill color
Color of the fill
Fill style
Style of the fill
Transparency

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Amount of transparency (0-100%) (0 to 100)

Pen color
Color of the outline
Pen style
Style of the outline
Pen width
Width of the outline in pixels (0 to ...)
Actions
OK
Click this button to accept the changes and close the dialog box.
Cancel
Click this button to discard any changes and close the dialog box.
Reset
Click this button to reset the object's properties to default values.

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Heat Map

A Heat Map object can be created on the canvas to display a plot of how well events fit the
model. The Heat Map tool in the Workspace toolbar is used to create the graph.

Save Image Flag

Save Image
Check to flag this object for saving its image.
Position and Size
X Position
Enter the x position in inches. (0.00 to 100.00)
Y Position
Enter the y position in inches. (0.00 to 100.00)
X Size
Enter the x size in inches. (0.25 to 100.00)
Y Size
Enter the y size in inches. (0.25 to 100.00)
Other Properties
Background
Choose the wrapper or background for the object.
Title Font
Font of the title

Actions
OK
Click this button to accept the changes and close the dialog box.
Cancel
Click this button to discard any changes and close the dialog box.
Reset
Click this button to reset the object's properties to default values.

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Edit Graphics for Heat Map: Surface

This set of properties appears on the Surface tab in the dialog used for editing a model state
graph. You can view and edit these properties by double-clicking a histogram or by right-
clicking a histogram and choosing Edit Graphics.

Enable Model Surface

Check to enable surface graphics for the model.
Surface Color
Choose the color that represents abnormal populations.
Base Color
Choose the color that represents very few abnormal populations.
Sensitivity Threshold
Lower values has higher sensitivity and false positives. (0.01 to 1.00)
Smoothing SD
Controls the smoothing of the model difference distribution. (0.00 to 5.00)
Actions
OK
Click this button to accept the changes and close the dialog box.
Cancel
Click this button to discard any changes and close the dialog box.
Reset
Click this button to reset the object's properties to default values.

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Edit Graphics for Heat Map: Dots

This tab is part of the Edit Graphic Options for Model Graph. It configures the display of dots.

Enable Dots
Check to enable or disable showing dots.
Auto Dot Size
Check to have the system try to find the best size of the dots.
Dot Size
Choose the appropriate dot size. (0.00 to 5.00)
Show All Dots
Check to show all the dots in the listmode buffer.
Desired Dot Number
Enter the specific number of dots to display. (0 to ...)
Probability of inclusion
Enter 1.0 for all dots, 0.05 for outermost 5% of dots. (0.00 to 1.00)

Actions
OK
Click this button to accept the changes and close the dialog box.
Cancel
Click this button to discard any changes and close the dialog box.
Reset
Click this button to reset the object's properties to default values.

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Edit Graphics for Heat Map: Zones and P-Values

This set of properties appears on the Zones et al. tab in the dialog used for editing a model
state graph. You can view and edit these properties by double-clicking a histogram or by
right-clicking a histogram and choosing Edit Graphics.

Zones
Show Model Zone Labels
Check to show the zone labels for the celltype.
Show Model Zone Boundaries
Check to show the zone boundaries for the celltype.
Label Font
Choose the font to use for the zone labels.
P-Values
Show P-Value 1
Check to show the first p-value for the celltype.
P-Value 1
Choose the first p-value grid line to display. (0.00000 to 1.00000)
Show P-Value 2
Check to show the second p-value for the celltype.
P-Value 2
Choose the second p-value grid line to display. (0.00000 to 1.00000)

Actions
OK
Click this button to accept the changes and close the dialog box.
Cancel
Click this button to discard any changes and close the dialog box.
Reset
Click this button to reset the object's properties to default values.

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Edit Graphics for Heat Map: Animation

This set of properties appears on the Animation tab in the dialog used for editing the model
heat map system. You can view and edit these properties by double-clicking or by right-
clicking in the plot area and choosing Edit Graphics.

Enable
Enables animation for the Heat Map.
CellType
Line Width
Choose line width for animation. (0 to 10)
Line Style
Choose the desired line style.
Color
Choose the color.
Region/Gate
Dot Size
Choose the appropriate dot size in points for region/gate animations. (0.00 to 6.00)
Actions
OK
Click this button to accept the changes and close the dialog box.
Cancel
Click this button to discard any changes and close the dialog box.
Reset
Click this button to reset the object's properties to default values.

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Region Label Properties

The properties of a region label can be edited by double-clicking the region label.

Name and Alias

Show Region Name
Show/hide region name
Region Name (Read only)
Name of the region
Show Region Alias
Show/hide region alias
Region Alias
Alias for the region

Statistics
Show Region Counts
Show/hide region counts in the region label
Show Region Percent
Show/hide region percent in the region label
Show Region Percent Total
Show/hide region percent total in the region label
Decimal Digits
Enter the number of decimal digits to display. (0 to 6)

Color and Font

Text Color
Color of the label's text
Label Font
Font of the label's text

Background Properties
Fill color
Color of the fill
Fill style
Style of the fill

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Transparency
Amount of transparency (0-100%) (0 to 100)
Pen color
Color of the outline
Pen style
Style of the outline
Pen width
Width of the outline in pixels (0 to ...)
Actions
OK
Click this button to accept the changes and close the dialog box.
Cancel
Click this button to discard any changes and close the dialog box.
Reset
Click this button to reset the object's properties to default values.

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Parameter Graph

A Parameter Graph object can be created on the canvas to display a single Parameter Profile
plot. The Parameter Graph tool in the Workspace toolbar is used to create the graph.

The plot shows the same information that appears in the Parameter Plots in the Cell Type
widget. The difference is that as a separate Parameter Graph object, the plot is not subjected
to the automatic sizing and hiding logic that apply to the Cell Type widget plots. This makes
the Parameter Graph useful for visualization of important parameters that you always want or
need to see.

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Zone Statistics Table

Each Cell Type in a GemStone model contains a Zone Statistics Table in the workspace. The
table contains a row of information for each "zone" that has been defined in the Cell Type.
For details on how to define and edit zones, see Edit Zones.
At the top of the object, the name of the cell type is displayed.
A checkbox in the upper-left corner of the object is used to flag the object to be saved as a
graphic image file. For additional information on saving graphics, see Batch Options panel.

The Name of each zone is displayed in the left column.

The Active column can be used to turn zones on and off quickly. When a zone is active
(checked), statistics are computed and zone-coloring is applied to dots. When inactive, a
zone is ignored by the analysis routines.
The Color for the zone is displayed and can be edited by clicking the color swatch.
Several statistics are displayed for each zone:
Percent: the percentage of events relative to this cell type that are contained by the
zone.
% Total: the percentage of events relative to all cell types that are contained by the
zone.
Events: the number of events contained by the zone.
% Minimum Purity: a probability value between 0.0 and 100.0 that the events are
actually contained by the zone. See for Edit Zones details.

See also:
Batch Options panel
Edit Zones

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Legend

The Legend object displays a list of the parameters in a model for one Cell Type. The color
associated with each parameter is displayed next to the parameter name.
The object can be added to a Cell Type workspace by clicking the Legend tool on the
Workspace toolbar, and then clicking and dragging to define an area where the legend
should be displayed.
A checkbox in the upper-left corner of the object is used to flag the object to be saved as a
graphic image file. For additional information on saving graphics, see Batch Options panel.

See also:
Workspace Names and Overview
Workspace toolbar

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Table

The Table object is used to display analysis results and keywords on the report. It is created
using the Table tool on the Workspace toolbar.

A table has a user-defined number of rows and columns, which can be changed by double-
clicking the object and editing the fields.
The cells in a table can contain text and keywords. Keywords must be surrounded with
square brackets to be interpreted as keywords and evaluated.

Decimal Places
You have control over the number of decimal places displayed by numeric keyword results
that you add to a table. Use the Edit menu Preferences command to display the
Preferences dialog. Decimal places are set in the General section.

Adding Text
Text can be typed directly into a cell in the table. Simply click the mouse in a cell and type
the text. Typical text editing keys can be used to delete, copy, and paste text.

Adding Keywords

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The easiest way to add keywords to the table is to select keywords from the list of available
information. This is done by clicking the cell to enter Edit mode, and then right-clicking to
display a menu of categories. Within the category sub-menus you will find all of the available
keywords that can be included.

The columns in the table will automatically size to fit the content.

See also:
Table Context Menu

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Table Context Menu

The Table Context menu appears when you right-click the title area of a table object.

Edit Properties
This command displays the Edit Properties dialog for the table, used to set the number of
rows and columns in the table.
Bring To Front
Move the table to the front of all graphic objects.
Send To Back
Move the table to the back of all graphic objects.
Delete
Delete the table.
Export Table
Save the table to disk. The standard File Save dialog is displayed to allow you to select a
location and name for the file. The file is saved in tab-delimited ASCII format.
Copy Table to Clipboard
Copy the table as tab-delimited text to the clipboard.

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Toolbars and Shortcuts

Main toolbar

The main toolbar contains a set of buttons that provide commonly used application actions.

Displays the Open Document dialog to open a GemStone document

from disk.
Displays the Save Document dialog to save the GemStone
document to disk.
Displays a dialog to select FCS files for analysis.
Displays the Edit Parameter Database dialog
Classifies the current data with the current model.
Optimizes the control points for all parameter profiles in the model
to best fit the current data.
Auto-analyzes the data.
Enriches the data in the packet to select for events that are
classified by the model.
Classifies the data in the entire FCS file with the current model.
Launches the online help system.

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Batch Controls toolbar

The batch toolbar contains a set of buttons to navigate through the selected batch of files.
The toolbar appears in the lower left of the GemStone workspace.

