ModFit LT User Guide

Copyright 1994-2017 by Verity Software House

All Rights Reserved.

Updated 24 January 2017

This guide is intended for ModFit LT version 5.0 and later minor versions.
 Table of Contents
ModFit LT User Guide ......................................................................................................... 7

License and Warranty ......................................................................................................... 8

Latest Versions .................................................................................................................10

Naming Convention ...........................................................................................................11

New Features ...................................................................................................................12

System Requirements ........................................................................................................24

Minimum System Configurations ..................................................................................24

Optimal Configurations................................................................................................24

Installation and Setup of ModFit LT ....................................................................................25

Registering your new software ...........................................................................................27

Tutorials...........................................................................................................................36

First Time Using ModFit LT .............................................................................................36

Manual analysis with ModFit LT .......................................................................................41

Using the Sync Wizard ...................................................................................................47

Using the Cell Tracking Wizard ........................................................................................56

Enhanced Cell Tracking Analysis......................................................................................63

Using Auto Linearity .......................................................................................................76

Databasing with ModFit LT .............................................................................................82

Optimizing Auto Analysis ................................................................................................90

Creating a report template..............................................................................................98

Field Testing ............................................................................................................... 102

Reference ....................................................................................................................... 104

Main Window .............................................................................................................. 104

Ribbon Bar .................................................................................................................. 107

Toolbar ....................................................................................................................... 111

Data Files .................................................................................................................... 113

Open FCS ................................................................................................................ 113

 File Open dialog box ................................................................................................. 114

Analysis Setup .......................................................................................................... 115

Change Data Set... ................................................................................................... 118

Recent Files list ........................................................................................................ 119

Analysis ...................................................................................................................... 120

Auto Analysis command ............................................................................................ 120

Auto Analyze and Auto Linearity ................................................................................ 121

Fit with Current Model command ............................................................................... 122

Auto Linearity........................................................................................................... 123

Choose Model command ........................................................................................... 124

Edit Properties for Cycle ............................................................................................ 127

Scale command ........................................................................................................ 129

Range command ...................................................................................................... 130

Fit Diagnostics command .......................................................................................... 133

Fit Diagnostics dialog ................................................................................................ 134

Sync Wizard ............................................................................................................. 136

Cell Tracking Wizard ................................................................................................. 149

Reports ....................................................................................................................... 175

Reports ................................................................................................................... 175

New command ......................................................................................................... 177

Open Reports command............................................................................................ 178

Report command ...................................................................................................... 179

Save command ........................................................................................................ 180

Save As command .................................................................................................... 181

File Save As dialog box ............................................................................................. 182

Print command ......................................................................................................... 183

Page Setup command ............................................................................................... 184

Zoom command ....................................................................................................... 185

Recent Reports list ................................................................................................... 186

 Edit 1P Graphics ....................................................................................................... 187

Edit 2P Graphics ....................................................................................................... 190

Report tools ............................................................................................................. 194

Batch Processing ......................................................................................................... 207

Batch Processing ...................................................................................................... 207

File Batch Settings .................................................................................................... 209

File Batch Toolbar..................................................................................................... 212

Choose Batch Dialog ................................................................................................. 213

Report Batch Settings ............................................................................................... 214

Report Batch Toolbar ................................................................................................ 216

Clipboard .................................................................................................................... 217

Copy command ........................................................................................................ 217

Paste command........................................................................................................ 218

Databases ................................................................................................................... 219

Databases ................................................................................................................ 219

Edit Properties for All Databases ................................................................................ 221

Edit Properties for Database ...................................................................................... 223

Edit Keyword Map .................................................................................................... 225

Database Viewer ...................................................................................................... 227

Add Record .............................................................................................................. 229

Database Wizard ...................................................................................................... 230

Exporting .................................................................................................................... 232

Export: Save PDF ..................................................................................................... 232

Export: Save Histogram Graphic ................................................................................ 233

Export: Save Report Graphic...................................................................................... 234

Logging ...................................................................................................................... 235

Edit Log ................................................................................................................... 235

Edit Properties for Log .............................................................................................. 236

Preferences ................................................................................................................. 238

 Auto Analysis Settings ............................................................................................... 238

Standards ................................................................................................................ 244

Properties for Standards............................................................................................ 245

Peak Finder Settings ................................................................................................. 246

Options and Configuration ......................................................................................... 250

Edit Parameter Database........................................................................................... 256

Edit Properties for Parameter..................................................................................... 259

Edit User Equations .................................................................................................. 261

Edit Properties for Equation ....................................................................................... 263

S-Phase Cutoffs command......................................................................................... 264

Settings Manager ..................................................................................................... 266

Research and Compliance Modes .................................................................................. 268

Research and Compliance Modes ............................................................................... 268

Setting Up Compliance Mode ..................................................................................... 270

21 CFR Part 11 Compliance ....................................................................................... 276

Customizing Models ..................................................................................................... 278

Model Descriptions ................................................................................................... 278

Colors command ...................................................................................................... 288

Quick Model Editor ................................................................................................... 290

Full Model Editor ...................................................................................................... 293

Edit Components ...................................................................................................... 295

Edit Properties for Component ................................................................................... 297

Edit Properties for Detail ........................................................................................... 300

Edit Ranges ............................................................................................................. 303

Edit Properties of Range............................................................................................ 305

Edit Analysis Results ................................................................................................. 309

Edit Properties for Analysis Result .............................................................................. 312

Edit Report Results ................................................................................................... 313

Edit Properties of Report Result ................................................................................. 315

 Equation syntax........................................................................................................ 316

Help ........................................................................................................................... 320

Check for Updates .................................................................................................... 320

Ask Tech Support ..................................................................................................... 321

Make a Suggestion ................................................................................................... 322

Report a Problem ..................................................................................................... 323

Application Info ........................................................................................................ 324

User Guide ............................................................................................................... 325

About command ....................................................................................................... 326

Appendices ..................................................................................................................... 327

Analysis Results ........................................................................................................... 327

Histogram and S-Phase Assessment .............................................................................. 330

DDE Support ............................................................................................................... 334

Strengths and Limitations of Automatic Analysis ............................................................. 335

Index ............................................................................................................................. 338

ModFit LT Help ModFit LT User Guide

ModFit LT User Guide

Copyright 1994-2017 by Verity Software House

All Rights Reserved.

Updated 24 Jan 2017

This guide is intended for ModFit LT version 5.0 and later minor versions.

Verity Software House

Phone: (207) 729 6767

Fax: (207) 729 5443

Email: verity@vsh.com

Web: www.vsh.com

© 1994-2017 Verity Software House. All Rights Reserved 7

ModFit LT Help License and Warranty

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 computer

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 computer for one user account.

Network versions of software require one serial number for each simultaneous use. Network
versions are intended for installation and use on a local area network; other uses are not
supported.

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

© 1994-2017 Verity Software House. All Rights Reserved 8

ModFit LT Help License and Warranty

limitation of implied warranties or liability for incidental or consequential damages, so some

of the above limitations or exclusions may not apply to you.

© 1994-2017 Verity Software House. All Rights Reserved 9

ModFit LT Help Latest Versions

Latest Versions

To find the latest versions for ModFit LT and the 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 verity@vsh.com 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.

The software also has a Check for Updates command in the Help menu. Use this command
periodically to see if there are updates for your software. The command can download and
install updates that are available, if you have administrative rights to your computer.

© 1994-2017 Verity Software House. All Rights Reserved 10

ModFit LT Help Naming Convention

Naming Convention

This manual and ModFit LT's analysis results use shortened forms of ploidy terms to improve
readability. We use the term "Diploid" when referring to "DNA diploid". Similarly, we use the
terms "Aneuploid" and "Tetraploid" when referring to "DNA aneuploid" and "DNA tetraploid"
respectively.

When using analysis results in publications or other contexts outside of the software, we
suggest that the explicit "DNA" terms be used. See "Convention on Nomenclature for DNA
Cytometry", Hiddeman et, al., Cytometry 5:445-446 (1984) for additional details.

© 1994-2017 Verity Software House. All Rights Reserved 11

ModFit LT Help New Features

New Features

Version 4.1

New Features:

Added [AutoLinearity()] DDE command handling.

Added equations and results for models generated by the Cell Tracking Wizard.

Added equations and results for the shape of the S-Phase components.

Added equations and results for the state of the G1 position in the sync wizard.

Equations added to custom models are now added to keyword results, allowing the values
to be added to the database.

When building custom models, it is now possible to have ranges "auto-position" to a

specific peak in the histogram.

Added a Copy to clipboard to the database.

Simple analysis estimates will now update as ranges are moved and will display the mean
and SD in the status bar or tool tip when moving a range.

The X-zoom graph can now be set to adjust to the Dip-G1 or the cell tracking parent peak
position using a new option in the Options and Configuration.

Automatic backup and recovery: We've designed a new automatic backup/recovery

system. The system will save a copy of your report into "My Documents/ModFit LT for
Win/AutoRecover" every 2-minutes by default. A property, "Save Recovery Backup", has
been added to the Options and Configuration to specify how often the backup is created. In
the event of a crash, power failure, or other abnormal termination, ModFit LT will check the
AutoRecover folder on next start-up and allow the user to recover any auto saved reports.

Automatic crash detection and reporting (WINDOWS ONLY): We've added a new crash
reporting system that creates a ZIP file with information that Verity can use to pinpoint the
crash location in the software. The Crash Reporter will offer to send a crash report to Verity,
as well as to restart ModFit LT after a crash. Users can choose whether to include the
application log (if active) and any auto-saved ModFit LT reports with the crash report. Crash
reports are saved into "My Documents/ModFit LT for Windows/CrashReports"

Added keyboard shortcuts for "Auto Analyze and Auto Linearity" and "Fit with current
model".

Added "Auto Analyze and Auto Linearity" to the Analysis menu.

Added options to customize the appearance of many of the report objects.

© 1994-2017 Verity Software House. All Rights Reserved 12

ModFit LT Help New Features

Improvements:

Added new logic that will detect when zooming is needed and apply zooming to
measurements in Accuri data files. The user is notified and given a chance to adjust the zoom
via a timed notification message.

Using a more accurate method for determining the zoom to percentile boundaries.

Made changes to when information about parameter names, transforms, zooming, etc. are
stored in the parameter database. An entry will only be stored if an edit is made and the user
answers yes to the question: "Do you want ModFit to remember these settings for
[Parameter Name]?". This change should prevent confusion regarding parameter display
properties and defaults.

Changes made to the graphs in the Analysis Setup dialog will now be reflected in the
report graphs, and the reverse.

The user login has been disabled by default, go to Edit -> Options and Configuration to re-
enable it if needed.

One can now correctly specify and use Gaussian components with multiple compartments.

One can now create a new model component from scratch rather than simply duplicating
an existing component.

The model's fit line preferences can now be edited via Edit Colors.

When a standard is added or edited so that its expected position is less than the peak
finder's Start At setting, the user will now be asked if that setting should be adjusted
automatically to allow a peak to be found at the expected position of the standard.

Clarified and added options that are shown when a sample analyzed by the synch wizard
with a floating G1 position and a peak is not found in the expected range.

The lower limit for AutoLinearity has been lowered so that it can be set to values as low as
1.80.

The debris component, aggregate component, and RCS calculation will now ignore events
in channel zero. This prevents issues when a large pile up of events is present. This change
will cause ModFit LT to get different results for the same analysis as previous versions.
However in most cases it will have little if any effect.

It is now possible to move an equation up or down in the equations editor.

Simplified the file and report batch options by removing the Auto Linearity checkbox. The
auto linearity option is now add as part of the analysis mode.

Added options to the full model editor to edit the fit line style and weight.

© 1994-2017 Verity Software House. All Rights Reserved 13

New Features ModFit LT Help

Added copy and paste context menu entry for objects on the report page.

The Y-zoom graph will now adjust its x scale to match the main analysis graph.

Bug Fixes:

Fixed a bug with the calculation of generation SDs and thus the Division Error Index when
using the proliferation wizard and ADC channels. This bug caused the SDs of the generations
to be incorrectly computed on linear values rather than log channels.

Proliferation analysis of DDE data will now use the correct number of decades to compute
the generation spacing.

Fixed a bug that prevented editing of parameters after reading a report that contained an
analysis of listmode data before opening a listmode data file or after opening another file
type (histogram, ASCII, DDE). This bug also caused the axes to be labeled "Channels"
instead of with the listmode parameter names.

Fixed a bug that causes the incorrect transform to be applied. A linear encoded FCS
parameter will indicate that it is being displayed as log and vice versa. If the user attempts to
analyze a linear FCS parameter in this case, LT would think it is a log parameter and the
analysis will fail. If the user tries to manually change to the correct transform the data will be
incorrectly transformed, for example linearizing a linear encoded FCS parameter. The
problem occurs when a parameter is loaded with one encoding (linear vs. log), and then
subsequently a parameter with the same $PnN and $PnS is loaded that is encoded in the
opposite manner.

Fixed a bug that occasionally caused the drawing of a visual artifact in the debris area of
the fitted histogram.

Fixed a bug that would cause a crash in Auto Scale-Y was turned off and a gate graph was
selected in the analysis setup dialog.

Fixed a bug that would prevent choosing a database with the same file name in a different
directory.

Fixed a bug that would cause a crash if the Cell Tracking Wizard was run on an ASCII file,
histogram file, or DDE data.

Fixed a bug that caused the parameter zoom to be incorrect when the parameter was
encoded as log in the FCS file and displayed as linear in ModFit LT.

Fixed a bug that caused a database entry to be added for both the initial analysis (auto or
fit with current) and the final analysis when auto linearity was checked in the file and report
batch properties.

14 © 1994-2017 Verity Software House. All Rights Reserved.

ModFit LT Help New Features

Fixed a bug that caused regions to obscure the data in PDFs generated from the report.
This also fixed errors that would be found on the generated PDFs when loaded into certain
PDF readers.

Fixed a bug that prevented the opening of listmode FCS data after doing a DDE connection
from WinList.

Fixed a bug that caused a crash if a 1024 res histogram was loaded and then DDE data
was pushed over from WinList.

Version 4.0

Major improvements:

New Analysis Setup dialog for listmode files

The gating and analysis parameter selection dialogs have been redesigned into one
streamlined dialog that provides access to all gates and gating tools with improved ease and
better graphics.

New region tools

Gating regions can now be created with a full compliment of region types, including freehand
regions.

Improved 2P graphics
We have overhauled the graphics for the 2P gating plots to provide better visualization and
publication quality.

Parameter selection
For listmode files, the parameters displayed on 1P and 2P plots can now be changed by
clicking on the axis label and selecting a new parameter from the list.

External keywords in XML files

ModFit can now read XML files containing additional keyword information and display those
keywords on reports and in the database.

Display log or linear axes

ModFit now supports both log and linear axes and presents results in the units displayed on
the axis.

Log axis based on FCS file keywords

The program now makes use of keywords in FCS files to determine whether an axis should
be display in log or linear.

Parameter zoom
When reading listmode files, ModFit can automatically zoom in on a portion of the data based
on several user-selectable methods.

© 1994-2017 Verity Software House. All Rights Reserved 15

New Features ModFit LT Help

Parameter database
User selection of parameter alias, zoom, transform, and scale are stored in a parameter
database.

PDF support
ModFit can now generate PDFs of the report, including during batch processing.

Compensation from keywords

When ModFit detects compensation information stored in FCS file keywords, it can now read
and apply that compensation automatically.

Auto-linearity access improved

Auto-linearity is now available via a tool button, file batch processing, and while using synch
wizard. There is also a keyword that indicates whether auto-linearity has been performed.

Choose Dataset for multi-dataset files

For listmode files containing more than one dataset, such as FC-500, Galios, and Navios files,
the program now allows selection of the dataset at any time via a command in the File menu.

Improved graphic output

The program now offers a Copy Special and graphics export preferences to allow output of
high quality images to clipboard and disk.

OLE support eliminated

OLE support has been eliminated from the program.

Bug Fixes:

If text in the Keyword object was edited by the user, the edits where not stored and did
not appear on the report. This has been corrected.

Fixed a crash that would occur if the graph object was edited before data was loaded.

File -> New will now completely clear the report and properly reset to the original state.

Ranges will no longer move to the top left of the graph and be stuck there if no peaks are
found.

The DDE example script would fail if an analysis had not been done before it was run.

ModFit will now correctly interpret keyword values sent from WinList via DDE.

Pasted images will now properly allow for transparent areas.

The axes can now correctly display tics for values less than 1.0.

Component colors will now be displayed correctly when Transparent is not checked.

16 © 1994-2017 Verity Software House. All Rights Reserved.

ModFit LT Help New Features

Version 3.2

This version supports native OSX on the Mac platform, and Vista on the Windows platform.

We have removed OLE automation support from this build. Please contact Verity Software
House tech support (tech@vsh.com) if you would like to see this added back in a future
release.

Graphics are now bitmap format instead of vector format for clipboard and disk files.

A number of bug have been fixed.

Version 3.1 Service Pack 3

22 September 2005

The program now supports high-resolution, digital data up to 32-bits. This includes support
for 18-bit FCS data files exported by BD's Diva software.

A "Convert" button has been added to the listmode Choose Parameter dialog. This button
displays the Log Conversion dialog that allows linear parameters to be converted into log
parameters. Use this option when reading Diva-generated files that were stored in linear, FCS
3.0 format. The dialog displays a list of all parameters in the file, allowing the user to select
parameters to convert to log for display and analysis. It also provides an option to convert to
4-decades or the "computed" decades for the native, linear resolution.

The program now supports FCS files containing more than one dataset. When a multi-
dataset file is opened, the program displays the Choose Dataset dialog to allow the user to
select the dataset which should be used for analysis. The selection remains in effect until it is
invalid or the user makes a new choice. The dialog can be redisplayed by enabling the "Multi
Dataset Prompt" property in the File Batch Settings dialog.

When the program is configured to base ploidy determination on an external reference or

an internal standard, several new results are reported. The results will display a "Reference
Diploid" value that shows the location where the Diploid G1 is expected to be. In addition,
each cell cycle shows a "DI based on reference:" statistic, which is computed as the G1 mean
of the cycle divided by the "Reference Diploid" value. For all cycles except Diploid, the typical
"DI" is also still reported.

© 1994-2017 Verity Software House. All Rights Reserved 17

New Features ModFit LT Help

A new function has been added to the equation processor. The "F" function can be used to
convert a keyword value into a floating-point number. This allows the keyword to be used in
calculations. The syntax is: F[expression], where expression is typically a keyword. For
example, the $P1B keyword stores the number of bits used to store a value for parameter 1
in an FCS file.
To compute the number of bytes used by parameter 1, the following equation could be used:

"Parameter 1 bytes = ";F[[$P1B]]/8

To display the same result as an integer: "Parameter 1 bytes = ";p[0];F[[$P1B]]/8

New internal keywords have been added to provide access to the External Reference Mean
[ExtRefMean], and the Diploid-to-Standard Ratio [DipToStdRatio].

The SetProperty DDE/OLE command has been enhanced for the ExternalReference
property.

DDE Syntax from WinList: [SetProperty(Property=value)]

OLE Syntax from VB: SetProperty(propertyString, valueString)

Supported properties and values:

AutoAggregates=On,Off

AutoDebris=On,Off

Standards=0,1,2

ExternalReference=position or

ExternalReference=position,dynamicRange

In this second format, the dynamicRange is used by ModFit LT to scale the position value to
it's histogram scale.

A new ploidy index of -1 has been added for hypo-diploid models. Ploidy index can be
displayed using the MF_PLOIDYINDEX keyword.

Bug fix: Program crashed when S-Phase component was changed to a single Gaussian.
This has been corrected.

Bug fix: The Mac version did not properly list files on some network drives. When the
network drive was selected, files in the application folder were displayed. Corrected.

Version 3.1 Service Pack 2

19Dec2003

18 © 1994-2017 Verity Software House. All Rights Reserved.

ModFit LT Help New Features

The Proliferation Wizard now detects conditions where there is little or no Parent
generation. The wizard locks the SD for the Parent to the last known good SD and alerts the
operator. This addresses problems that could occur in analyzing a sequence of proliferation
data files where the Parent generation is not present as the experiment progresses.
(11Dec2003 VI 1115)

The X-zoom graphic is now placed behind the main graph by default, instead of on top of
it. It can be moved to be on top of the main graph by selecting the main graph, choosing the
Report->Send to back command, and then selecting and moving the zoom graph.

Implemented "battery" usage system. (26Sep2003)

Bug fix: Corrected crash that could occur when an older version of the proliferation model
was opened in a report and used for analysis in a newer version of the program. (10Dec2003
vi 1115)

Version 3.1 Service Pack 1

30May2003

The program now supports 3 gates and displays gated data in the second and third gates.
See the Listmode appendix for more details.

Prolif wizard now supports export of "partition" information. Options for export appear on
the Other tab of the prolif wizard dialog. A new tutorial describes how this feature can be
used with WinList to provide enhanced proliferation analysis.

The Proliferation wizard now supports a "floating" model option for the daughter
generations. On the Generations tab, you can choose between the "Standard" and "Floating"
model options. In the standard model, the mean of each generation is dependent on the
previous generation, based on the spacing value. In the floating model, a range is associated
with each generation and the mean is allowed to 'float' in the model. This option should
ONLY be used with well-defined, visibly-separated generations.

Added a Lock SD option to the Parent Tab of the Proliferation Wizard. This option is useful
for proliferating samples in which the parent population becomes indistinct. When the option
is enabled, the SD for the parent population can be edited. All generations use the same SD
as the parent, regardless of the state of the Lock SD option.

The status bar now displays low and high X and Y values as gate regions move or resize to
allow for more precise positioning. You can also display the position of a region by clicking
and holding on the region without moving it.

When the program opens, it is now positioned and sized to the location and size which
were last used.

© 1994-2017 Verity Software House. All Rights Reserved 19

New Features ModFit LT Help

The registration dialog now displays a confirmation dialog when a valid unlock code is
entered.

The User Login name is now stored in a keyword, MF_USERLOGIN, which can be displayed
on reports and stored in the database.

The program now uses the Unbiased Summation method for reducing histogram resolution
to 256 channels. This will likely improve RCS values for histograms that have native
resolution greater than 256-channels. It may also result in slightly different locations for
peaks found by the Peak Finder.

After selecting a batch of data files or reports, the program now automatically displays the
Edit Properties for Batch dialog to allow batch properties to be adjusted prior to opening the
first file.

Options and Configuration now provides separate options for auto-scaling X and Y axes to
allow greater control.

Bug fix: User edits to the X axis label are now left in place when new data files are opened.
If first characters of the label are the default label "Channels", the program will auto-label the
axis. Otherwise, the user label is left alone.

Bug fix: Graphics now update correctly for first-time DDE connection with Winlist on PC.

Bug fix: The program could crash if gating resulted in zero events in the histogram. The
crash would occur when trying to fit the histogram. This condition could also occur with DDE
connections to WinList.

Bug fix: The Proliferation Wizard did not reposition the parent range if the parent position
was adjusted in the dialog when editing the model. This led to cases where the model would
fail to fit some files if the parent population moved beyond the scope of the range.

Bug fix: program could crash if low or high channels for zoom graphics were greater than
256 channels AND preference settings for Desired Resolution were 256. Crash would occur
when a new data file was read into the report.

Bug fix: program could crash with file or report paths greater than 120 characters.
Revisions now support paths to 250 characters.

Bug fix: When batch analyzing files using the "Fit with current model" option, the program
would slow down as it progressed through the batch. An internal array was not properly
cleared in this scenario.

Bug fix: With auto-scaling disabled, user-defined scale didn't apply when advancing to next
batch file while in scale view. This has been corrected.

Version 3.1

20 © 1994-2017 Verity Software House. All Rights Reserved.

ModFit LT Help New Features

Several new keywords have been documented.

The Edit Keywords dialog now uses an Add button instead of a Copy button. The added
equation will have a standard default value.

OLE features have been enhanced to provide more extensive OLE Automation capabilities.

A Reset button has been added to the batch controls.

A new AutoAnalysis setting has been added to make all G2M's dependent. By default, this
setting is enabled. This is a SIGNIFICANT CHANGE from previous versions that greatly
improves operator-to-operator consistency.

The logic that detects shifting of positions of internal standards has been improved.

Crashes that could occur if AutoAnalysis could not create any cell cycles in the model have
been corrected.

Options and Configuration now has an Overrides section that can apply settings to models
in reports as they are loaded.

Models now have the ability to ignore internal standards in the generation of debris and
aggregate components. By default, internal standards are now ignored by these components.