Edit properties for batch

processing.
Choose a file in the batch.
Go to the first batch file.
Go to the previous batch
file.
Redo the current batch
file.
Go to the next batch file.
Go to the last batch file.
Auto-advance through the
batch.
Stop auto-advancing.

See also:
Overview: Batch Processing
Overview: Workspace Names and User Interface

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Workspace toolbar

The workspace toolbar appears on the left edge of the Cell Type canvas. It contains buttons
to create and manipulate objects in the Cell Type canvas.

Selection Use this tool to select, move, and size objects in the canvas.
Zoom Use this tool to define a portion of the canvas to zoom-in on.
Click and drag to surround the area that you want to zoom.
Cancel zoom Click this tool to cancel out of zoom and return to normal
viewing.
Text Click and drag to define an area for the text. A dialog will be
displayed to input the text.
Line Click and drag to define the end points of the line.
Arrow Click and drag to define the end points of the arrow.
Rectangle Click and drag to define the outline of the rectangle.
Ellipse Click and drag to define the bounding rectangle for the ellipse.
1P Histogram Click and drag to define the bounding rectangle for the 1P
histogram.
2P Histogram Click and drag to define the bounding rectangle for the 2P
histogram.
Parameter Click and drag to define the bounding rectangle for a
Graph parameter graph.
Overlay plot Click and drag to define the bounding rectangle for the overlay
plot.
Heat Map plot Click and drag to define the bounding rectangle for the heat
map plot.
Legend Click and drag to define the bounding rectangle for the
legend.
Table Click and drag to create a table object.
Save Graphics Use the tool to save graphic images for objects that are
enabled for saving.
To enable an object for saving, check the box in the upper-
right corner of the object.
To save graphics, click the Save Graphics tool button.
Delete object Click this button to delete the selected objects from the
canvas.
Note: Some objects are not user-deletable.
Utility Actions Click this tool button to select one of the utility actions from a
list.
Choose Use this tool to select a parameter to include in the current
Parameter Cell Type.
Once a parameter is selected, it appears in the Cell Type
widget for the Cell Type. It can also be displayed in 1P and 2P
histograms.

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See also:
Overview: Workspace Names and User Interface

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Parameter Profile toolbar

The Parameter Profile toolbar is located on the right side of each Parameter Profile in the Cell
Type widget. It provides tools useful for working with the parameter profile.

Selection Use this tool to select, move, and size objects in the
Parameter Profile.
Zoom Use this tool to define a portion of the object to zoom-in
on.
Click and drag to surround the area that you want to
zoom.
Cancel zoom Click this tool to cancel out of zoom and return to normal
viewing.
Estimate Y When this tool is clicked, GemStone uses a peak-finding
Positions routine to analyze the parameter. It then adjusts the
parameter value (Y-axis) for each Control Point in the
parameter profile.
Estimate Line When this tool is clicked, GemStone adjusts the line-spread
Spread for each Control Point in the parameter profile.
Estimate X When this tool is clicked, GemStone adjusts the state index
Positions (X-axis position) of each Control Point in the parameter
profile. The positions are adjusted to provide the best fit of
the data currently displayed.
Edit Graphics Click this tool to edit the graphics settings for the
Parameter Profile.
Enable Profile: This button toggles the Enable Profile property for the
On parameter profile.
When enabled (on), the parameter profile is an active part
of the model and becomes part of the selection process for
events in the cell type.

Enable Profile: Turn Enable Profile property off (disabled) to eliminate the
Off parameter profile from the model selection process.

See also:
Overview: Workspace Names and User Interface
Cell Type Properties panel

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1P Graphics toolbar

The 1P Graphics toolbar is located on the right side of each 1P histogram object. It provides
tools useful for working with the object.

Selection Use this tool to select, move, and size objects in the object.
Zoom Use this tool to define the portion of the object to zoom-in on.
Click and drag to surround the area that you want to zoom.
Cancel Click this tool to cancel out of zoom and return to normal viewing.
zoom
Edit Edits the graphics settings for this object.
Graphics
Create 1p Use this tool to define a 1P region on the histogram. The region
Region can be used for gating or to color unclassified events.
After selecting this tool, click and drag on the histogram to define
the region.
Delete Click this tool to delete the histogram.
object
Create Click this tool to create a floating dialog copy of the histogram
dialog object. The dialog will create an independent duplicate of the
histogram in a floating window that can be moved outside of the
workspace.

See also:
Overview: Workspace Names and User Interface
1P Histogram

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2P Graphics toolbar

The 2P Graphics toolbar is located on the right side of each 2P histogram object. It provides
tools useful for working with the object.

Selection Use this tool to select, move, and size objects in the object.
Zoom Use this tool to define the portion of the object to zoom-in on.
Click and drag to surround the area that you want to zoom.
Cancel Click this tool to cancel out of zoom and return to normal viewing.
zoom
Edit Edits the graphics settings for this object.
Graphics
Swap Click this tool to reverse the parameters on the X- and Y- axes.
axes
Save Use this tool to save the graphics to disk in one of the graphic
graphics formats available. The standard File Save dialog will be displayed.
Enter a file name and choose a graphics format, then click Save.
Conform This tool toggles "Conform mode" on and off. By default, Conform
On mode mode is off.
When Conform mode is on, a region that is being moved will
"conform" its shape to the boundary of other region(s) that it
Off bumps into.
There are several important considerations when using Conform
mode.
1. You must not move the region by the region’s label in this
mode. Instead, drag the region by dragging its interior.
2. Conform mode works best with free-hand regions that are
created with many vertices.
Free-hand Use this tool to define a free-hand region on the histogram. The
Region region can be used for gating or to color unclassified events.
After selecting this tool, click and drag on the histogram to define
the region.
Rectangle Use this tool to define a rectangular region on the histogram. The
Region region can be used for gating or to color unclassified events.
After selecting this tool, click and drag on the histogram to define
the region.
Ellipse Use this tool to define an elliptical region on the histogram. The
Region region can be used for gating or to color unclassified events.
After selecting this tool, click and drag on the histogram to define
the region.
Bike-chain Use this tool to define a bike-chain region on the histogram. The
Region shape of this region resembles the chain of a bicycle, with one end
larger and the other smaller. The region can be used for gating or
to color unclassified events.
After selecting this tool, click and drag on the histogram to define
the region.

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Delete Click this tool to delete the histogram.

object
Create Click this tool to create a floating dialog copy of the histogram
dialog object. The dialog will create an independent duplicate of the
histogram in a floating window that can be moved outside of the
workspace.

See also:
Overview: Workspace Names and User Interface
2P Histogram

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Overlay Graphics toolbar

The Overlay Graphics toolbar is located on the right side of each parameter overlay object. It
provides tools useful for working with the object.

Selection Use this tool to select, move, and size objects in the object.
Zoom Use this tool to define the portion of the object to zoom-in
on.
Click and drag to surround the area that you want to zoom.
Cancel Click this tool to cancel out of zoom and return to normal
zoom viewing.
Edit Edits the graphics settings for this object.
Graphics
Animation Use this tool to turn animation on or off for the overlay
Start graph.
When animation is started, a parameter "ribbon" is shown
for several seconds, and then it is hidden and the next is
Stop shown. This continues for each parameter, and the process
repeats starting with the first parameter again.
When animation is stopped, all parameter ribbons are
shown at the same time.
Pause or This tool only has an effect when Animation is on for the
Pause Continue overlay graph.
Animation When the graph is animating, click the Pause button to
pause an animation.
Continue When the graph is paused, click the Continue button to
resume the animation.
Save Use this tool to save the graphics to disk in one of the
graphics graphic formats available. The standard File Save dialog will
be displayed. Enter a file name and choose a graphics
format, then click Save.
Text Click and drag to define an area for the text.
A dialog will be displayed to input the text.
Line Click and drag to define the end points of the line.
Arrow Click and drag to define the end points of the arrow.
Rectangle Click and drag to define the outline of the rectangle.
Ellipse Click and drag to define the bounding rectangle for the
ellipse.
Create Click this tool to create a floating dialog copy of the overlay
dialog graph object. The dialog will create an independent
duplicate of the overlay in a floating window that can be
moved outside of the workspace.

See also:

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Overview: Workspace Names and User Interface

Parameter Overlay Plot

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Shortcuts

GemStone supports many keyboard shortcuts for commands and other actions. The table
below describes these and their uses.

Shortcut Available Description

Ctrl+N (PC) Always Creates a new Model Document.
Command+N See New Document for details.
(Mac)
Ctrl+O (PC) Always Opens a Model Document.
Command+O See Open Document for details.
(Mac)
Ctrl+S (PC) Always Saves a Model Document.
Command+S See Save Document for details.
(Mac)
Ctrl+F (PC) Always Displays the Select FCS Files dialog to
Command+F choose data files to open.
(Mac) See Select FCS Files for details.
Ctrl+Y (PC) Always Synthesizes data.
Command+Y See Synthesize Data for details.
(Mac)
Ctrl+X (PC) Always Exports Model Data.
Command+X See Export Model Data for details.
(Mac)
Alt+F4 (PC) Always Exit the program.
Command+Q
(Mac
Ctrl+D (PC) Always Displays the Edit Parameter Database
Command+D dialog.
(Mac) See Edit Parameter Database for details.
Ctrl+A (PC) Always Classifies data.
Command+A See Classify Data for details.
(Mac)
Ctrl+B (PC) Always Optimizes the model.
Command+B See Optimize Model for details.
(Mac)
Ctrl+E (PC) Always Enriches for data that matches the model.
Command+E See Enrich Data for details.

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(Mac)
Ctrl+R (PC) Always Classify the entire file.
Command+R See Classify File for details.
(Mac)

Esc Always Interrupts the current command process.