The Full Model Editor now displays the component ID when showing the list of components
in the model.

Bug fix: Sync and Prolif wizards didn't clean out analysis keywords from database or
keyword objects. Corrected 30Jul2001 (vi 969)

Bug fix: Debris component could report negative percentages and areas in unusual cases.
Corrected 30Jul2001. (vi 999)

Bug fix: The program did not read INCLUDE equation files from the model's path if a model
was opened in the Full Model Editor. Corrected 26Jul2001.

Bug fix: A crash occurred when a model component (other than the last component) was
deleted from a model with the Full Model Editor. Corrected 25Jul2001 (vi 1010)

Bug fix: A crash in peak finder could occur when a histogram had no events. This unusual
case could occur in a DDE connection with WinList when the histogram in WinList is gated
and shows no events. Corrected 25Apr2001

Bug fix: The program can now identify hypo-diploid conditions where the hypo-diploid peak
is at a lower channel than an internal standard.

Bug fix: The program now defeats S-phase cutoff warning when in OLE automation mode.
Corrected 4Sep2001 (vi 294)

Bug fix: A custom model in saved report did not "fit" when the report was re-opened.
Corrected 4Sep2001 (vi 975)

© 1994-2017 Verity Software House. All Rights Reserved 21

New Features ModFit LT Help

Version 3.0

AutoDebris™ component handles Paraffin, Fresh and Frozen preparations.

AutoAggregates™ provides a more robust system for handling higher aggregate forms.

AutoLinearity™ determines the best linearity setting for G2M and aggregates automatically,
eliminating a major source of analysis variability.

Cycle Analyzer™ features an enhanced PeakFinder to work with extensive variety of

histograms, and uses a new rule-based system for ploidy classification.

Auto-analysis now handles complex histograms such as near diploid, hypo-diploid,

multiploid, tetraploid plus aneuploid, and more.

Choosing a model uses a revised dialog to make model selection much more flexible and
open-ended. Models are created dynamically instead of being stored on disk.

The peak finder system has been completely revised to provide a much more robust,
statistical method for identifying peaks in histograms.

The new database system includes a database viewer, expanded keyword list, and support
for multiple databases active at one time.

Quick Model Editor allows quick changes to current model, with quick access to
AutoDebris, AutoAggregates, G2M, S-Phase, and Linearity.

Full Model Editor provides a complete model construction system. Use existing models as
base for edits or start from scratch to build custom models for novel applications.

Dynamic model creation system builds models on the fly for auto and manual analysis.

New batch analysis controls provide efficient and sophisticated data file and report
processing. The new batch toolbars automatically appear when multiple files are selected,
with VCR-style buttons to allow go-to-first-file, repeat, next file, auto-advance, and go-to-
last-file.

Powerful batch configuration options are available for processing data files and reports.

User Defined Keywords allow you to create custom equations that can be displayed on
reports and stored in databases.

Revised report format includes new results for events in cell cycles, average cycle events
per channel, and an improved layout for results.

New graphics include a legend of model component colors, report objects that zoom in on
portions of the histogram, and a report representation of the Fit Diagnostics dialog.

Export histogram and report graphics to disk as PICT, metafile, or bitmap.

22 © 1994-2017 Verity Software House. All Rights Reserved.

ModFit LT Help New Features

Histograms can now be analyzed at 1024 channels, and support has been added for 16-bit
FCS files, multiple histogram FCS format, and ASCII data file format.

New Auto-analysis options allow you to set tetraploid threshold and determine how ploidy
will be determined.

The Proliferation Wizard features new results for Upper Generation Proliferation Index
(UGPI) and Precursor Frequency.

New Internet-based registration system allows quick registration and access to Verity
support.

Three levels of user-access to program features allow better control over program
configuration options.

HTML-based online help provides more efficient searching and viewing of documentation.

An event logging system has been added to assist technical support in tracking down
problems.

Many reported problems with printers have been corrected.

Many reported problems from version 2.0 have been corrected.

© 1994-2017 Verity Software House. All Rights Reserved 23

ModFit LT Help System Requirements

System Requirements

Minimum System Configurations

PC: Windows 7, Windows 10 or greater with 50MB free disk space for program files.

Macintosh: OSX 10.8.5 or greater with 50MB free disk space for program files.

Optimal Configurations

Faster CPUs with more cores and greater RAM will improve performance.

© 1994-2017 Verity Software House. All Rights Reserved 24

 ModFit LT Help Installation and Setup of ModFit LT

Installation and Setup of ModFit LT

Before you begin, make sure the computer meets the System Requirements.

You must have administrative rights to the computer during installation of the software.
After installation, the program can be run by users with restricted access to the computer.

You will need to register the software with Verity Software House in order to unlock the
program on the computer. This can be done using the online registration process or by
contacting technical support.

If your program is an update version, you will need the serial number for the previous
version in order to register and unlock the software.

Windows Stand-alone Version

Installation

Log on to your computer with an account that has Administrator privileges. You must have
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 ModFitLTSetup.exe to install the program and required support
libraries. 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.

Using the software

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
ModFit LT 5.0.

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 Installation and Setup of ModFit LT ModFit LT Help

Macintosh Stand-alone Version

Installation

Log on to your computer with an account that has Administrator privileges.

If you have an installation CD, insert the CD into the CD-ROM drive. If you downloaded the
software, extract the installation files from the download into a temporary folder.

Run the ModFit LT Installer. Follow the directions on the screen to complete the installation.

Using the software

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 ModFit LT application in the Applications/ModFit
LT folder.

26 © 1994-2017 Verity Software House. All Rights Reserved.

ModFit LT Help Registering your new software

Registering your new software

Why You Should Register

With this version of ModFit LT, 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. An unregistered installation will be fully
functional for 30-days, and then become a "trial version" with limited functionality until it is
registered..

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 Register button on the dialog
box that is displayed when the program starts up.

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Registering your new software ModFit LT Help

Click the Next button to begin the registration process.

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

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ModFit LT Help Registering your new software

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
appear; they are case-sensitive. If you have lost or misplaced the Serial number or CD Key,
contact Verity technical support at tech@vsh.com 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.

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Registering your new software ModFit LT Help

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.

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ModFit LT Help Registering your new software

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.

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Registering your new software ModFit LT Help

Follow instructions on screen to complete the process.

To register your software using another computer...

When the installation computer does not have internet access but another computer does,
you can use the fourth option in the list and click Next to begin the process.

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ModFit LT Help Registering your new software

On the installation computer, write down the Computer ID displayed in the

Register Software dialog.
Next, go to the computer that has Internet access, launch a browser, and navigate to
www.vsh.com.

Click the Register Software link.

If you already have a web account, login with your user name and password. If you do not
have a web account, create a new account that will be associated with this registration.

After you login, the Software Registration page will be displayed on the web site. Fill in
the information in the form on this page to register the product. If you are registering an
update version, you will need the serial number of the previous version to complete the
registration. If you are registering an additional user license, you will need the serial number
of another copy of the program that is registered to your institution.

Getting an Unlock Code is a two-step process. First, you fill in and submit the registration
form on the web site. This sends a registration request is sent to Verity. Your Unlock Code is
then emailed to you, so make sure you provide a valid email address.

When you receive your Unlock Code via email, paste or type it into the Unlock Code field in
the Registration dialog box on the installation computer. When possible, use Copy and Paste
to do this, to ensure that the characters are correct. Then click the OK button.

Your program will become fully functional once the Unlock Code is entered correctly.

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Registering your new software ModFit LT Help

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

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ModFit LT Help Registering your new software

Verity Software House

PO Box 247, Topsham, ME 04086
USA

Email: verity@vsh.com

Web: www.vsh.com

Phone: (207) 729 6767

Fax: (207) 729 5443

© 1994-2017 Verity Software House. All Rights Reserved 35

ModFit LT Help Tutorials

Tutorials
First Time Using ModFit LT

This short tutorial will introduce you to the major features of the program. It just takes a few
minutes.

Start the program by following instructions in the Installation and Setup section.

Click the OK button on the opening dialog, if it appears.

The ModFit LT ribbon bar appears at the top of the application window. Buttons are grouped
to give you quick access to the most common commands in ModFit LT.

Opening a data file

Click the Open FCS button.

The Open File dialog is displayed. With this dialog, you can navigate to a folder in which you
have stored the data you want to analyze with ModFit LT. We will just open a sample file for
now.

Select Sample1.fcs from the Samples folder located where ModFit LT is installed. Click
Open to load the file.

ModFit LT reads the sample file and displays the histogram on the report page. You are now
ready to analyze the sample.

Auto-analyzing the data

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ModFit LT Help Tutorials

Click the Auto button.

ModFit performs its automatic analysis of the sample. It scans the histogram for peaks, and
based on the sizes and positions, the program attempts to determine the number of cell-
cycles in the sample. Then, it creates a model to use with the sample and performs a non-
linear least squares analysis of the data.

Review the analysis

The program displays the analyzed histogram and the computed percentages for various
populations. All of the elements on the report page are moveable, and most can be re-sized,
allowing you to customize the look of your reports.

Review the results of the analysis to see the type of information ModFit LT provides. See the
section on Analysis Results for more on this topic.

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Tutorials ModFit LT Help

Adding text to the report

Select the Insert tab of the ribbon bar to display tools to add elements to your reports.

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ModFit LT Help Tutorials

Click the text tool in the report toolbox.

The Edit Text dialog appears. Here you can enter any text you want to add to the report. In
addition, you can store comments in a database so they can be easily retrieved without
retyping them. See Report Design for more on using comments and moving objects on the
report.

Type a comment, and then click OK.

The comment is displayed on the report. You probably need to move it to another location.

Click and hold the mouse button down on the text you just added. Drag it to a new
location and release the mouse button.

All report objects can be moved in the same manner.

Auto-naming and saving the report

This is a good time to save this report.

Click the Save button on the Home tab of the ribbon.

The Save Report dialog will appear with a suggested name for the report, based on the
name of the FCS data file that was analyzed. Navigate to a location where you want to save
the report and click Save.

From the Home tab, click New. This option clears the report from the screen.

Retrieving the report

When you want to retrieve a report from disk, you simply re-open the report.

From the Report group of the Home tab, click Open.

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Tutorials ModFit LT Help

The Open Reports dialog is displayed, showing the reports in the current folder. Here you
can navigate to a new folder if you need to.

Select the report you just saved. (Hint: Look for the name of the data file, "Sample1.FCS".
There should be a report with "Sample1" as the first part of the name.)

Click Open.

The report will be loaded, and you should see things just the way they appeared when the
report was saved.

You can also use the Recent Reports menu in the File menu to select from recently saved
reports.

Welcome to the world of modeling with ModFit LT.

See also:

Creating a report template

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ModFit LT Help Tutorials

Manual analysis with ModFit LT

If ModFit LT has difficulty detecting peaks or identifying the ploidy patterns of a sample using
automatic analysis, you can use manual analysis to analyze the data.

Manual analysis involves several steps. First, you open the sample you want to analyze. Next,
you use the Choose Model dialog box to select a model that matches your assessment of the
sample. Once the model is selected, you need to position ranges over important peaks, and
finally tell the program to analyze the sample.

When to Use Manual Analysis

Here is an example.

Click the Open FCS button on the Home tab of the ribbon.

Open the file "Problem1.fcs" from the Samples folder.

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Tutorials ModFit LT Help

There are several problems with this histogram, the most important being that its peaks are
very broad and indistinct. In order for Auto Analysis to succeed, the important peaks in the
histogram must be correctly identified. Peaks are shown by the small black triangles under
the graphic. Depending on your Peak Finder settings, the program probably missed one or
both of the dominant peaks in the histogram.

This is a circumstance where you must use manual analysis to model the sample. Even if the
peaks are all detected, you can still continue with this tutorial.

Choosing a Model

Click the Choose Model button on the ribbon bar and a dialog is
displayed.

This dialog allows you to define the model that you want to use for the histogram. Based on
the options you choose, the program will construct a model to match those assumptions. This
is a DNA Aneuploid sample, so the selections we make in the dialog need to reflect that.

Enable AutoDebris by checking the AutoDebris checkbox.

Enable AutoAggregates by checking the AutoAggregates checkbox.

Disable Apoptosis by clearing the check in the Apoptosis checkbox.

Set Linearity to 2.0. This is the default G2/G1 ratio.