PgUp/PgDn When a Opens and activates the previous or next
Parameter Profile Parameter Profile in the Cell Type Widget.
is selected

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File Menu
New Document

File menu
When selected, GemStone will discard the current model and GemStone document from
memory and create a new, empty GemStone document.
A GemStone document contains the Cell Types, parameter profiles, and objects on the Cell
Type canvas.

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Open Document

File menu
When selected, this command will display the standard File Open dialog to allow the user to
select a previously saved GemStone document file. GemStone will open the selected file and
display the Cell Types, parameter profiles, and other objects contained in the document.

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Save Document

File menu
If the document has been saved previously, this command will resave it without displaying a
dialog. The previous version of the document will be overwritten.
If the document has not been saved, this command performs identically to the Save
Document As command.

See also:
Save Document As

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Save Document As

File menu
When selected, this command will display the standard File Save dialog to allow the user to
save the current GemStone document to a file on disk. GemStone will save the Cell Types,
parameter profiles, and other objects contained in the document.

Error! Hyperlink reference not valid.

Error! Hyperlink reference not valid.

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Select FCS Files

File menu
This command allows the user to select data files to be analyzed by GemStone. The Select
Files dialog is displayed. This is a special File Open dialog designed to make selection of FCS
files efficient and easy.

Favorites
A group of controls at the top left of the dialog allows you to store and select file folders that
you frequently visit as "favorites". Use the drop-down list to select a folder you have
previously stored as a favorite. Use the star button to store a folder as a favorite, and use the
trash can button to delete a path from the favorites list.
File Folder Tree
A tree control on the left of the dialog allows you to navigate to the FCS files that you want
to analyze. Double-click a folder in the tree or click the "+" symbol next to it to expand its
contents. The files contained in the selected folder will be displayed in the file list.
Files of Type
This dropdown control displays the current file selection filter. Click the down-arrow to view
and select another filter, or type a selection filter into the control directly. Use "*" to display
all files in the selected folder.
File List
The list in the center of the dialog displays the files in the selected file folder that match the
selection filter in the Files of Type control. Normally, the list shows only the names of the

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files. However, you can use the Columns button to select additional information to display in
the list. The Show Details option must be enabled in order to view the additional columns
of information.
Show Details
When this option is enabled, the File List will display additional columns of information
about the files. Use the Columns button to select the information you want to display.
Sorting
When this option is enabled, you can click on the labels at the top of columns in the File List
to sort the files based on the information in that column. When the option is unchecked, files
are listed alphabetically by file name.
Open
This command adds the selected file(s) to the File Batch and closes the dialog.
Add
This command adds the selected file(s) to the File Batch and keeps the dialog open for
further file selection.
Close
This command closes the dialog.
Select All
Click this command to select all of the files in the File List.
Columns
This command displays the Edit Batch Columns dialog to choose the columns of information
you want to display in the File List.
Information
This command displays the Edit FCS Information dialog, which displays keyword information
from the selected FCS file.

See also:
File Database panel
Edit Batch Columns
Edit FCS Information

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Edit FCS Information

This dialog displays information about an FCS file. It can be displayed by selecting a file in
the Select FCS Files dialog and clicking the Information button.

See also:
Select FCS Files

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Close FCS File

File menu
Use this command to close the current FCS file. The command closes the file and removes its
keywords from memory. This is useful when constructing models that will be used to
synthesize experimental data.

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Synthesize Data

File menu
Models are most often used to analyze datasets. However, one of the best ways to evaluate
a model is to use it to synthesize an experimental dataset. The Synthesize Data command is
used to generate data based on the current model.
Before choosing this command, construct a model or open one from disk. This model will be
used as a basis for the synthesized data.
When the command is executed, a dialog is displayed to allow you to choose the number of
events to synthesize. Enter the value and click OK to synthesize the data.
The model itself determines the characteristics of the synthesis process. The same
probabilistic processes that are used for classifying real data with a model are put to use to
synthesize events. If the model contains multiple cell types, the synthesis routines will
apportion events accordingly. The better your model describes the real world process, the
more realistic the synthesized data will appear.
After synthesis, the generated data appears in the model's Parameter Profile plots and other
graphics. Statistics and zones act on the synthesized data, too.
The synthesized data can be exported to an FCS file using the Export Data command.

Before After

See also:
Export Data

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Export Model Data

File menu
This command exports the current packet of FCS data in memory to a new FCS file. It is most
useful in conjunction with the Synthesize Data command to store experimental datasets
created by GemStone for other software to read and interpret.
Only those parameters displayed in one or more Cell Type objects will be included in the
exported file.
The standard File Save dialog is displayed, allowing you to navigate to a location and enter a
file name for the export data. Files are stored in FCS file format.

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Manage Settings

File menu
This command displays the Settings Manager dialog, which can be used to import, export,
and clear settings for GemStone.

Pathways
Import or Export Settings Path
This path is the location that Export Settings or Import Settings will act on. Click the path to
choose a different location.
Recent Paths
This control stores the most recent locations used for importing or exporting settings.
Selecting a path from this list will set the Import/Export Settings Path.

Settings to Act On
This section displays a list of settings that can be imported or exported. The table below lists
each setting and a description. Check the box next to the settings that you want to import or
export before using the Export, Import, or Clear Settings buttons.
Setting Description
All Application Settings All configuration files and settings. This option includes a
number of settings that are not listed as individual options in
the list below.
When this option is selected, all other options are disabled.
Preferences User preferences set up with the Edit->Preferences dialog.
Color Palette Palette of colors set up with Edit->Color Palette.
User Commands Custom commands created with the Advanced Editing option
in the Batch Options panel.
Batch Files Files in the batch list.
Batch Columns Columns for the batch list database.
Parameter Database Database of parameters that GemStone has read.
FCS Selected Fields in the Select FCS Files dialog.
Information

Actions
Export Settings
After checking the settings you want to saved, click Export Settings to store the settings on
disk. The Choose Folder dialog will appear to allow you to select a location for the settings. A
confirmation dialog is displayed after the settings have been exported.
Import Settings

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After checking the settings you want to import, click Import Settings to read the settings
from disk. The Choose Folder dialog will appear to allow you to select a location to read the
settings from. A confirmation dialog is displayed after the settings have been imported.
Clear Settings
After checking the settings you want to clear, click the Clear Settings button to delete the
settings file(s) from disk. A confirmation dialog is displayed to confirm that you really want to
clear the settings. Click OK to continue to clear the settings files, or Cancel to escape.
After clearing settings, GemStone will close to complete the action.

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Print All Cell Types

File menu
This command prints the Cell Type canvas for each Cell Type defined by the model. The
canvas is scaled to fit on the current printer page.
The standard Print dialog is displayed to allow the selection of print options.

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Exit (Quit)

File menu
This command closes the program. It is Exit on Windows versions, and Quit on the Mac.

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Edit Menu
Edit Parameter Database

Edit menu
This command displays the Edit Parameter Database dialog, used to edit properties of FCS
file parameters and to control how GemStone interprets parameters.
Each time GemStone reads an FCS file, it interacts with the Parameter Database. If a file
contains a parameter that GemStone has never seen before, a record is added to the
Parameter Database with information from the file. When GemStone reads that parameter
from another file, it will use the record from the Parameter Database to determine how the
parameter will be interpreted and displayed. Records in the database can be edited to control
what the parameter display name will be, what color and transformation it will use, and much
more. The database even allows you to map any number of FCS parameter names to a single
record in the database. See How to Remap Parameters in a Model for a discussion on this.

Fields
Live
This column displays "On" for parameters that are contained in the FCS file that is currently
opened. It displays "Off" for parameters in the database that are not present in the current
FCS file.
Unique Name
This editable field is used as the display name for the parameter. By default, it shows the
$PnS keyword value, but it can be edited to any text you want to use for the parameter
name.
Each record in the database must have a unique name. If you type the same name as
another existing record, GemStone will ask if you want to merge the information from the
record you are editing into the existing record. This is an easy way to map more than one
FCS parameter to the same record in the database.
Param Index
If the parameter is "Live", this is the ordinal index of the parameter in the FCS file. If the
parameter is not contained in the current FCS file, this field displays -1.
Allow Normalization
Normalization is a process that spans files that the program reads. It allows GemStone to
adjust the mean linear intensity for selected parameters to be approximately equivalent
across files. There are two aspects to normalizing parameters. First, in the Batch Options
panel, an option other than Initialize needs to be selected. This tells GemStone that you want
to normalize or set normalization targets. Secondly, enable the Allow Normalization flag for
each parameter that you want to normalize. Use the Edit Properties for Parameter dialog to
view or edit the Target Mean Linear Intensity.
Auto Load
When this option is enabled, GemStone will automatically add a Parameter Profile to the
model to display this parameter when it is found in an FCS file.

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Transform
Select the transformation that should be used to display the parameter: Linear, Log, or
HyperLog. Additional properties for the transformation are available by clicking the
Properties button.
Abbreviation
Enter an abbreviation to use for the parameter. The abbreviation is used as a label on the
parameter ribbons in Parameter Overlay plots.
Color
Choose a color to use for this parameter. The color is used, by default, for the parameter
when displayed in Parameter Overlay plots.
Description
This option field can be used to add notes or to describe the parameter.

Actions
Properties
Click this button to display additional properties for the selected parameter record. The Edit
Properties for Parameter dialog is displayed.
New
Click this button to create a new parameter entry manually. The system will prompt for the
Unique Name and other information about the parameter.
Delete
This action will delete the selected record from the database.
OK
Click this button to close the dialog.
Open
Use this action to open a previously saved Parameter Database file from disk. The standard
File Open dialog is displayed. Navigate to the file and click Open. The selected database file
replaces the database in memory.
Save
Use this action to save the current Parameter Database to a file on disk so that it can be read
at a later time or by other users. The standard File Save dialog is displayed. Navigate to the
desired location, enter a file name, and click Save to store the file.
Delete All
This action removes all records from the database.