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ModFit LT Help Tutorials

Set Internal Standards to 0. There are no internal standards in this file.

Set Number of cycles to 2. We want to model 2 cell cycles in this data file: a Diploid and
an Aneuploid cycle.

For the Model template, choose "Dip Ane". This tells the program the ploidy to assign to
each cycle.

For the Range positions, choose "Compute range positions". If you use this dialog to
modify a model, you might choose "Use current range positions" so that the program did not
move existing ranges.

Click OK to confirm your choices and close the dialog.

Adjusting Ranges

The program will construct the model and try to position the model ranges over detected
peaks. It switches to the Range view automatically. You can switch to this view anytime by
clicking the Range button on the ribbon.

In the Range view, you can position ranges over the peaks in the sample. You move a range
by clicking on the range label, and dragging it to a new location with the mouse button
down. When finished, you release the mouse button. To adjust the width of a range, click the
black handle and drag to choose a new size. If a range is already correctly positioned and
sized, do not move or resize it.

Make sure the Debris range is located over the starting point of debris at the left edge of
the histogram. This range is always used to identify the start of debris. The right side of the
range should be placed at approximately two-thirds of the histogram scale.

Next, ensure that the Dip G1 range is positioned over the first peak, the DNA Diploid G0-
G1. It does not need to be stretched to encompass the peak as long as it is centered on the
peak. You should see a red fill in the first peak that indicates the range estimate of the size
of the peaks.

Move the An1 G1 range over the center of the second peak, the DNA Aneuploid G0-G1.
This peak will fill with yellow to indicate the range estimate.

Notice that as you move a range, the left pane on the status bar displays information about
the range position. The left and right values are in the units shown on the X-axis; the top is
in number of events. When the mouse button is released, the status bar indicates the fitted
estimates for the range.

Move the Dip G2 range over the portion of the histogram that is about twice the position
of the Dip G1 range. This is the approximate position of the Diploid G2-M.

Move the An1 G2 range to about twice the position of the An1 G1 range. This is the
approximate position of the Aneuploid G2-M.

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Tutorials ModFit LT Help

Fitting the Model

With the ranges in position, you are ready to analyze the sample.

Click the Fit button on the ribbon to perform the non-linear least squares fit. The
results will be similar to the one shown here.

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Review the analysis to ensure that the model correctly matches the sample. If it does not,
you should make sure you selected the correct model and that its ranges are correctly
positioned.

Summary

The manual analysis mode should be used when the peak finder or automatic analysis mode
fails, or you need to make your own decisions about how to model a sample. It enables user-
intervention at the key points in the analysis: model selection and range positioning.

Models are selected using the Choose Model option on the Home tab of the ribbon bar.

Ranges used to identify peaks must be positioned so that each range identifies a unique
peak; they should not overlap. For example, the Dip G1 range is used to identify the Diploid
G0G1 peak, and no other peak range should be placed over the same peak. You should not,
for example, place the An1 G1 range over the same peak as the Dip G1 range. The Debris
range is the only exception. Since it does not identify a peak, it should be stretched across
about two-thirds of the histogram.

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Tutorials ModFit LT Help

See also:

Model Descriptions

Choose Model command

Ranges command

Using the Sync Wizard

Using the Proliferation Wizard

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ModFit LT Help Tutorials

Using the Sync Wizard

This tutorial introduces the use of the "Sync Wizard", used to analyze data with perturbed
populations. The Sync Wizard allows you to specify important aspects of your data and
constructs a model to match. Once the model is created, you can use the Sync Wizard to run
through a set of files, adjusting the model as needed.

You will learn how to start the Sync Wizard, how to adjust the settings used to create the
model, and how to analyze your data samples with the model created. Best of all, you will
see how easy it is to use the Sync Wizard to model synchronized cell lines.

Let's dig in.

Starting the wizard

Start ModFit LT.

Click the Open FCS button on the ribbon bar. The Open dialog box will appear.

The files you will use in this tutorial are stored in a folder called "Sync" in the Samples folder
under the ModFit LT program folder.

Navigate to the Sync folder and open "Cho074dn.fcs". This is the control population, and
is the first file you will analyze.

Click the Sync Wizard button on the Home tab of the ribbon bar. The Sync Wizard dialog
will appear.

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Tutorials ModFit LT Help

Setting up the model

Notice the tabs across the top of the dialog. These tabs are labeled with the categories you
can adjust in the model.

The options on the Start tab let you create or edit a model, and also provide an alternate
path to open a data file. Since you have already opened the file we want to use and are
ready to create a model, you do not need to make any changes to the Start tab.

Click the G0-G1 tab.

This tab displays your data file so that you can tell the Sync Wizard where the G0-G1 peak is.

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ModFit LT Help Tutorials

The program positions the red cursor over the first peak it finds in the sample. In this case, it
is correctly positioned. If, however, you need to adjust it, you simply click the red triangle
and drag it over the G0-G1 peak. If you would rather type in a position, you can edit the G0-
G1 channel edit box directly.

Now you need to decide whether to let ModFit LT adjust the G0-G1 position during the
modeling process, or to lock the G0-G1 position. Here is how to decide.

If your sample has a clearly defined G0-G1 peak and you want the program to look for the
best peak position over a range of channels, choose "Adjust automatically." The Sync Wizard
will create a range, "G0G1", with boundaries above and below the peak position, centered on
the peak. It will look for the peak within the "G0G1" range. If your G0G1 peaks shift from file
to file, as long as they fall within the range the program should find them. If the G0G1 peaks
shift too much, you can adjust the position or size of the "G0G1" range to account for the
shift. In fact, the Sync Wizard will prompt you if it cannot find a peak in the range.

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Tutorials ModFit LT Help

If the peak is not clearly visible or you do not want the program to scan for the G0-G1 peak,
you should choose "Lock the position". This setting prevents the program from adjusting the
position.

Select "Adjust automatically" for this sample.

Click the G2-M tab.

This tab displays your data file so that you can tell the Sync Wizard what the G2/G1 ratio is.

If you know the G2/G1 ratio already, you may enter it directly into the G2/G1 ratio edit box.

Enter 1.92 in the G2/G1 ratio edit box.

If you have a sample with a visible G2-M peak, you can click the red triangle and position it
over the G2-M peak. As you move the triangle, the channel position and G2/G1 ratio values
update automatically.

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ModFit LT Help Tutorials

The model created by the Sync Wizard will multiply the G0-G1 peak position and standard
deviation by the G2/G1 ratio appearing on this tab to compute the G2-M mean position and
standard deviation. The position of the G2-M peak is always, therefore, dependent on the
position of the G0G1 peak. Similarly, the standard deviation of the G2-M peak is dependent
on the standard deviation of the G0G1 peak.

Click the S-Phase tab.

This tab allows you to select what shape you want to use to model S-Phase, how many of
them you want to use, and how to space them if you use more than one. Let's change the
setting to 5 equally spaced rectangles.

Change the Number of Compartments to 5.

Click the Other tab.

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Tutorials ModFit LT Help

This tab allows you to enable debris and aggregate subtraction if needed. The default state
of no debris or aggregates is fine for this example.

Finally, click the Report tab.

Here you will find options that let you fine tune the information reported by the Sync Wizard
model.

Running the model

You are now ready to do the analysis.

Click the Analyze button on the Synchronization Wizard dialog.

The Sync Wizard creates the model you just designed, sets some ranges on the data, and
fires off the analysis of the sample. You should see results similar to these:

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Notice that the S-Phase is reported both as a total percentage and for each of the rectangle
compartments in your model. You could at this point add comments, print and save your
report. You do not need to do that for the tutorial.

You will also notice a "Wizard" toolbar is now docked at the bottom of the report.

The first button on the toolbar lets you open and analyze a data file in a single operation.
The second re-displays the Synchronization Wizard dialog so that you can edit the model.
And the third lets you exit the Sync Wizard. These options are also available on the Home
tab of the ribbon.

Adjusting the model

Let's suppose you are interested in just three S-Phase compartments: early, mid, and late S-
Phase. Using 3 compartments will make the model more conservative in S-Phase. A more
conservative model avoids over estimating the S-Phase population. You can edit the model to
do this quite easily.

Click the second button on the Wizard toolbar. The Synchronization Wizard dialog is
displayed.

On the Start tab, the Wizard has switched from "Create" to "Edit the current synchronization
model" automatically.

Click the S-Phase tab.

Change the "Number of Compartments" from 5 to 3.

Click the Analyze button. The sample is analyzed again, this time reporting on just 3
compartments in S-Phase.

Opening and analyzing new data files

Let's run through several more samples to see what happens to the data.

Click the first button on the Wizard toolbar. The Open dialog appears.

Open the next file in the sequence from the Sync folder: cho075dn.fcs. As soon as the
file is loaded, the Sync Wizard analyzes it and the new results displayed.

Continue opening each subsequent sample. The S-Phase and G2-M populations begin to
increase while the G0-G1 peak diminishes.

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Tutorials ModFit LT Help

When you have finished with the Sync Wizard, click the third button on the Wizard toolbar.
The Wizard will ask whether you are finished using the Wizard. Choose Yes to exit the
Wizard.

Summary

The Synchronization Wizard is a tool designed to allow you to easily analyze samples with
perturbed S-Phase and G2-M populations. It is divided into "tabs" that allow you to describe
your data and choose options to use in modeling your data.

Once you have created a model with the Wizard, the Wizard toolbar is displayed. It provides
shortcuts to opening and analyzing samples, adjusting the Sync Wizard settings, and quitting
the Sync Wizard.

Control the model's flexibility by adjusting the shape used and number of compartments in S-
Phase. The default setting, 3 rectangles, is good for most cases. The trapezoid shape more
closely approximates the shape of the perturbed S-Phase distribution, but it can provide the
model too much flexibility.

A Few Additional Notes

If you save a report with the Wizard toolbar displayed and the Wizard active, you will save
the current state of the Sync Wizard model in the report. In this way you can create your
own "default" settings for the Synchronization Wizard. Simply re-open the report to return to
the saved state.

If you allow the model to "Adjust automatically", you can switch to the Range view and
adjust the G0-G1 range so that it encompasses the range of channels over which the
program can scan for the G0-G1 peak. If the Sync Wizard does not detect any peaks under
the G0-G1 range and it is set to "Adjust automatically", it will prompt you with several
choices: to move the range, to lock the position, or to cancel the analysis.

See Also:

Sync Wizard: Start tab

Sync Wizard: G0G1 tab

Sync Wizard: G2M tab

Sync Wizard: S-Phase tab

Sync Wizard: Report tab

Sync Wizard: Other tab

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Using the Cell Tracking Wizard

This tutorial introduces the use of the Cell Tracking Wizard, used to analyze cell tracking dye
samples.

You will learn how to start the Cell Tracking Wizard, how to adjust the settings used to create
the model, and how to analyze your data samples with the model created. The primary job is
to identify the location of the parent population and confirm a few other settings. Once these
simple decisions are made, the Wizard constructs a model that easily analyzes your cell
tracking samples.

Starting the wizard

Start ModFit LT.

Click the Open FCS button on the ribbon bar. The Open dialog box will appear.

The files you will use in this tutorial are stored in a folder called "Proliferation" in the Samples
folder under the ModFit LT program folder.

Navigate to the Proliferation folder and open "nonprol.fcs". This is the first file you will
analyze, the "parent" population.

Choose "FL2-Height Log" as the parameter to analyze.

Click OK in the Analysis Setup dialog; we will not define any gates for this example.

Click the Cell Track Wizard button on the Home tab of the ribbon bar.
The Cell Tracking Wizard dialog will appear.

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Setting up the model

The tabs across the top of the dialog are labeled with the categories you can adjust in the
model.

The options on the Start tab let you create or edit a model, and also provide an alternate
path to open a data file. Since you have already opened the file we want to use and are
ready to create a model, you do not need to make any changes to the Start tab.

Click the Parent tab.

This tab displays your data file so that you can tell the Cell Tracking Wizard where the Parent
population peak is. The program automatically positions the red cursor over the right-most
peak it finds in the sample.

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Click the red triangle and drag it over the center of the Parent peak, if necessary.

If you would rather type in a position, you can edit the Parent channel edit box directly.

This tab also allows you to "lock" the standard deviation (SD) of the Gaussian component
used to model the parent population. This option is most useful when analyzing samples in
which there is a great deal of proliferation. In these cases, the parent population is often
difficult for the model to resolve, and the modeling process broadens the SD of the parent
too much. For this example, leave this option unchecked.