See also:
Edit Properties for Parameter
How to Remap Parameters in a Model

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Edit Properties for Parameter

This dialog is displayed to edit the properties for a parameter record in the Parameter
Database dialog. It provides access to all user-editable properties and displays a number of
read-only properties for the parameter. To display the dialog, select a parameter in the Edit
Parameter Database dialog, and then click the Properties button at the bottom of the dialog.

Name
This field displays the name that will appear on axis labels and other locations in the
program. This field can be edited to any text you want to display.
Abbreviation
The abbreviation for the parameter is used in Parameter Overlay plots to identify parameter
ribbons. In general, it should be a short, concise name.
Allow Normalization
Enables normalization for this parameter. Normalization adjust the intensity of the parameter
in subsequent data files to be approximately equivalent to the target mean intensity.
Normalization is a process that spans files that the program reads. It allows GemStone to
adjust the mean linear intensity for selected parameters to be approximately equivalent
across files. There are two aspects to normalizing parameters. First, in the Batch Options
panel, an option other than Initialize needs to be selected. This tells GemStone that you want
to normalize or set normalization targets. Secondly, enable the Allow Normalization flag for
each parameter that you want to normalize.
Target Mean Linear Intensity
When the Allow Normalization option is enabled, this field is used to specify the mean
intensity that the parameter should be normalized to when a new file is read.
Auto Load
When this option is enabled, GemStone will automatically add a Parameter Profile to the
model to display this parameter when it is found in an FCS file.
Auto Zoom
When this option is enabled, GemStone will automatically zoom-in on the parameter when it
is displayed. The auto-zoom will set the lower end of the parameter display to the first
channel which has events, and the upper end of the display to the last channel that has
events.
Always Update Transform
When this option is enabled and the HyperLog transform is used, GemStone will adjust the B-
coefficient and the negative boundary of the HyperLog transformation whenever a file is
read. The data in the file are used to determine the best negative boundary and B-coefficient
for the transformation.
Color
Choose a color to use for this parameter. The color is used, by default, for the parameter
when displayed in Parameter Overlay plots.

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Fluorochrome
This combo box provides another mechanism for assigning a color to the parameter. If you
select a fluorochrome from the list, GemStone will set the Color field to the emission
wavelength color of the fluorochrome. This is a quick way to match parameter colors to the
fluors they represent.
SD for Log
This value is used as the default Line-spread Value for this parameter profile for parameters
that use a Log or HyperLog transform.
%CV for Linear
This value is used as the default Line-spread Value for this parameter profile for parameters
that use a Linear transform.
Description
This option field can be used to add notes or to describe the parameter.

Status
Name
This field displays the internal name used to identify the parameter. It is based on keyword
values from the FCS file.
Live
This field displays "On" for parameters that are contained in the FCS file that is currently
opened. It displays "Off" for parameters in the database that are not present in the current
FCS file.
Param Index
If the parameter is "Live", this is the ordinal index of the parameter in the FCS file. If the
parameter is not contained in the current FCS file, this field displays -1.
Mean Transform Intensity
This is the mean intensity of the parameter on a scale of 0.0 to 100.0, referred to as
Transform units.
Mean Linear Intensity
This is the mean intensity of the parameter in linear units.
Short Name
This is the value of the $PnN keyword in the FCS file.
Long Name
This is the value of the $PnS keyword in the FCS file.
Lookup Param Name
This is the name that is used to look up the parameter in the parameter database.

Editors

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Edit Transformation
Click this button to display the Edit Properties for Linear Transformation dialog. This dialog
allows selection of a transformation, and setting of properties related to the transformation.
Edit Conditions
Click this button to view or edit conditions used to select this parameter. Conditions can be
created to map different parameter names to a common, database parameter record.

OK
Click the OK button to accept changes and close the dialog.
Cancel
Click the Cancel button to cancel changes and close the dialog.
Reset
Click the Reset button to set all properties back to default values.

See also:
Parameter Database

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Properties for Linear Transformation

This dialog is used to select a transformation to be used when displaying a parameter.

Properties related to the transformation can also be set. The dialog is accessed from the Edit
Properties for Parameter dialog box, via the Edit Parameter Database dialog.

Editable Settings
Transform
Choose Linear, Log, or HyperLog. The selected transform is used whenever this parameter is
displayed: in Parameter Profile plots, in 1P and 2P histograms, and in Parameter Overlay
plots.
Calculated Decades
When this option is enabled, GemStone will calculate the number of decades displayed by
Log and HyperLog transformations based on the dynamic range of the parameter. For
example, if a parameter has a range of 0 – 262144, GemStone would calculate the number of
decades as log(262144), or approximately 5.42 decades.
Decades
If the Calculated Decades option is not enabled, this value is used for the number of decades
in the Log and HyperLog transformations. Data values are scaled to this setting.
HyperLog B Coefficient
The B Coefficient defines the size of the linear portion of the HyperLog transform. A value of
10, for example, indicates that there will be 10 linear channels above and below 0. Best
visual results are obtained by using multiples of 10 for the B Coefficient. See also the Always
Update Transform property in the Edit Properties for Parameter dialog, which tells the
program to recalculate the B Coefficient each time a file is read.
Max Linear Range
This field normally displays the dynamic range of the linear parameter values, based on the
$PnR keyword for the parameter. When Linear is selected for the Transform, this field
determines what the high value on the axis is set to. Data values are scaled to this new
range, not clipped.
Negative Percent
This value determines how much of the HyperLog axis will be apportioned to negative values.
Zooming Settings
High Linear Range
This field sets the high linear value used for zooming. By default, it is the full dynamic range.
Low Linear Range
This field sets the low linear value used for zooming. By default, it is the lowest value that
can be displayed on the axis.
Current Settings
Min Linear Range

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This field displays the lowest linear value for the current transformation.
Normalization Factor
This field displays the normalization factor that was applied to this parameter. Normally, it is
1.00. However, if Allow Normalization is enabled for the parameter in the Edit Properties for
Parameter dialog, the value will show the actual factor used to normalize the data to the
target linear mean intensity.

See also:
Edit Properties for Parameter

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Edit Parameter Conditions

This dialog is used to set up conditions for mapping more than one FCS parameter name to a
common, database parameter. This allows files with different parameter names to be
analyzed by the same model, and for the parameter axis label and transformations to be
common across the files.

There is one entry in the list for each parameter name variant that will be mapped to this
database record. You can create new conditions by clicking the New button, or remove a
condition by selecting it in the list and clicking the Delete button.
To view or edit details for one of the conditions, select the entry in the list and click the
Properties button. This will display the Edit Properties of Parameter Condition dialog.

See also:
Edit Properties for Parameter
Edit Properties of Parameter Condition

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Edit Properties of Parameter Condition

This dialog is used to edit the Short Name and Long Name for a parameter that is to be
mapped to a common, database parameter.
GemStone allows files with different parameter names to be analyzed by the same model,
and for the parameter axis label and transformations to be common across the files. This is
accomplished by creating "conditions" for each of the parameter names that will map to a
common parameter. The conditions simply define two keyword values from the file that will
be used to identify the parameter and map it correctly.

Short Name
The field should contain the value for the $PnS keyword for this parameter in the FCS file.
Long Name
The field should contain the value for the $PnN keyword for this parameter in the FCS file.

See also:
Edit Properties for Parameter
Edit Properties of Parameter Condition

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Edit Color Palette

Edit menu
This dialog is used to edit the colors that are used by GemStone for parameter and zone
coloring. You can define the number of colors in the palette, and select the colors and color
names you want to use. The colors in the palette are used when initially creating objects. In
most cases, you can choose any color you want for an object after you have created it.
The list contains the colors that have been already defined in the palette. Colors and color
names can be directly edited in the list. Simply click the color wheel next to a color to select a
new color. Click on a color name to edit the text.

Edit
This command edits the properties of the selected color in the list.
New
Use this command to create a new entry in the color list. Once the entry has been added, the
color and color name can be edited.
Delete
Use this command to delete the selected color from the color palette.
OK
Click OK to accept the changes and close the dialog.
Open
Use this command to open a color palette file from disk, previously saved using the Save
command. The standard File Open dialog is displayed, allowing you to select the file you
want to open.
Save
Use this command to save the color palette file to disk, so that it can be shared with others
or re-opened at another time. The standard File Save dialog is displayed, allowing you to
navigate to the appropriate location and to enter a name for the file.
Properties
Use this command to edit properties of the palette. The Properties for Color Palette dialog is
displayed.

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Properties for Color Palette

This dialog allows properties of the color palette to be edited. The palette contains a set of
colors that are used by GemStone for parameter and zone coloring.

Number of Colors
This field determines the number of colors initially created in the palette.
Create Method
The field determines the method used to create the color palette: Cube Root or Rainbow.

See also:
Edit Color Palette

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Edit Logging System

Edit menu
This command displays a dialog to view the application log and to edit properties of the
logging system.
When the logging system is enabled, GemStone records log entries for the operations that it
performs. It is most often used to trouble-shoot a problem, but it can also provide a record of
user interactions with the program.

If you want to...

Enable logging Click the Properties button to display the Edit Properties dialog.
Clear the log Click the Delete All button.

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View All Application Information

Edit menu
This command displays a dialog that contains all information that is available to the database
and reporting. The dialog information is read-only.

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Edit Information Equations

Edit menu
Information Equations are user-defined mathematical expressions that can be evaluated and
stored in the information database that GemStone maintains. Equations allow you to
generate additional statistics to extend the information that can be extracted and stored by
the program. Any equations that you create are available to the database and to reporting
objects like the table through the information system.
The equations you write are stored as part of the GemStone model document, so they are
unique to each model. You can store a set of equations using the Save button and then
import those equations into another model using the Open button.
A dialog is displayed, showing a set of example equations on the left, and the list of
equations already defined on the right.

Anatomy of an Equation
Each equation has at least two fields that can be edited: Description and Format. In
addition, the equation will have additional Properties that can be edited if the equation
contains placeholders for some of its arguments (e.g. "%1"). The placeholders in an equation
are used to reference keywords and other elements in the information system.
For example, let's examine the "Simple Ratio" equation that is the first in the example list. It
has a Description, "Simple Ratio", which can be edited to be anything you want. To edit the
description, click the mouse in the Description field, and edit the text.
The Format portion of the equation is where the equation expression is actually defined.
The format can contain numbers and mathematical operators, but it can also contain special
functions and placeholders. Function are found in the examples in the tree on the left of the
dialog and listed in Equation Functions. Placeholders are the parts of the equation format
that use the percent symbol (%) and a number.
In the example equation, the Format field uses two placeholders: %1 and %2. As the name
implies, a placeholder identifies part of the equation that will be replaced by an element that
GemStone has read from a data file or that it has computed. For example, with our Simple
Ratio equation, if we want to create a ratio between the number of events in Zone 1 and
Zone 2, we would tell GemStone to replace %1 with the Zone 1 events and %2 with Zone 2
events. This is done by selected the equation in the list and then clicking the Edit button.

In the Edit Equation dialog, right click the Information field to display the menu of choices
available to fill-in the placeholder. In our example, you would choose
CellType1_Zone1_NumberOfEvents for %1 and CellType1_Zone2_NumberOfEvents for %2.

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Each placeholder in an equation must be associated with an information field in this manner
before the equation can be properly evaluated.

Suppose that you want to create a custom equation that multiplies the mean of a parameter
in Zone1 by 4.56, and then divides by the mean of another parameter in Zone1. The Format
field would look like this:
%1*4.56/%2
After typing the equation into the Format field, you would click the Edit button and select the
statistics to fill-in for %1 and %2.

Suppose that you wanted to use a function in this equation, to take the square root of the
result. The format field would look like this:
FSQRT(%1*4.56/%2)
For a list of functions, see Equation Functions.

If you want
to...
Add a new Select an example equation from the tree on the left and click Insert or
equation Add to copy the example to the equation list. Edit the format field to
define the equation expression. Then use the Edit button to select fields
that will be evaluated by the equation (see Edit Equation).
Edit Select the equation in the list on the right and click the Edit button. In
placeholders in the Edit Equation dialog, right click in the Information column to
an equation display the choice of items. Select the keyword or statistics you want
from the choices shown.
Remove an Select the equation in the list on the left and click the Delete button.
equation
Save a set of Click the Save button, navigate to a location on disk, and save the

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equations equation file.

Open a set of Click the Open button, navigate to the location where the equation file
equations was previously saved on disk, and open the equation file.

See also:
Edit Equation
Equation Functions

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Edit Equation

This dialog is used to edit the placeholders in an equation. Placeholders are the parts of the
equation format that use the percent symbol (%) and a number. Each placeholder must be
associated with a piece of information, which is used when the equation is evaluated.

To select the information associated with a placeholder, right click the Information field to
display the menu of choices available to fill-in the placeholder. An expanding menu of choices
is displayed. Choose the category to expand the appropriate submenu, then select the
specific item from the choices.

Each placeholder in an equation must be associated with an information field in this manner
before the equation can be properly evaluated.

See also:
Edit Information Equations
Equation Functions

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Equation Functions

These functions can be used when writing equations using the Edit Information Equations
dialog.

Indexing
Indexes for FCS parameters start at 1 and represent the parameter order in the FCS file. For
example, if a file has six parameters, they are referenced as P1 through P6 in equation
functions. Parameters do not need to be included in the model to be referenced by an
equation. The best way to determine the parameter index for a specific parameter is to add
one to the Associated Parameter field in the Parameter Profile properties panel.

For example, if the Associated Parameter is 4, the parameter indexing would add one for an
index of 5.

Indexing for gate and regions starts at 1. The expression "G1" refers to the gate for Cell Type
1, and region R1 is the first region index.
Indexes for the Cell Types defined in a model also start at 1. However, there is a hidden Cell
Type that contains all unclassified events. This is referred to as Cell Type 0, and it can be
referenced in equation functions as "C0".

Parameter Scales
GemStone keeps track of several different scales for parameters at the same time. The table
below describes each scale and gives the symbol to use to access the scale in equation
functions.
Scale Symbol Description
Linear L Raw, linear data values from the data file for the
parameter. These values are in the ADC range of the
instrument.
Compensated C Compensated values, on the same scale as Linear.
Transformed P Internal floating point scale.
Log or HyperLog transforms have been applied, if the
parameter is displayed using one of these transforms. If
compensation is defined for the parameter, the
Transformed value is in compensated data space.
Linear value 0.0 maps to Transformed value 0.0.
Linear max value maps to Transformed 100.0.

Functions

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This system is designed to provide a flexible but efficient framework to calculate any kind of
statistic we want from the data. This ambitious goal is achieved by an extensive syntax.
Let's begin with the simplest of the summation functions, the gated sum:

GSUM({GIDn{,Cq}}{;IDm{;IDp})
where
{ } indicate an optional argument
GIDn = Rn or Gn
n represents a gate or region index, starting with 1
C = cell type
q = 0 (unclassified), 1, 2, ..., number of cell types
ID = L, C, P
m and p represent a parameter index, starting with 1.
The number of IDn arguments limited to two.
The IDm and IDp are optional.

With no arguments, GSUM becomes a simple count.

The first optional argument to the GSUM function, GIDn, identifies a region or gate. If a gate
or region ID is specified, only the events in that gate or region are considered. If the gate or
region ID is omitted, then all events are considered in the sum.
For example:
GSUM()
returns the number of events
GSUM(G1)
returns the number of events in gate G1
GSUM(R1)
returns the number of events in region R1

A semi-colon (;) is required between the optional gate and the next argument.
The GSUM function can have up to two ID arguments that identify a parameter of interest.
There are 3 scales for parameter data: L for linear, C for compensated, and P for
transformed. These are described in the Parameter Scales section above. For example, L1,
C1, and P1 all related to parameter 1 in the data file. The scaling is different for each of
these representations of parameter 1.

GSUM(;L1)
returns the sum of linear intensity values for parameter 1
GSUM(;C1)
returns the sum of compensated intensity values for parameter 1
GSUM(;P1)

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returns the sum of transformed intensity values for parameter 1

If two parameter ID arguments are specified in the GSUM function, the result is the products
of the event values for the specified parameters. For example,

GSUM(R3;P3;P3)
finds the sum of the square of transformed parameter 3 values that are in region R3.

Gated cross products (cc) are computed in this manner:

GSUM(R3;P3;P4)
returns the correlation coefficient for parameters 3 and 4 events that are in region R3.

A simple linear mean is evaluated as,

GSUM(G3;L1)/GSUM(G3)
returns the linear mean for parameter 1 events that are in gate G3.

Other types of intensity related summation functions compute the mode and median:

GMOD(;P2)
computes the mode for parameter 2 in transformed space.
GMED(G3;P2)
computes the median for parameter 2 events that are in gate G3. The result is in
transformed space.

Quadrant statistics include

GQ1(G3;P2)
returns the location at which 25% of parameter 2 events gated on G3 fall below and
75% fall above.
GQ3(G3;P2)
returns the location at which 75% of parameter 2 events gated on G3 fall below and
25% fall above.

Percentage calculations are trivial. For example, would be given by

GSUM(G3)*100/GSUM()
returns G3 events as a percent of total events
GSUM(R3)*100/GSUM(G2)
returns R3 events as a percentage of G2 events.

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Any expression can be transformed between linear units and transformed units:

FLToT(Pn,<Exp>)
converts a value on the Linear scale to a value on the Transformed scale for the
specified parameter n.
FTToL(Pn,<Exp>) (Transformed to linear)
converts a value on the Transformed scale to a value on the Linear scale for the
specified parameter n.

For example, suppose P2 is a parameter displayed on a log scale. Then,

FTToL(P2,GSUM(G3;P2)/GSUM(G3))
finds the geometric mean for parameter 2 gated on G3. The result is expressed in
linear units that relate to the log axis displayed for the parameter.

Some of the more commonly used functions are also included.

GeoMean(G3;P2)
is a convenient short-hand for FTToL(P2,GSUM(G3;P2)/GSUM(G3))
LinMean(G3;P2)
is equivalent to GeoMean(G3;P2)
LinMedian(G3;P2)
is short-hand for FTToL(P2,GMED(G3;P2))
LinMode(G3;P2)
Is short-hand for FTToL(P2,GMOD(G3,P2))

VAR(G3;L3;L3)
computes the variance of parameter 3 in linear space.
VAR(G3;L3;L4)
computes the covariance of parameters 3 and 4 in linear space.

SD(G3;L3)
computes the standard deviation for parameter 3 events in gate G3 in linear space.
QSD(G3;P2)
computes the quadrant variance for parameter 2 events in gate G3 in transformed
space.

CV(G3;L3)
computes the coefficient of variation for parameter 3 gated on G3 in linear space.