Click the Generations tab.

Normally, you will not need to make adjustments on this tab. It allows you to review and edit
the number of generations to model and the type of model to use.

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The wizard provides two variations for modeling the generations: Standard and Floating. In
the Standard mode, the mean position of the Gaussian for each generation is dependent on
the position of the parent generation. For example, if the parent population is located at
channel 2000, the first generation will be located at half the parent location, or 1000. Each
generation will be at the location that is half the location of the previous generation.

The floating option allows the mean of each generation to be computed independently of the
other populations. This option provides much greater flexibility to the model and will work
well for samples with visually distinct generations. The floating option is not recommended
for samples where generational boundaries are not obvious to the eye.

For our example, we do not need to adjust any additional settings.

Running the model

You are ready to analyze the first sample with your model.

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Click the Analyze button on the Cell Tracking Wizard dialog.

The wizard creates the model and analyzes the sample.

Percentages and means are reported for the parent and all generation populations. At this
point you could add comments, print and save your report. You do not need to do that for
the tutorial.

You will also notice the wizard toolbar is now docked at the bottom of the report.

The first button on the toolbar lets you open and analyze a data file in a single operation.
The second re-displays the Cell Tracking Wizard dialog so that you can edit the model. And
the third lets you exit the Cell Tracking Wizard. These options are also available via the cell
track wizard button on the ribbon bar.

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Opening and analyzing new data files

After setting up the model on a non-proliferating sample, you are ready to open and analyze
the samples collected at subsequent time points. For our tutorial, we have one proliferating
sample.

Click the first button on the wizard toolbar. The Open dialog appears.

Open the next file, "prol.fcs". As soon as the file is loaded, the wizard analyzes it and the
new results are displayed. We now see some interesting distributions and the percentages for
each generation. Also reported are some indices to help quantify the extent of proliferation in
the sample.

Adjusting the model

You may be interested in trying several other options in the Cell Tracking Wizard, found in
the Other tab. To try these, you need to display the dialog.

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Click the second button on the wizard toolbar. The Cell Tracking Wizard dialog is displayed.

On the Start tab, the Wizard has switched from "Create" to "Edit the current cell tracking
model" automatically.

Click the Other tab.

Enable the Background option. This will use a rectangle to subtract debris in the sample.

Enable the Asymmetric cytokinesis option. This option will interpolate along the
background rectangle to set the standard deviations for the generations. It yields an analysis
result that quantifies the degree of error the cells made in dividing in half.

The other edit field in this group defines the starting generation for "true proliferation." This
value is used in the calculation of Upper Generation Proliferation Index and the Precursor
Frequency. For a description of the use of Precursor Frequency, see "A flow cytometric
method to estimate the precursor frequencies of cells proliferating in response to specific
antigens", Givan, A., et al, Journal Of Immunological Methods Vol. 230, 1999 Elsevier Science
B.V.

Click the Analyze button. The sample is analyzed again, with slightly different results.

When you have finished with the Cell Tracking Wizard, click the third button on the wizard
toolbar. The Wizard will ask whether you are finished using the Wizard. Choose Yes to exit
the Wizard.

Summary

The Cell Tracking Wizard is a tool designed to allow you to easily analyze samples prepared
with cell tracking dyes. It is organized by categories into tabs which allow you to describe
your data and choose options to use in modeling your data.

Once you have created a model with the Wizard, the Wizard toolbar is displayed. It provides
shortcuts to opening and analyzing samples, adjusting the Cell Tracking Wizard settings, and
quitting the Wizard.

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Enhanced Cell Tracking Analysis

Features in ModFit LT and WinList provide unparalleled capabilities to analyze cell

proliferation.

ModFit LT performs sophisticated least squares analysis in just a few clicks of a mouse and
reveals the secrets of the proliferation data: proliferation index, non-proliferative fraction,
and precursor frequency, and fractions for each proliferating generation. The proliferation
results can be exported so that other applications can extend the analysis.

Enter WinList. NStat regions can import the proliferation results from ModFit LT, and then
partition the generations according to the proliferation analysis. This enhancement sets the
stage for exciting new analysis capabilities.

This tutorial will explore these new features to examine a CFSE-stained sample containing
several other markers.

The big picture

From the Cell Tracking Wizard in ModFit LT...

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... into WinList for exciting, new explorations of proliferation.

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Getting started

To start, we will use the Cell Tracking Wizard in ModFit LT and export its results. Then, you
will set up WinList to import those results into an NStat region.
You will need both ModFit LT and WinList for this tutorial. WinList must be version
9.0 or greater.
1. Launch WinList and pass through the opening dialogs.

2. Launch ModFit LT and pass through the opening dialogs.

3. Using ModFit LT's Open FCS command, navigate to the Samples folder located in the
WinList application folder. Select "CFSE sample.fcs" and click Open. Note, the dialog may
look different than the one shown below, depending on the version of ModFit LT you are
using.

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4. Choose "FL1-H-CFSE" as the analysis parameter, and click OK.

5. Do not create any gates for this file; we'll do that later in WinList.

6. Click the Cell Tracking Wizard button on the ribbon bar.

7. In the Cell Tracking Wizard dialog, click the Generations tab and choose the "Floating"
model option from the drop-down list. Click the Other tab, and enable Export partition
table and Prompt for file name checkboxes. These options will cause the program to
create the export file after analysis and allow you to enter a name for the export file. Finally,
click the Analyze button to perform the analysis.

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8. When the analysis is complete, the Choose Location to Save Partitions dialog will be
displayed. You can save the file to any location. For this tutorial, save the export file to the
desktop so that it's easy to find later.
When you choose the Export option for the Cell Tracking Wizard, the program
determines the boundaries between adjacent generations and writes that information
to the export file. The boundaries are the points of intersection between the Gaussian
components in the model.

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Setting up WinList

9. Activate WinList and click the Open FCS button on the ribbon bar.

10. Navigate to the Samples folder located in the WinList application folder. Select the "CFSE
sample.fcs" file and click Open.

11. In the Create Histograms dialog, select CFSE in the X Axis list and CD69 PE in the Y
Axis list. Click Add to List two times. This will create two copies of CFSE vs. CD69 PE. Click
OK to close the dialog.

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You can change the graphical display for the histograms you've just created. Right
click on the histogram and choose Graphics. Try out the different display options to
see what you like best..
For this analysis we need to use a standard Log transform for the X and Y axis, not
HyperLog. The exported information from ModFit LT is in Log units.

12. Move the mouse over the CFSE label on the X-axis and click the small triangle that
appears. Select Log from the list of transformations. Repeat this for the CD69 Y-axis.

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13. Click on the title bar of the first dot plot to activate it. Click the NStat region tool in the
histogram toolbar.

14. In the NStat Properties dialog, click the Import button.

15. Navigate to the Desktop folder and select CellTrackingWizardPartitions.txt and click
Open.

16. Edit the NStat rows to 1 and check the Show Vertices, Results, and Enable gates
checkboxes. The number of columns should already be 10 from the Import. Click OK.

Isolating the generations

At this point, you have completed the basic import process. Let's extend the WinList analysis
to explore other markers as a function of the proliferation analysis.

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17. Click the title bar of the second histogram, and click the NStat region tool button to
create another NStat. For this one, set NStat rows to 2 and NStat columns to 1. Check the
Enable gates option and uncheck other options. Click OK.

Now, one of the histograms contains an NStat that shows the proliferation partitions, and the
other histogram has an NStat that will allow us to define the CD69 negatives and positives.

18. Drag the region by the label to adjust the second NStat so that it approximately divides
the two major populations in the second histogram, as shown.
You can change the graphics for any histogram by choosing Graphics from the
right-click context menu for a histogram.

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To analyze and visualize the generations better, we will create a calculated parameter using
the FCOM function. This function creates a new parameter that will allow us to look at each
population independently. It uses gates as input, and we'll use the gates from the NStat
regions we just created.

Let's create a few FCOM calculated parameters to help isolate the generations.

19. Click the Add Parameter button on the main ribbon bar. Alternatively, you can right-
click in the data source and choose Add Parameter from the context menu.

20. Click the Add button in the Edit Calculated Parameters dialog.

21. Type Proliferation in the Name edit box. Type or copy this expression into the
Equation edit box, and then click OK.

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FLOG(FCOM(G1,G2,G3,G4,G5,G6,G7,G8,G9,G10)+0.5)*256/FLOG(1024)+FR
ND(3)

This expression deserves some explanation. Let's break it apart, starting with the FCOM
portion. FCOM is a "gate combination" function. It takes any number of gates as arguments;
the result of the equation depends on how many gates are in the expression. With a single
gate, there are 2 possible event classifications: an event is "in" or "not in" the gate. With two
gates, there are 4 possible event classifications. With the ten gates in our expression, FCOM
can produce 1024 different event states.

However, since these gates do not overlap at all, most of the 1024 possible states have no
events in them. In fact, the only states that contain events are the 10 single-positive event
states, resulting in values of 1, 2, 4, 8, 16, 32, 64, 128, 256, and 512. If we plotted the result
of the FCOM function as a histogram, we would see 10 spikes of events, and the spikes
would be exponentially spaced.

Our goal is to see these 10 spikes evenly spaced across the histogram. This is where the
FLOG function comes in. By taking the log of the FCOM output and scaling it by 1024, we
redistribute the 10 FCOM values evenly across 256 channels. The final portion of the
expression, "+FRND(3)", changes the spikes of events into Gaussian distributions so that
they can be better visualized.
Take a look at the WinList reference sections on Add Parameters and FCOM for
details on the complete set of WinList functions.
22. Once again, click the Add button in the Edit Calculated Parameters dialog.

23. Type CD69 Expression in the Name edit box. Type or copy this expression, and click
OK.
(FCOM(G11)+0.5)*256/2+FRND(3)

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24. Click OK.

CD69 expression as a function of proliferation

We're now ready to explore this data file in really interesting ways. Wouldn't it be nice to see
CD69 as it relates to proliferation, viewing each generation as a discrete cluster? Could we
quantify the CD69 expression of CD69-positive cells for each generation? Yes and yes.

25. From the Insert tab on the ribbon bar, click the Density plot. This creates a Density
plot in a default location on the data source. Click the X Axis label and choose Proliferation.
Click the Y Axis label and choose CD69 PE.

Let's create another histogram to isolate the CD69 positives and negatives.

26. Right-click on the histogram that we just created and choose Clone from the context
menu. A duplicate copy of the histogram is created.

27. Change the Y Axis to show CD69 Expression.

In the graphics we can see the generations separated into discrete populations along the X-
axis. That's the first FCOM function we created. The Y-axis shows the continuous CD69
parameter in one histogram and discrete subsets of CD69 positives and negatives in the
other histogram. Regions can be created to quantify the subsets.

You can, of course, explore how other parameters relate to cell proliferation. Simply create
additional histogram to display the parameter combinations you are interested in.

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Making life easier

You have done the real work in setting things up with this tutorial. If you save this setup as a
WinList protocol or protocol bundle, the calculated FCOM parameters, NStat regions, and
selected histogram can be recalled very easily from disk.

When you analyze new data files with ModFit LT's Cell Tracking Wizard, you can export new
partition files. Then, open the same data file into your WinList protocol and import the
partitions into the NStat region as we have demonstrated in this tutorial. All of the calculated
parameters will update based on the new data and partitions.

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Using Auto Linearity

Purpose:

The purpose of this tutorial is to demonstrate the appropriate use of ModFit LT linearity
controls. The goals of the tutorial are listed below.

1. Define linearity and its potential effects on cell cycle analysis.

2. Show an example of a fit with an inappropriate linearity setting.

3. Demonstrate the manual method of adjusting linearity.

4. Discuss the linearity configuration settings.

5. Discuss a recommended approach to modeling with an optimized linearity setting.

Definition and Potential Effects

We define linearity as the observed ratio between the DNA Diploid G2M and G0G1 positions.
Theoretically, this value should be 2.0, but it can vary from this ideal value.

Linearity affects cell cycle analysis in several ways. The first effect is that for models that
have indistinct G2M peaks, the program uses the linearity setting to position the G2M in the
fitting process. For example, if a G1 peak is found at channel 100, the G2M position would be
at 200 with the linearity setting of 2.0. If the linearity setting were 1.9, the G2M position
would be 190.