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Additional Examples
GSUM() Total number of events
GSUM(G1) Number of events in gate G1
GSUM(R1) Number of events in region R1
GSUM(C1) Number of events in CellType 1
GSUM(C0) Number of unclassified events (Cell Type 0 is
the Unclassified Cell Type).
GSUM(C0,R2) Number of unclassified events in region R2
GSUM(;L1) Sum of linear intensity values for parameter 1
GSUM(G1;L1) Sum of linear intensity values for parameter 1
that are in gate G1
GSUM(G1)/GSUM(R1) Ratio of the sum of gate G1 events to R1 events
MEAN(G1;P1) Mean of parameter 1 events that are in gate G1
GMOD(G1;P1) Mode of parameter 1 events that are in gate G1
GMED(G1;P0) Median of parameter 1 events that are in gate
G1
VAR(G1;P1) Variance of parameter 1 events that are in gate
G1
SD(G1;P1) SD of parameter 1 events that are in gate G1
CV(G1;P1) CV of parameter 1 events that are in gate G1
GQ1(G1,P1) First quartile of parameter 1 events that are in
gate G1
GQ3(G1,P1) First quartile of parameter 1 events that are in
gate G1
CC(G1;P1,P2) Correlation Coefficient of P1 vs. P2 events that
are in gate G1
FLtoT(parameter, value) Converts linear value 100 into transformed units
for parameter 1
FLtoT(P1,100) Converts linear value 100 into transformed units
for parameter 1
FTtoL(P1,GSUM(G1;P1)/GSUM(G1)) Geometric mean for parameter 1 events that are
in gate G1
GeoMean(G1;P1) Simple form geometric mean for parameter 1
events in gate G1
FLOG(10) Natural log of 10
FL10(1) Log base 10 of 1

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FEXP(3) Exponent of 3
FE10(3) Exponent base 10 of 3
FSQR(4) Square of 4
FCUB(3) Cube of 3
FBND(arg,lower,upper) Evaluates the first argument to see if it is
between lower and upper. Returns the first
argument if it is between the others, returns
lower if arg is less then lower, or returns upper
if arg is greater than upper.
FPWR(10,3) Returns 10 raised to the 3rd power.
FSQRT(5) Returns the square root of 5.

See also:
Edit Information Equations
Edit Equation

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Show Tool Button Labels

Edit menu
This command toggles the display of text labels for the main toolbar on and off.

Labels On

Labels Off

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Edit Preferences

Edit menu on PC, Application menu on Mac

This command displays a dialog that allows all preferences for the program to be edited.

Select an item in the tree on the left side of the dialog to display the properties for that
object in the pane on the right. Preferences are automatically saved as defaults for new
objects, and they are remembered after the program quits.

File System Preferences

General Preferences

Graphics Preferences
Cell Type Graphics Preferences
Control Point Graphic Preferences
2P Graphics Preferences
1P Graphic Preferences

Merging File Preferences

Minimization Preferences
Miscellaneous Preferences
Parameters Preferences
Workspace Preferences

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Preferences
File System Preferences

Packet Size
Enter the number of events from the listmode file to keep in memory.
Auto Compensate
When enabled, GemStone will automatically decode compensation keywords in FCS files and
apply the compensation described in the keywords. Currently, files created by BD Diva
software, Accuri software, and Beckman Coulter files are supported. If an FCS file does not
contain compensation information, this setting will have no effect.
Hardware Compatible Comp
If compensation is applied by the Auto Compensate logic, this option forces the
compensation to be a subtraction similar to hardware compensation.
Read All Keywords
Enable this option to read keywords from all datasets.
Desired Dataset
If the FCS files you work with contain more than one dataset, this value allows you to select
which dataset to read. If you want the program to prompt you to select a dataset when there
is more than one, set Desired Dataset to 0. To automatically select a specific dataset, enter
the 1-based dataset index. For example, to read the 2nd dataset in a file, enter the value 2.
Dataset Descriptive Keywords
Enter keywords that you want the program to read and present to you from each data source
to help select the one you want to open. Use commas between keywords.

HyperLog
Percent Negative Boundary
This value controls how GemStone determines the negative boundary for the HyperLog
transform. Using the default value of 3.0 as an example, the program finds the channel at
which 3.0% of events are in lower channels. If the channel at that location is a negative
channel, the channel is used as the lower boundary of the histogram. If the channel is
positive, a secondary calculation is used to determine the lower boundary.
Smaller values allow more negative channels to be shown.

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Negative To Positive Ratio

This ratio controls the relative portions of the axis that are devoted to negative and positive
channels. The default value of 0.20 allows up to 20% of the axis to be devoted to negative
channels and 80% to positive channels.

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General Preferences

Specimen Keyword
This keyword is used on the Cell Type widget and other objects to identify the data file that is
currently opened.
Number of decimals
This property determines the number of decimal places that will be displayed for numbers in
tables.

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Graphics Preferences

These preferences are common to the graphic objects on the canvas and reporting areas.
Background for Drag Objects
Select the background to use for objects on the canvas.
Title Font for Drag Objects
Select the font used for titles.
Axis Num Major Tics
Select the number of major tics for axes.
Axis Pen Weight
Enter a pen weight for the axes.
Axis Pen Color
Select a color for the axes.
Axis Number Font
Select a font for numbers on the axes.
Axis Number Color
Select a color for the numbers on the axes.
Axis Label Font
Select a font for the axis labels.
Axis Label Color
Select a color for axis labels.
Background Color
Select a background color for plots.
Show X Grid Lines
Enable to show X grids.
Show Y Grid Lines
Enable to show Y grids.
Grid Line Width
Enter a line width.
Grid Line Style
Choose a line style for grids.
Grid Color
Choose a color for grids.
Unclassified Color
Select a color for unclassified events.

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Cell Type Graphics Preferences

Enable Dots
Check this option to enable dots in the cell type parameter plots.
Dot Size
Type a default dot size.
Show All Dots
Enable to show dots for all events in the packet.
Desired Dots
Select a desired number of dots to display. If Show All Dots is enabled, this setting is
ignored.
Show Unclassified Events
Enable this option to show unclassified events in addition to the classified events.
Show Fidelity Plot
Enable this option to show the state index fidelity plot by default.

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Control Point Graphic Preferences

Size
Set the size in pixels for control points.
Pen Width Selected
Select a pen width for control points that are selected.
Pen Width Not Selected
Select a pen width for control points that are not selected.
Pen Color
Select a pen color for control points.
Fill Color Selected
Select a fill color for control points that are selected.
Fill Color Not Selected
Select a fill color for control points that are not selected.
Tip Color
Select a fill color for the triangular tips of control points.
Transparency
Choose a transparency setting for the control point fill.

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2P Graphics Preferences

Enable Dots
Check this option to enable dots in the 2P plots.
Dot Size
Type a default dot size.
Show All Dots
Enable to show dots for all events in the packet.
Desired Dot Number
Select a desired number of dots to display. If Show All Dots is enabled, this setting is
ignored.
Show Unclassified Events
Enable this option to show unclassified events in addition to the classified events.
Enable State Vectors
Enable the display of state vectors.
Enable Surface
Check this option to display the surface graphics.
Probability Mixing Color
Choose a color that will be used to for the densest portions of the surface plot (the highest
probability). The default color is white.
Number of Vectors
Select a desired number of state vectors to display.

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1P Graphic Preferences

Fill Style
Choose a fill style for 1P histograms.
Transparency
Enter a transparency for filled 1P histograms (0 = solid, 100=transparent)
Resolution
Enter the number of channels for 1P histograms.
Show Peaks
Enable this option to show the peaks that were found by the peak finding routine. Peaks are
used by optimization routines to locate populations and position control points.
Peak P-Value
Enter a P-value filter to apply to the set of peaks that were found for the parameter.

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Overlay Graphic Preferences

Show X Grid Lines

Check to show the X grid lines.
Show Zone Boundaries
Check to show the zones boundaries on the overlay.

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Parameter Graphics Preferences

Enable Surface
Check this option to display the surface graphics.
Probability Mixing Color
Choose a color that will be used to for the densest portions of the surface plot (the highest
probability). The default color is white.

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Heat Map Preferences

This dialog allows all preferences for the program to be edited. The current section allows
editing of Heat Map Graphics properties.

Chi Multiplier
This value is used to scale the Y-axis distribution of the Heat Map over 100 units. The value
is 5. (1.00 to 20.00)
Actions
OK
Click this button to accept the changes and close the dialog box.
Cancel
Click this button to discard any changes and close the dialog box.
Reset
Click this button to reset the object's properties to default values.

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Merging File Preferences

Auto Create Model Resolution

User higher values for more parameters. Higher values slow down the merging process. (2 to
...)
Auto Create Model Threshold
Controls the spot size for the merging process. A normal value is close to 1. (0.00 to 10.00)
Auto Calculate
If true, will automatically calculate the resolution and threshold to satisfy the desired yield.
Desired Yield
If Auto Calculate is true, finds the threshold and resolution to give a desired yield. (0.00 to
100.00)
Maximum Auto Create Resolution
Maximum resolution to try for the Auto Create Model option. (2 to ...)

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Minimization Preferences

Delta Param
Choose the fraction of the parameter to change.
Termination Criteria
When the response function changes by less than this value, the system terminates the
minimum search routine.
Max Function Evaluations
Choose the maximum number of function evaluations.

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Miscellaneous Preferences

Client Timeout
Enter the number of seconds to wait until a battery request times out.
Battery Server Port
Enter the port number used to connect to the battery server. This value must match the port
that the battery server is configured to use.
User Login Option
Select the user login option you want to use when the program starts up.
User Group
Select the default user group for the user login dialog. The user group controls access to
program features.
Show Banner
Enable this option to show the banner dialog when the program is launched.

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Parameters Preferences

Estimated %CV for Linear Parameters

Enter the default estimated %CV for linear parameters.
Estimated SD for Log Parameters
Enter the default estimated SD for log parameters (e.g. 10.0). (0.10 to 50.00)
Estimate Min Response Change
Enter the estimated minimum response changes. Lower values produce more accurate
estimates.