The second major effect is on the aggregation model component. This model component
assumes, for example, that doublet positions are twice singlet positions and triplet positions
are three times the singlet positions. ModFit LT uses the linearity setting to transform the
data so that it is perfectly linear prior to creating the theoretical shape of the aggregate
distribution. Thus, linearity can also affect the positions of computed aggregates and affect
the model fit and S-Phase estimate.

How important is the linearity setting in estimating the S-Phase fraction? It can change S-
Phase estimates significantly, as shown in the next graph.

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In this particular example, a linearity setting of either 1.90 or 2.1 results in approximately
twice the aneuploid S-Phase estimate as a value of 1.99.

Therefore, it is important to optimize this setting for a particular histogram and model in
order to obtain accurate and consistent S-Phase estimates.

Example of inappropriate linearity setting

Here is a report demonstrating a model that used an inappropriate linearity setting.

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Notice that the Aneuploid S-Phase is quite high and the reduced chi-square is 1.97 for this
analysis.

In order to better appreciate the linearity effects, we also show a scaled view of the graph.
This allows us to easily see the Diploid G2M peak and aggregates.

Notice that the diploid G2M model component has disappeared - overwhelmed by the
aggregate component.

Automatic linearity detection from Quick Model Editor

Let's use a feature in ModFit LT that is designed to automatically find this optimal linearity
setting.

Click the down-arrow on the right of the Fit button on the ribbon bar and choose Auto
Linearity to trigger the command.

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With this method, the program models the data with a set of linearity settings, searching for
the one that results in the lowest reduced chi-square. The message status bar in the lower-
left corner indicates its progress.

After it has completed this analysis, it re-fits the histogram with the optimized linearity
setting. For this example the optimized linear setting is 1.96.

The advantage of using the automatic linearity detection is that it potentially eliminates an
important factor in S-Phase estimate variability.

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We can also automate the entire process with batch processing.

Automatic linearity with Batch Processing

When you are processing a batch of files, you can set the batch processor up to perform
auto-linearity for you. You can set this up in the Edit Properties for File Processor dialog,
found on the Options tab of the ribbon.

The first property in the dialog includes an option "Auto Analyze and Auto Linearity". Use
this choice if your data files generally auto-analyze well with ModFit LT. Similarly, there is a
choice to "Fit and Auto Linearity", which can be used for files that require manual analysis.

This is a powerful technique that enables optimal linearity detection automatically to be done
in background.

Linearity configuration settings

How does the program determine the starting and ending linearity value to test in the auto
linearity detection system? These settings are stored in the Options and Configuration section
of the program. This dialog can be displayed if you have administrative access to the
program by choosing the Configuration command from the Options tab of the ribbon bar.

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ModFit LT contains reasonable default values for these settings. If your laboratory has a
smaller range of possible linearity values, you can save considerable time by making the Low
linearity and High linearity settings more restrictive.

A recommended approach to modeling with an optimized linearity setting

Our recommended approach to the routine analysis of DNA files is as follows. First, find a
good average linearity setting for your laboratory and enter it into the Auto Analysis Settings
option in the program. You may also need to tailor other aspects of the auto analysis system
as well.

Second, use batch file processing with "Automatic Analyze and Auto Linearity" as much as
possible.

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Databasing with ModFit LT

Research and Compliance Mode note:

In RUO mode, all users can create and edit databases.
In Compliance mode, databases can only be created and edited by ModFitAdmins
and ModFitEditors.
This section introduces the concepts and functions of ModFit LT's database features. It is
more of a discussion than a tutorial, however there are examples to clarify some aspects of
the system.

ModFit LT has a simple, powerful database engine that allows you to store large amounts of
data easily in one or more database files. You can view databases from within the program,
manually add, delete, and replace records, and select the fields you want to store in each
database. The databases are created in a simple, text format known as tab-delimited text,
which can be read by common database and spreadsheet programs. These features give you
a great deal of flexibility in choosing what information to store, where to store it, and how to
integrate the information into other database and information systems.

How do you create a database?

It is best to run an analysis of a data file before you create or edit a database. Open one of
your data files and perform an Auto-Analysis. This will fill the program with the information
you can choose to store in your database. You can set up a separate database for each kind
of analysis you do, one database for everything you do with the program, or anything in
between.

The fastest way to create a database from your results is to use the Database Wizard,
found on the Edit tab of the ribbon.

The wizard helps you to select a database (or create a new one) and it adds fields for all of
the results that are shown on the current report. This is by far the fastest and easiest way to
set up a database.

If you want to select additional fields or have greater control over the database structure,
you can click the Databases on the Edit tab of the ribbon. The Edit All Databases dialog
will display.

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There is one database created by the program automatically. This database will not store
anything until you tell the program what information you want to save in the database. To do
so, you need to select the database in the list and click the Edit button. You can also double-
click the item in the list to do this.

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The Edit Keyword Map dialog is displayed. The left side of this dialog presents the
information available to store, and the database fields are shown on the right. Initially, the
right-hand list is empty.

What can you store in the database?

ModFit LT allows you to database virtually any statistic it generates, as well as keyword
information from the files that you analyze. The tree on the left of the dialog can be
expanded to display branches of keywords. To open or close a branch of the tree, double
click the label or click the arrow next to the label. Each branch of the tree contains a set of
related keywords.

To add an item to your database definition, select it in the tree on the left and click the Add
button. A field will be appended to the database. You can also select more than one Keyword
at a time and click Add to create multiple fields at once.

NOTE: Because of limitations imposed by common database and spreadsheet programs, you
may want to restrict the number of fields you create. Some programs will not read a text
database with more than 255 fields of data.

The field names initially assigned when you click the Add button are based on the keyword
added.

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You can edit the names for the fields after they are added to the list, if you wish. To do so,
click once on the field name to select it, then after a brief pause, click slowly again. An edit
control will appear around the field name, allowing you to type a new name for the field. You
can also use the Tab key to move from one field to the next if you need to edit the names of
several fields.

To remove one or more fields from the database, select the fields in the list and click the
Delete button.

To replace an existing field with a new keyword, select the new field in the Keywords list and
the one to replace in the list. Then, click the Replace button. The name of the field will not
change, only the keyword used to evaluate the field.

To change the order of fields in the database, select one or more fields in the list and then
use the Up and Down buttons to move the fields.

When you have finished selecting information to store in the database, click the OK button to
close the Edit Keyword Map dialog.

What files are created?

When you select keywords to store in a database, ModFit LT creates three or four files on
disk to manage the database. The location of the files depends on the operating system, as
shown in the table below. You can choose another location, as long as you have read/write
permissions to that folder.

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Operating System Default Location

Windows 7, 10+ C:\Users\[user name]\Documents\ModFit LT for Windows

Macintosh OSX /Library/Application Support/ModFit LT for Mac

A file is created to store a "map" of keywords to database fields. The map file name uses the
name of the database object and appends ".map" to it. For "Database1", the map file is
named "Database1.map".

Next, the program creates the actual database file. This is the tab-delimited, ASCII text file
that you may open in other database or spreadsheet programs. The database file name uses
the database object name and appends ".txt" to it. This file is readable in virtually any
program that can open unformatted text. The first line of the file contains the names of the
fields separated by tabs; it is known as the database header or definition. Each subsequent
line contains a record of information.

If the database had been created previously, ModFit LT will automatically create a backup of
the database before overwriting the existing one. The backup file will have the same name as
the database, with "backup" appended to it.

Finally, the program creates a text file called a "key" file. This is a text file that explains the
database and mapping files. You can open this file in a text editor to review the structure of
the database you have created. Its name is based on the database object name, with
"key.txt" appended to it.

How do you activate the database?

Once a database has been defined, there are several additional properties you can specify.
These properties determine whether the database is active, whether it will prompt you before
saving a record, whether or not you want to see the records in a database viewer, and where
you want to store the database.

To edit these properties for a database, select the database in the Edit All Databases
dialog box and click the Properties button at the bottom of the dialog. The Edit Properties
for Database dialog will be displayed.

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The properties are enabled or disabled with simple check boxes. You can use the Choose
button to use an existing database that you have defined previously. You can change the
location of the database using the Path button.

In order to store records of information in a database, it must be "active". To make a

database active, check the Active checkbox in the Edit Properties dialog. Database records
are saved after Automatic Analysis or Execute Model commands are performed. Additionally,
you can save a record at any time by clicking the "+" button on the database viewer.

If the Prompting checkbox is checked, ModFit LT will display a message each time it is
about to save a record to the database. You can choose at that time whether or not you
want to save the record. If Prompting is disabled, the database will save a record without
prompting.

To view the database while you work with ModFit LT, check the Viewer checkbox. A
database viewer window will be created and displayed. The database viewer can be moved,
resized, and scrolled horizontally or vertically. It has a toolbar that provides some additional
manual database functions. See the topic Database Viewer for more information.

The size of the font used for the database viewer can be set with the Viewer font property.

To select an existing database on disk, click the Choose button. A file selection dialog box
will appear, allowing you to navigate to the existing database and select it. Linking to an
existing database will rename the database in memory to match the one selected on disk,
and the associated database map on disk will be loaded as well.

To change the location of a database, click the Path button. A path navigation dialog will
appear. Navigate to the new location.

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How do you rename or create additional databases?

To rename a database, display the Edit All Database dialog box by selecting Edit Databases
from the Edit menu. Click once to select the database you want to rename. After a brief
pause, click again on the object to display an edit box in the Name column. Type the new
name for the database.

The name you type must contain only alphabetic, numeric, or space characters, and must be
at least 4 characters shorter than the maximum file name length for the operating system
you are using. If a file with the new name already exists, you will be given a chance to
overwrite the existing file, use the existing file, or cancel the operation.

When you want to create an additional database, click the New button. This will create an
empty database with a new name.

Each database has its own map of keywords to fields, and its properties determine whether it
is active, prompting, and showing a viewer.

Errors with databases

Since the database system involves creating and manipulating files on disk, there are
conditions where errors can occur. If, for example, one of the critical database files is moved
or deleted, the program will not be able to use the database. When the program runs into a
problem like this, it displays a dialog box of choices.

When you see this dialog, it means that the database "txt" file and the "map" file are out-of-
sync with one another. They may have a different number of fields, a different order,
different fields defined, or one of the files may be missing.

If you choose the first option, the map file is emptied and partially recreated with the fields
defined in the database header. In this case, you need to recreate the keyword-to-field
"map" using the Edit Keyword Map dialog.

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If you choose the second option, all records in the database txt file are emptied and a new
"header" is written into it to match the database map file.

The third option leaves the map and the txt files out-of-sync and deactivates the database,
allowing you to investigate the problem before any action is taken.

See also:

Edit Databases

Edit Keyword Map

Edit Properties for All Databases

Edit Properties for Database

Database Viewer

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Optimizing Auto Analysis

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins have permission to perform them.
This section describes how to configure Auto Analysis to work with your data files. Three
configuration dialogs are used to set up the program for Auto Analysis: Peak Finder Settings,
Auto Analysis Settings, and Options and Configuration dialogs. By modifying the content of
these three dialogs, you can optimize Auto Analysis to handle your data sets.

There are two major benefits to configuring the Auto Analysis system correctly. First, it will
reduce the amount of time you have to spend analyzing each file. Of equal importance, you
will increase user-to-user consistency of analysis results. The time you spend configuring the
system can save analysis time in very few analyses.

Prerequisites

In order to complete this process, you need a data set of twenty or more data files of the
same cell type that have been acquired according to the same protocol. Your protocol for
sample preparation, staining, and acquisition should be well defined and strictly followed.
Since different types of samples may have different characteristics and preparation
procedures, you may need to configure the program differently for each type of sample you
analyze.

You will need to log in to the program as an Administrator in order to set up the program and
modify the settings. As a Basic or Editor user, you cannot access the dialogs that configure
the Auto Analysis system.

Setting Options and Configuration

From the Options tab, choose Configuration. This will display a dialog containing a
number of configuration settings. Most of these settings do not need to be changed from the
program default settings.

One set of values you may need to adjust is found under the section AutoLinearity
Settings. The Low and High linearity values define the range over which the program can
adjust the linearity during AutoLinearity adjustments when the AutoLinearity option is
enabled. You should enter the acceptable range for G2/G1 ratios for your data set.

Review other settings in this dialog. For details on each setting, see Options and
Configuration.