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Workspace Preferences

Grid Snap
Enable this option to snap drag objects to the grid.
Grid Size
Set the size of grid elements, in pixels.
Angle Snap
When rotating, snap objects to increments of the specified angle.
Angle Snap Size
Set the angle snap, in degrees.
Copy DPI
Choose a desired number of dots per inch (DPI) for copying graphics to the clipboard.
Saving Format
Select a file format to use when saving the graphics to disk.
Default Page Width
Enter a size for the canvas in inches.
Default Page Height
Enter a size for the canvas in inches.
Auto-expand Parameter Plots
Select the number of parameter plots to keep open as new plots are added to the model.

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Analysis Menu
Classify Data

Analysis menu
Use the Classify Data command to model the data in memory with the Probability State
Model. This command is typically used when opening a new FCS file into a GemStone
document to apply the model to the new FCS data. A related command, Classify File,
performs the same functions but on the entire FCS file.
When classifying data, GemStone uses the Probability State Model to assign each event to a
Cell Type, and within a Cell Type, to a particular state on the progression axis. Virtually every
decision in the classification process is probabilistic.

See also:
What is a Probability State Model?
Auto Analyze
Batch Processing

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Optimize Model

Analysis menu
Use the Optimize Model command to adjust the positions and line-spreads of Control Points
based on the FCS data that is currently displayed. This is a useful for making small
adjustments to the model due to slight intensity shifts from one file to another.
The process makes adjustments to all Parameter Profiles in all Cell Types. GemStone uses
peak-finding routines to determine the best intensity positions for the Control Points. It
performs statistical fits on peaks to determine line-spread adjustments.
Optimize Model can be time-consuming. A progress meter is displayed on the status bar to
let you see how the optimization is proceeding. You can halt the process by pressing the Esc
key on the keyboard.

See also:
What is a Probability State Model?
Auto Analyze
Classify File
Batch Processing

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Auto Analyze

Analysis menu
Auto Analysis is a more sophisticated version of Optimize Model. It is used to automatically
tune the positions of Control Points to the data file that is currently opened. Like Optimize
Model, it affects all Parameter Profiles in all Cell Types.
Auto Analyze simulates the approach that you might take to adjust a model manually. It
starts by turning off all of the Parameter Profiles so that nothing is being fit by the model.
Next, it searches for Parameter Profiles that are using the "Constant" profile, and it optimizes
the position and line spread for each. Once all Constant profiles are adjusted, it uses the
Enrich Data command to select as many events as possible into the model. Then, one by one
it visits the remaining Parameter Profiles and optimizes the positions of the Control Points.
Once it has completed these steps, the complete model is analyzed once again with all
profiles active.

See also:
What is a Probability State Model?
Classify Data
Classify File
Batch Processing

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Enrich Data

Analysis menu
This command is typically used during the model-building process to select events from a
data file that match the model. Before using the command, you should make sure that the
only parameters with Match Status enabled are those that use "Constant" profiles. Constant
profiles are sometimes referred to as "selection" profiles, because they are used to select
subsets of events from the data file. They are the most useful for the Enrich Data because
they are so simple and robust.
The Auto Analyze command uses this command as part of its analysis process.

See also:
What is a Probability State Model?
Auto Analyze
Batch Processing

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Classify File

Analysis menu
Use the Classify File command to model the all of the data in the file with the Probability
State Model. A related command, Classify Data, performs the same functions but only on the
data in memory.
When classifying data, GemStone uses the Probability State Model to assign each event to a
Cell Type, and within a Cell Type, to a particular state on the progression axis. Virtually every
decision in the classification process is probabilistic.

See also:
What is a Probability State Model?
Auto Analyze
Classify Data
Batch Processing

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Fidelity Analysis

Cell Type Properties panel, Utility Actions

For each event it analyzes, GemStone keeps track of the probability that the event has been
correctly classified. This allows it to evaluate the classifications for an entire Cell Type and to
report an additional statistic: State Fidelity Index. The State Fidelity Index is the average
probability for all events in all states that the events are correctly classified, expressed as a
percentage. This value is show in the Cell Type Properties panel. The closer the value is to
100, the more likely it is that events are correctly classified.
The State Fidelity Index is more than a fancy statistic, though. It is the basis for an objective
assessment of how well the markers in the model are working to define the progression. The
Fidelity Analysis is that assessment, and with it we can determine which markers are the
most important and which are least important. We now have a means for objectively
determining the best markers to use in a panel. In this case, "best" means the markers that
allow the model to produce the highest State Fidelity Index for each cell type.
The Fidelity Analysis command is found in the Cell Type Properties panel's Utility Actions. To
perform the analysis, simply select the command from the list of Utility Actions.
Here is what the Fidelity Analysis does. First, it determines which Parameter Profiles in the
Cell Type are active and use a something other than a Constant profile. Then, it methodically
evaluates all combinations of these non-constant profiles, performing a Classify Data
command on each combination. It stores the State Fidelity Index for each of these
combinations.
When the analysis is complete, it presents a table of the results. The table is organized
starting with each Parameter Profile alone, then every combination of two parameters, and
so on. The rows are sorted from lowest State Fidelity Index to highest. This allows you to
evaluate the relative importance of each parameter in each combination. From this, you can
determine which parameters are most vital to your panel of markers.

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In the example results shown above, we can see that the best State Fidelity Index our model
achieves is 5.82, using TDT, CD10, and CD20. Notice, that TDT alone is reaching a higher
index than CD10 and CD20 together. This shows us that TDT is by far the most important
marker for this panel and this model.
When you close the window, GemStone allows you to save the table of results to a text file
on disk. The text file is a simple, tab-delimited file that can be imported into spreadsheets,
statistic software, and databases for further analysis and storage.
As you might guess, the parameter profile selected for each parameter has an impact on how
important the parameter is in the Fidelity Analysis. In general, the more the profile changes
over the progression, the more important it is. To illustrate, here are a few profiles with
synthesized data. We analyzed each one separately to determine its relative Fidelity Index.

For a profile that increases or decreases continuously over the progression and varies over
the full dynamic range for the parameter, we see a very high Fidelity Index of about 90%. In
our example, we created the profile with a very small line-spread, which also improves the
fidelity score.

If we increase the line-spread of this parameter profile to 5.0, the score drops all the way to
8%. So, not surprisingly, we see that line-spread plays an important roll in the likelihood that
we are correctly classifying the events. Larger line-spread means more uncertainty.

At the other extreme, a constant parameter profile always yields a Fidelity Index of 1.0. Since
all events in the progression have the same likelihood of falling into any of the states on the

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progression, we have virtually no confidence that any given event is classified into the correct
state.

A typical Step-Up profile with a line-spread of 3.0 produces a fidelity index of around 8%.

If we combine two profiles that have transitions in different portions of the progression, the
Fidelity Index of the model as a whole improves to ~11. This illustrates the synergistic nature
of parameter profiles in defining the progression.

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Use the Fidelity Analysis feature to determine objectively which markers are really adding
information in your panel.

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 Reference

Battery Menu
Battery Menu

The Battery menu is used to manage the "software batteries" that GemStone uses. Software
batteries encode a certain amount of "charge". With each charge, you can open one data file
for analysis.
The program keeps track of the files you have opened and allows you to re-open files without
using a battery charge. You can purchase additional charges when you need to open
additional data files, and you can transfer charges to other users.

See also:
Overview: Software Batteries
How to Work with Software Batteries

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Battery Status

Battery menu

This dialog displays the status of the software battery currently installed for GemStone. See
Battery Menu for a description of the battery system.

Application Info
Application name
This field displays the name of the application.
Computer ID
This field displays the computer ID. The battery system uses the Computer ID when ordering
or requesting batteries to identify the computer that the battery is for.

Battery Info
Battery ID
This field displays the ID of the current battery.
Charge
This field displays the charge remaining on the current battery.
Install-by date
This field displays the date by which the battery had to be installed. Batteries must be
installed prior to their Install-by date in order to operate correctly.

OK
Click this button to close the dialog and accept changes.
Cancel
Click this button to cancel the dialog.
Buy
Click this button to order additional software battery charges from the Verity Software House
web site. See Buy Now for details.
Charge
Click this button to download prepaid battery credits from the Verity Software House web
site. See Charge Battery for details.

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 Reference

Buy Now

Battery menu
Use this command to order additional software battery charges from the Verity Software
House web site.
In order to use it, your computer and GemStone need to have internet access. Firewalls can
sometimes interfere with this function.

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Charge Battery

Battery menu
Use this command to add charges to your GemStone software battery. The command
initiates the Charge Battery Wizard, which will guide you through the process of getting
charges for your battery.

See also:
Overview: Software Batteries
How to Work with Software Batteries

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 Reference

Charge Battery Wizard

This wizard is displayed by selecting Charge Battery from the Battery menu of the
application. The wizard guides you through the process of getting charges for your software
battery.

For a discussion of how to use the wizard, see How to Work with Software Batteries,

See also:
Overview: Software Batteries
Check Balance
Upload Credits

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GemStone User Guide

Check Balance

Battery menu
This option requires that you have a member account on www.vsh.com. Visit the web site to
create a member account if you have not already done so.
This command allows you to check to see how many battery charges remain in your Verity
Software House web account. In order to use it, your computer and GemStone need to have
internet access. Firewalls can sometimes interfere with this function.

Web Login
Enter the login name for your Verity Software House web account.
Password
Enter the password for your Verity Software House web account.
Remember Password
Enable this option if you want the password to be remembered on this machine.

Click the OK button to initiate the balance check. The program will communicate with
www.vsh.com to determine the balance and display the results.

Use the Charge Battery command to download some or all of the available charges.

See also:
Upload Credits

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 Reference

Upload Credits

Battery menu
This option requires that you have a member account on www.vsh.com. Visit the web site to
create a member account if you have not already done so.
This command uploads battery credits from your local battery to the Verity Software House
web site. In order to use it, your computer and GemStone need to have internet access.
Firewalls can sometimes interfere with this function.
If the command is successful, all battery charges on the computer will be uploaded to the
specified user account on the Verity Software House.
This command is useful for several purposes. When credits are uploaded to Verity Software
House, you can easily transfer them to another user or download them to another computer
that you are using. You can use the Upload Credits command before reformatting your hard
drive or reload your operating system to preserve your available charges. After reformatting,
you can download the credits using the Charge Battery command to restore charges on your
computer.

Web Login
Enter the login name for your Verity Software House web account.
Password
Enter the password for your Verity Software House web account.
Remember Password
Enable this option if you want the password to be remembered on this machine.

When you click OK, the program will communicate with www.vsh.com to upload the credits.
The results of the upload will be displayed.

See also:
Check Balance
Charge Battery

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Server Menu
Server Menu

This menu provides commands for Battery Server functions. It only appears in Battery Server
versions.

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 Reference

Properties for Battery Server

Server menu
This dialog is informational and does not provide any editing of properties. It is most useful
for viewing the server name, IP, and port address.

Server host name

This is the network name of the server computer.
Server IP Address
This is the IP address of the server computer.
Server Port
This field specifies the IP Port that the battery server uses to communicate with client
applications. The default value is generally not used by other well-known applications. If
there is a port conflict with another application, a value between -1 and 65535 can be
entered. Use -1 to allow the program to find an available port.
Client applications need to know the port that is used by the server. It must be entered in the
Preferences dialog.
Server Password
Enter a password that client applications will use to authenticate their requests for battery
charges.
Maximum Battery Request
Enter the maximum number of charges that can be requested at a time by a client
application.
Server Timeout
This field sets the number of seconds that the server will wait before communication with a
client times out. Use a higher value to allow more time for slower network connections.

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Transfer Charge

Server menu
This command is used to fulfill an offline battery request from a client application. The
information used to fill in this dialog comes from the client computer and is displayed to the
user in the Charge Battery dialog box.
Fill in the fields with the information from the client computer, and then click OK to generate
a set of codes that the user will use to add the charges to the client computer.

Program
Choose the program that the user has installed on the client computer.
Transaction Code
Enter the transaction code from the client computer Initiate Offline Transfer dialog.
Computer ID
Enter the computer ID from the client computer Initiate Offline Transfer dialog.
Charge To Transfer
Enter the amount of charge to transfer to the client computer.
User (optional)
Enter the name of the user, or leave blank. This field will be stored in the transaction log.
OK
When you click OK, the program will attempt to draw the requested number of charges from
the server battery. If charges are available, a dialog will be displayed containing a set of
codes. Print or send these codes to the user, who will then need to enter them in the Charge
Battery dialog.
Cancel
The button closes the dialog without taking any action.

See also:
Charge Battery

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 Reference

Help Menu
Help Index

Help menu
This option displays this online User Guide in your browser. The User Guide is fully indexed
and searchable, and contains tutorials and reference sections to provide a comprehensive
explanation of GemStone features and functions.

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Ask Tech Support

Help menu
This command interacts with your email application to create a new message to Verity
Software House Tech Support. A new message is created with the appropriate "To" email
address and some information about your application in the body of the email.
Fill in the email message with your contact information and the question(s) you want to ask.
A member of the Tech Support team will respond to your question as quickly as possible.

See also:
Make a Suggestion
Report a Problem

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 Reference

Make a Suggestion

Help menu
This command interacts with your email application to create a new message to Verity
Software House Tech Support. A new message is created with the appropriate "To" email
address and some information about your application in the body of the email.
We value your suggestion very highly, and make every attempt to incorporate changes into
the software that address suggestions from our clients.
Fill in the email message with your contact information and your suggestion for the program.
A member of the Tech Support team will respond to your email as quickly as possible.

See also:
Ask Tech Support
Report a Problem

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GemStone User Guide

Report a Problem

Help menu
This command interacts with your email application to create a new message to Verity
Software House Tech Support. A new message is created with the appropriate "To" email
address and some information about your application in the body of the email.
When reporting a problem, please try to include as much detail as possible about the
conditions that caused the problem. If at all possible, please try to reproduce the problem
and list the steps that will cause it to occur. While it is sometimes possible to correct
problems that are not reproducible, it is much more likely that we can fix a problem that we
can reproduce.
A member of the Tech Support team will respond to your email as quickly as possible.

See also:
Make a Suggestion
Ask Tech Support

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 Reference

Application Info

Help menu

This command displays a dialog that contains information about the program and its
installation. It is useful when interacting with tech support.

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About

Help menu on PC or Application menu on Mac

This command displays the application banner dialog box. The dialog contains information
about the application. It also provides access to the Registration or Battery system,
depending on the type of installation you have.
The phone number for Verity Software House is shown, as are hyperlinks to www.vsh.com
and the general email account.

See also:
Registering your new software
Battery Menu

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 Index
1 Edit Batch File Columns 135
1P Animation Options 204 Edit Canvas Properties Dialog 180
1P Graphic Preferences 290 Edit Color Palette 266
1P Graphics Toolbar 236 Edit Control Point 169
1P Histogram 200 Edit Equation 273
2 Edit FCS Information 249
2P Graphics Preferences 289 Edit Graphics for Heat Map Animation 222
2P Graphics toolbar 237 Edit Graphics for Heat Map Dots 220
2P Histograms 207 Edit Graphics for Heat Map Surface 219
A Edit Graphics for Heat Map Zones and P-
About 323 Values 221
Animation for 2P Histograms 214 Edit Information Equations 270
Application Info 322 Edit Logging System 268
Ask Tech Support 319 Edit Parameter Conditions 264
Auto Analyze 301 Edit Parameter Database 257
B Edit Parameters 152
Batch Controls toolbar 232 Edit Preferences 281
Batch Levels panel 139 Edit Properties for Parameter 259
Batch Options panel 141 Edit Properties for Relation 173
Battery Menu 308 Edit Properties for Zone 154
Battery Status 309 Edit Properties of Condition 265
Buy Now 310 Edit Relations 172
C Edit Zones 153
Cell Type Canvas 178 Enrich Data 302
Cell Type Properties panel 148 Equation Functions 274
Charge Battery 311 Exit Quit 256
Charge Battery Wizard 311, 312 Export Data 252
Check Balance 313 Export Database 136
Classify Data 299 F
Classify File 303 Fidelity Analysis 304
Close FCS File 250 File Batch Context Menu 134
Creating a Model for a Multi-Tube Panel 74 File Database panel 133
E File System Preferences 282
Edit Actions 145 Frequency Plot 174
Edit Background Page Properties Dialog G
181 Gating and Gate Expressions 157

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General Preferences 284 Model Building 103 56
Global Properties panel 146 Model Building 104 67
Graphic Options for 1P Histograms 202 Model Building 201 88
Graphic Options for 2P Histograms 210 N
Graphic Options for 2P Vectors 213 New Features in GemStone 9
Graphic Options for Parameter Profile New Protocol 243
Animate 190 O
Graphic Options for Parameter Profile Open Database 138
Confidence Limits 188
Open Protocol 244
Graphic Options for Parameter Profile
Optimize Model 300
Means 187
Other Graphic Options 191, 205
Graphic Options for Parameter Profile
Model 189 Other Overlay Properties 196
Graphic Options for Parameter Profiles Overlay Graphic Options 195
185, 186 Overlay Graphic Preferences 291
Graphics Preferences 285 Overlay Graphics toolbar 239
H Overview Building a Model 31
Heat Map 218 Overview Software Batteries 33
Heat Map and Animated Regions 96 Overview Using a Model 32
Heat Map Preferences 293 Overviews 25
Help Index 318 P
How To Remap Parameters in a Model 101 Parameter Graph 225
How to Use Batch Processing 107 Parameter Graphics Preferences 292
I Parameter Overlay Plot 193
Import and Export Settings 253 Parameter Profile 159
Installation and Setup of GemStone 15 Parameter Profile Descriptions 164
L Parameter Profile Properties panel 160
Latest Versions 7 Parameter Profile toolbar 235
Legend 227 Parameter Profiles Widget 147, 182
License and Warranty 3 Parameter Properties 262
M Parameters Preferences 297
Main toolbar 231 Print Cell Types 255
Make a Suggestion 320 Properties for 2P Surface 209
Merging File Preferences 294 Properties for Color Palette 267
Minimization Preferences 295 Properties for Open Protocol 144
Miscellaneous Preferences 296 Properties for Param State and SD 171
Model Building 101 36 Properties of Control Point 170
Model Building 102 46 Properties of Frequency Plot 176

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 GemStone User Guide

Properties of Parameter Profiles Widget T

184 Table 228
Properties of the Battery Server 316 Table Context Menu 230
Q Transfer Charge 317
Quick Start 5 Tutorials 35
R U
Reference Section 131 Upload Credits 314
Region Label Properties 216, 223 V
Registration 19 View Information 269
Report a Problem 321 W
S Welcome 1
Save Database 137 What is a Probability State Model? 27
Save Document As 246 Work with Battery Server 124
Save Protocol 245 Work with Software Batteries 115
Select FCS Files 247 Workspace Overview 26
Server Menu 315 Workspace Preferences 298
Shortcuts 241 Workspace toolbar 233
Show Tool Button Labels 280 Z
Synthesize Data 251 Zone Statistics Table 226
System Requirement 13

327