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Make any adjustments required to the values. You may want to save these settings to a file
that can be manually loaded back into the program. To do this, use the Save button at the
bottom of the list in the dialog. When you need to re-open the saved settings, use the Open
button. This also allows you to transfer settings from one computer to another.

Click OK to close the dialog and accept the changes you have made.

Reviewing a Data Set for Important Peaks

Next, the peak finder needs to be tested for your data set.

Click the Open FCS button on the Home tab. Navigate to the location of your data set and
select twenty or more of the files to be analyzed. Click the Open button.

The program will open the first file in the set. If the file is a listmode file, you will need to
select the parameter to analyze.

There are two things to notice at this point. At the bottom of the application window, the
program now displays a batch navigation toolbar. We will use this to review the data set.
Secondly, the first histogram has one or more peaks identified with black triangles along the
x-axis. These are the peaks that the program's Peak Finder has identified. When the program
is correctly configured for your data set, it will identify the vast majority of the internal
standards and G1 peaks for cell cycles in your data set. It will also identify many of the well-
defined G2M peaks, but these are not as critical as the G1 peaks. It should not identify many
false peaks, that is, portions of the histogram that you do not consider to be real peaks.

To review the data set, we need to tell the program what to do as we navigate through the
batch of files. Click the left-most button on the batch control toolbar to display the Edit
Properties for File processor dialog.

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Set the File processor as follows: No analysis, do not save or print reports, do not prompt for
database or files. Then click the OK button to close the dialog. This tells the program that all
we want to do is open the data files as we navigate through the batch.

Now, here is the process for peak finder review. For each file in the batch, record whether
the program has identified all of the internal standards and the G1 peaks for each cell cycle.
If the program misses an important peak, note the locations of peaks omitted. The major
reasons peaks are missed are that they are too small, too wide, or they do not represent a
normal, Gaussian distribution. All of these parameters can be adjusted in the program's peak
finder. Also note the locations for any false peaks. For missing peaks and false peaks, try to
note the characteristics of the peak, e.g. short, wide, shoulder.

Make notations for the files, and then click the "Next" button on the batch toolbar.

Repeat this process for each file in the data set.

Configuring Peak Finder Settings

Review the notations you made for the data set. If the program correctly identified 95% or
more of the peaks in your test data set, you probably do not need to make any adjustments.

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If the Peak Finder did not perform as well as it should, here is how to make adjustments to
the settings.

Hopefully, there are some common characteristics to the files that caused problems for the
Peak Finder.

Use the batch navigation buttons on the batch toolbar to re-open a histogram that is
representative of the mistakes the Peak Finder made.

Next, choose Peak Finder from the Options tab. The Edit Properties for Peak Finder
settings dialog is displayed.

Click the Show button at the top of the list of options. This will display a dialog that allows
you to see the current histogram, and to understand the characteristics of each peak.

As you move your mouse over the histogram, vertical lines will be displayed showing the
boundaries for a peak that the program found. The lower portion of the dialog shows a
number of peak filters, as well as the statistics for the peak your mouse is over.

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Example of Peak Finder with a "missing peak"

Move the mouse over a peak that the program did not identify. Look at the lower portion of
the dialog for one or more of the settings that do not show the word "Pass". Adjust the
sliders for the failing settings until all of the settings show "Pass" for the peak.

If no vertical lines appear for a peak, the peak does not qualify as a statistically valid peak.
This kind of error cannot be corrected in the Peak Finder.

As you make adjustments, sweep your mouse across the histogram to review the status for
all of the peaks. If you find that false peaks now "pass", you may need to compromise on
your adjustments. If, however, the false peaks are very small, the program may exclude
them from Auto Analysis for not passing other thresholds, so you should not worry about
them at this point.

Once you have made adjustments that satisfy the histogram, click OK to close the Peak
Finder dialog and return to the Edit Properties for Peak Finder settings dialog. You may at

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this point choose to save these settings to a file using the Save button at the bottom of the
list. Alternately, you may want to write down the new settings. Since this process is
experimental and may require several iterations, it is good to keep track of each set of
adjustments.

Click OK to close the dialog.

Now you need to try out the new settings on the data set again. Move to the first file in the
batch. Make notations for missing peaks and false peaks, and then move to the next file.

Repeat this process of reviewing and adjusting until the Peak Finder is tuned for your data
set.

Adjusting Auto Analysis Settings

The final phase in optimizing Auto Analysis for your data makes use of the Auto Analysis
Settings dialog. This dialog controls how the program interprets the peaks in a histogram.

From the Options tab, click Auto Analysis. The Auto Analysis Settings dialog will be
displayed.

The first three options are straightforward. We recommend that you enable AutoDebris and
AutoAggregates, as these components are robust and automatic in the way they handle
debris and aggregation in samples. The Apoptosis option should be disabled for Auto Analysis
unless you have a series of files in which you want to model apoptosis as a Gaussian
distribution below the first cycle's G1 peak.

For Linearity, either leave the value at 2.0 or enter the average G2/G1 ratio of a data set.
The G2/G1 ratio is easily computed by dividing the mean channel of a G2 peak by the mean
channel of the G1 peak of the same cycle. Typical ranges are 1.95 to 2.0.

In the Standards and Reference section, enter the number of internal standards your samples
contain. If you have internal standards, you should click the Edit button to set the properties
of the standards. The most important property to set for each standard is its expected
channel position. By entering an expected position, the program can make better decisions
about when peaks shift and where the first cycle begins.

If you want to make use of an external reference standard for determining the location of the
Diploid population, you should enter its channel location. This is only required if you want to
base the Diploid position on the external reference.

The Ploidy Determination section provides a number of interesting possibilities for assigning
ploidy labels to peaks. We will examine these in detail.

First, the Maximum cell cycles entry can in most cases be set to Unlimited. However, if you
want to restrict Auto Analysis to creating models with a limited number of cell cycles, select
the number of cycles in this drop down listbox.

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The Diploid determination list presents 4 options for determining which cycle is labeled
"Diploid". The first option, "First cycle is Diploid", simply assumes that the first peak, after
any internal standards and apoptosis, is the Diploid G1 peak. This option should be used for
paraffin samples. When you use this option, you do not need to enter a value in the related
field, Diploid-to-Standard ratio.

You can also choose to base the Diploid cycle decision on the position of one of the internal
standards or an external reference channel position. In these cases, you need to enter the
expected Diploid-to-Standard ratio. For example, if your 2nd internal standard appears at
channel 40 and you expect the Diploid G1 to be at channel 50, you can choose "Based on
Standard2" for Diploid determination and enter 1.25 for the Diploid-to-Standard ratio. (Note:
The value 1.25 in this example is for PI-stained trout red blood cells in relation to PI-stained
human Diploid cells. For DAPI staining or other internal standard cells, the ratio will be other
values.)

The G1 threshold field determines how big a peak has to be in order for it to be called a G1
peak. This is a relative height after adjustment for aggregates. In essence, it acts like
another filter in the Peak Finder specifically for G1 peaks. If you have a number of small
peaks that Auto Analysis is calling G1 peaks and you do not want them classified that way,
increase the G1 threshold about 2 or 3 units at a time until they are not longer considered G1
peaks.

The Peak Location Range field determines how much of a shift from the expected locations
you will allow. For example, if the value is set for 10 (percent) and the Diploid is expected to
occur at channel 50, the program will look for a peak between channel 45 and 55. Generally,
values between 5 and 10 are best for this field.

The S-Phase group does not require adjustments for most users. The settings are based on
industry-standard recommendations and practices, so if you change these settings, it will be
difficult or impossible to compare results in your lab with results of other labs.

The Tetraploid group allows you to set the threshold at which the program will call a peak a
Tetraploid G1 instead of a Diploid G2M. Recommended values range from 15 to 25, and you
may need to experiment with these values on your data set.

When you finish making your first adjustments to the settings, use the Save button to save
the settings to a file on disk. As with other configuration dialogs, you may need to go through
several iterations before you find the best settings for your data set.

Choose OK to close the dialog.

Testing the Auto Analysis settings

At this point, you are ready to test the full system.

Choose File Batch Settings from the Edit tab. Note that this is the same as clicking the Edit
Batch button on the batch control toolbar.

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Change the settings to Auto-analyze each data file that you open. Click OK to close the
dialog.

Click the "go to first" button on the batch control toolbar. The first file will be opened and an
Auto Analysis will be performed. Make a notation to indicate whether the program correctly
analyzed the data. Pay particular attention to the ploidy classifications for each cycle and the
graphical representation of the fit.

Advance to the next file. After the program auto-analyzes the file, make notations about the
success or failure of the program. Repeat this process for the entire data set.

After all files have been processed with Auto Analysis, review your notations. The goal is to
have the program work automatically for greater than 95% of the files. If you need to make
additional adjustments, repeat the process of reviewing and adjusting until you achieve the
best results.

When you have the settings that work best, display the Auto Analysis Settings dialog once
again. Save the settings to a file using the Save button. Click OK to close the dialog.

Summary

The process of tuning Auto Analysis for your own data sets involves three dialog boxes:
Options and Configuration, Peak Finder Settings, and Auto Analysis Settings. After setting up
configuration options, an iterative process is used to find the best Peak Finder Settings for a
data set. As the final step, a similar iterative process is used to tune the Auto Analysis
Settings to a data set.

Use a data set of samples from the same cell or tissue type. As you perform this process for
a data set, save your settings for each dialog box so that they can be reloaded at any time.

You should perform this process for different sample types if the preparation process or the
data files themselves have difference characteristics from the data set initially used to
optimize Auto Analysis.

You will always find some files in a data set that are unusual when compared with the rest.
Do not try to optimize Auto Analysis for unusual data, or you will find that it fails more often
than it works. Instead, base your decisions on the typical data in a data set, and you will find
the system has a much higher success rate.

See also:

Options and Configuration

Peak Finder Settings

Auto Analysis Settings

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Creating a report template

One of the best ways to save time with ModFit LT is to design one or more reports to use as
templates and save them to disk. You can open a template and use it as the starting point of
your analysis, with your customized comments, graphics, and layout.

In this tutorial, we will design a simple report using ModFit LT's built-in text and graphics
tools.

Launch ModFit LT.

Click the Open FCS button on the Home tab. Choose one of the sample files, and then
click Open to load it.

You will see a histogram, but no results. In order to display the results, we have to analyze
the data.

Click the Auto button to automatically analyze the sample. Once finished, ModFit LT will
show us the results in textual format.

Suppose that we want to create a report that looks something like the one pictured below.

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We have added four objects to this report and moved things around a bit. The top of the
report contains a circle, arrow, and title text, and there is a new free-form text object in the
lower half of the report.

We will build a report template like this simple example.

Building the example

Adjust the Zoom so that you can see the whole report page on the screen.

Click the histogram graphic and drag it down the report page about two inches.

Move the legend down about two inches.

Click the results text and drag it down the page, as well. Align the top of the text with the
top of the graphic.

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From the Insert tab, click the Ellipse tool . This will insert an ellipse object onto
the report page.

Move and size the ellipse to resemble the example.

Double-click the new ellipse object to edit its properties. Change the Fill Color to a color
you like, and click OK.

Click the Arrow tool on the Insert tab . An arrow will be inserted into the report.

Drag the arrow so that its tip is next to the right hand side of the circle. Then, click and
drag the other end of the arrow across the page to form a horizontal line.

Click the Text tool on the Insert tab . The Edit Text dialog will appear. Click the
Properties button.

Click the Label Font to display the font dialog. Increase the font size to 26. You can
change the font and color as well. Click OK when you are done adjusting the font.

Type "My Very Own DNA Report" (or some other title that suits you) into the text edit box,
then click OK. The text is inserted into the report.

Move and resize the text so that it is positioned correctly over the arrow.

OK, we have just one more item to add.

Click the Text tool once again. In the Edit Text dialog, type a comment that you might
make about the data you analyze. Then click OK.

Move this last text to the lower portion of the report page.

Great. You are done. All that is left is to save the report under some name that we will
remember.

Click the Save As button in the Report group on the Home tab of the ribbon. In the File
Save dialog, enter the name "PRACTICE". Click the Save button.

Using the template

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Wonderful. The report has been saved. So how does one use a report as a template? It is
really quite simple.

When you launch ModFit LT, open the report you want to use. In our example, that is
"PRACTICE". Once the report is loaded, open a new data file, and analyze it. All of the results
in the template reflect the new sample.

If you save reports for each sample, you would save the report using the Save As button on
the ribbon. Your template will remain untouched as long as you save each new analysis
under a new name.

If you want to protect your reports from someone accidentally overwriting them, you can
make them "read-only". If someone accidentally tries to save a report by the same name,
ModFit LT will require the user to choose a new name for their report.

Note: If you have saved reports with a previous version of ModFit LT, you should load them
into this version of the program and save them again to upgrade them to the latest version
format.

Summary

You can design report templates that include graphics, text, and the layout you want.
Templates can be opened in ModFit LT and used as the starting point for the analyses you do
with the program. They can save you time and energy, since you design them only once and
use them over and over again.

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Field Testing

NOTE: In Compliance Mode, this section can only be performed by ModFit Administrators.

This section provides a step-by-step process for testing the functions of ModFit LT in your
lab. It uses the sample data files shipped with the program, and allows us to compare the
results you see in your lab with those we have on file at Verity.

In order to perform this process, you must log in to the program as an Administrator. This is
required because you will need access to the Auto Analysis Settings and Peak Finder Settings
dialog boxes.

Please be sure to follow each step carefully.

If the program is not already running, launch ModFit LT by double-clicking on its icon. Click
the OK button on banner page.

Choose Manage from the Options tab of the ribbon.

Click the Reset button. The program will offer to restart, choose Yes.

The program is now in its default configuration.

Click the Open FCS button on the Home tab. Open SAMPLE1.FCS from the Samples
folder where the program was installed.

Click the Auto button.

From the File tab menu, choose the Export->Save PDF command.

Click the Open FCS button and load in SAMPLE2.FCS.

Click the Auto button.

From the File tab menu, choose the Export->Save PDF command.

Click File button and load in SAMPLE3.FCS.

Click the Auto button.

From the File tab menu, click the Print command.

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Click File button and load in SAMPLELM.FCS.

Choose P5: FL2-A for the analysis parameter. Click OK.

Click the Auto button.

From the File tab menu, choose the Export->Save PDF command.

Email the PDF reports to tech@vsh.com.

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Reference
Main Window

ModFit LT offers two interfaces for the main window. The Ribbon Bar provides easy access
to the most common commands without having to drill down in menus. The Toolbar and
menu interface is a good option for users who want to keep the legacy version of the
interface.

The program must be restarted when changing from one to the other.

To switch to the Toolbar and menu interface, select the Options tab on the ribbon bar and
click the Switch to menu-toolbar button.

To switch to the Ribbon Bar interface, choose Switch to ribbon bar interface in the View
menu.

Main window with Ribbon Bar

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Main window with Toolbar and Menu

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Ribbon Bar

The Ribbon Bar provides easy access to the most common commands without having to drill
down in menus. It is divided up into tabs that group commands based on function.

File tab

Command Description
New Create a new, empty report
Open Report Open a report from disk
Save Report Save a report to disk with its current name
Save Report As Save a report with a new name or location

Open FCS Open an FCS data file for analysis

Change Dataset Select a different FCS dataset

Export Export PDF and graphics

Print Print the report

Page Setup Set the page size and margins

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Recent Files Shows a list of recently-opened data files

Recent Reports Shows a list of recently-opened reports

Exit (Quit) Closes the program

Home tab

Clipboard Report Analysis View

Paste Open Open FCS Report
Copy New Auto Analyze Scale
Save Auto with Auto Linearity Range
Save As Fit with Current Model Zoom
Auto Linearity
Sync Wizard
Cell Track Wizard
Choose Model

Insert tab

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Text Shapes Graphs

Text Line X Zoom graph
Keywords Arrow Y Zoom graph
Rectangle Fit Diagnostics graph
Ellipse

Edit tab

Setup Model Database QC

Logging Quick Editor Edit Databases Fit Diagnostics
Gates Full Editor Add Record S-Phase Cutoffs
Analysis Colors Database Wizard

Options tab

Interface Batch Settings

Switch to menu-toolbar File Batch settings Auto Analysis settings
Report Batch settings Peak Finder settings
Options and Configuration

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User Equations
Manage settings
Select Shared Folder
(Only ModFitAdmins in
Compliance mode)

Help tab

Online Learn
Check for Updates Application Info
Visit website User Guide

Ask Tech Support About

Make a Suggestion

Report a Problem

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Toolbar

The toolbar is displayed across the top of the application window, below the menu bar. It can
be moved by clicking and holding the mouse down on a blank portion of the toolbar, then
dragging to a new location. The toolbar provides quick mouse access to common ModFit LT
functions.

The buttons appearing in the Toolbar are shown below along with an explanation of the
functions they perform.

Click To

Open an FCS data file for analysis. This is a

split button, which also contains a command
to open reports..

Perform an automatic analysis.

Adjust the scaling of the data, and the lower

and upper boundaries of the analysis.

Select a model to be used for manual

analysis.

Adjust the range positions on the data.

Analyze the data with the current model,

scaling, and range positions.

Perform auto-linearity on the current data

with the current model.

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Evaluate the fit for S-Phase variability and

compare with Consensus Conference
recommendations.

Edit and lay out the report.

Automatically name, then save the report.

Print the report.

Select a zoom factor for viewing the report

page on the screen.

Select a tissue type for the data sample. By

selecting a type, you apply the cutoffs for
that type. You must enter your own cutoff
values in the S-Phase cutoffs dialog.

This text indicates whether the program is

running in "Compliance" or "RUO" mode.

Display the ModFit LT User Guide.

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Data Files
Open FCS

Use this command to open a data file. Choosing this option will display the File Open dialog
to select a file to open.

A data file is a FCS (Flow Cytometry Standard) format histogram or listmode data file. It can
also open some formats of ASCII-encoded histogram files. You can load one data file at a
time into a report document.

If a listmode file is selected, the Analysis Setup dialog is displayed to allow selection of the
analysis parameter and to set up gates.

A batch of files may be selected at one time. When more than one data file is selected, the
File Batch Settings dialog is displayed to allow you to set batch processing options. Then, the
File Batch Toolbar is displayed at the bottom of the application window. This toolbar will
allow you to navigate through the selected files, and provides additional batch processing
options.

To select a batch of files, click the mouse on the first item you want to select and drag the
mouse over sequential files in the list. Alternately, you can click the first item, hold down the
Shift key and click the last item to select a group of files. Hold down the Ctrl key to select
non-adjacent files in the list.

See also:

Batch Processing

Shortcuts

Keys: CTRL + F on PC; Command + F on Mac

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File Open dialog box

This dialog is used to open both reports and data files in ModFit LT. This is the standard File
Open dialog box for the operating system, so its appearance and features are different on
Windows and Mac versions.

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Analysis Setup

If ModFit LT detects that the file you select is an FCS listmode file, it will present a dialog
that allows you to choose which parameter you want to analyze and which parameters to use
for gating.

The dialog contains a tree on the left side showing available gates and the Analysis
Population. A graphical view of the selected item is shown on the right side of the dialog.

Analysis Population

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The first item displayed is the Analysis Population. Choose the parameter you want to
analyze by clicking the label on the X axis and selecting a parameter from the list.

You can right-click the X-axis and choose Edit X Parameter to change the transformation
used for the parameter. See Edit Properties for Parameter for additional info.

Gates

After you choose the Analysis parameter, you can create gates. Use the Add Gate button to
create a new gate, and the Remove Gate button to remove an unneeded gate. Gates are
logical "AND" gates, which means that events must satisfy all active gates to display in the
analysis histogram.

Select a gate item in the tree to display the gate in the right-hand panel of the dialog. With
the item selected, click the X- and Y-axis labels to pick the gating parameters for the plot.
Use the region tools on the right to define a gating region on the plot.

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Graphics for the gating plot can be edited by right-clicking the plot area and selecting Edit
Properties. See Edit 1P Graphics and Edit 2P Graphics for additional details.

Define each gate for your analysis. The active gates will appear on the report page and can
be modified there once created.

The Auto Layout Gates option can be enabled to automatically organize gates on the
report. Uncheck this option if you create your own arrangement for gates.

Click OK to finish the gate and analysis population selection.

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Change Data Set...

This dialog is used to choose a "data set" for some FCS files. For example, Beckman Coulter
files from FC500, Navios, and Gallios instruments commonly create 2 data sets in each FCS
file: one in FCS2 format and the other in FCS3 format.

If the file you select contains more than one data set, the Change Data Set dialog will be
displayed.

Select the data set from the list of choices, then click OK. The program will re-read the FCS
file, extracting the data from the selected dataset for gating and analysis.

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Recent Files list

This menu is a convenient list that shows the most recent data files that have been opened in
ModFit LT. Select a file from the list to open the file for analysis.

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Analysis
Auto Analysis command

Use this command to automatically analyze the current data file.

The program uses the settings from the Auto Analysis Settings dialog, along with Peak Finder
Settings, to analyze the data file.

What Does Auto Analysis Do?

When you load a data file, ModFit LT identifies peaks it finds in the sample. Automatic
analysis looks at the peak positions, standard deviations, and other information about the
sample and attempts to match the data to a ploidy-specific model. The model is constructed,
ranges are positioned around the peaks, peak estimates are computed, and a non-linear least
squares analysis is performed on the data. In addition, ModFit LT tests the S-Phase
measurements for statistical confidence if the S-Phase assessment system is activated.

When automatic analysis finishes, ModFit LT displays the analysis results in both graphical
and textual form on the report.

See also:

Strengths and Limitations of Automatic Analysis

Shortcuts

Keys: CTRL + A on PC; Command + A on Mac

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Auto Analyze and Auto Linearity

Use this command to automatically analyze the current data file and then to perform Auto
Linearity on the file.

See Auto Analysis and Auto Linearity for additional details.

Shortcuts

Keys: CTRL + Shift + A on PC; Command + Shift + A on Mac

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Fit with Current Model command

Use this command to apply a model, with current scaling and range positions, to the
histogram data. This performs a non-linear least squares analysis of the data.

If you use Automatic Analysis, you will not need to use this command. It is done for you.

Use this command after you have opened a data file, chosen a model, and positioned the
model's ranges. If you change the model using the Quick Model Editor, the Full Model Editor,
or by positioning ranges, you need to use this command to re-fit the data with the model.

See also:

Choose Model

Ranges

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Auto Linearity

This command initiates the auto-linearity process with the current model and data. Auto
linearity adjusts the G2M and aggregate components based on iterative fitting process.

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Choose Model command

Use this command to select a model to use in the manual analysis of the data. A model
consists of a set of components, which are linked together based on the choices you make in
this dialog.

Selecting a model does not actually perform an analysis. After choosing a model, you need to

position its ranges and click the Fit button to analyze the data with the model.

The left portion of the dialog contains a tree diagram of the model. The top of the tree is the
Manual Analysis branch, and the choices you need to make when selecting a model are
described below. Also shown in the tree is a branch for each cell cycle you want to model.
The number of cycles you see will depend on what you choose for "Number of cycles" in the
dialog.

After making your initial model selections with the Manual Analysis branch selected, you can
fine-tune the model by selected each cycle branch and adjusting its properties. See the Edit
Properties for Cycle dialog box for details on cycle properties.

AutoDebris

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To enable debris modeling, check this option. The AutoDebris component can model debris
from fresh, frozen, pulverized, and paraffin samples. If the sample shows little or no debris,
you may choose to disable this option.

AutoAggregates

Check this option to enable a model component that automatically fits most forms of
aggregation. Aggregates can form in the sample as cells and cell fragments stick together.
Because all sizes and shapes of cells can form aggregates, the AutoAggregate component is
a continuous distribution. Its shape is unique for each data file, and is based on probabilities
of cells in each channel sticking together. To disable the AutoAggregates component,
uncheck this option.

Apoptosis

Note: this is not the recommended approach to use for Apoptosis. Secondary markers are
much more robust and accurate.

Check this option to model an apoptosis distribution in the sample. A Gaussian will be used to
model a peak, and a range to identify the peak will be displayed.

Linearity

Enter a linearity value for the sample. This value can be determined by dividing the G2M
position for a cycle by the G1 position for the same cycle. The program will use the linearity
value for computing positions for G2M populations that are not clearly defined peaks, and for
the AutoAggregate model component.

Standards

Use this option to select the number of internal standards that appear as distinct peaks in the
sample.

Number of cycles

Choose the number of cell cycles that appear in the sample. Each cell cycle contains model
components for G1, S-Phase, and G2M populations.

Model templates

After choosing the number of cycles above, select a model template that best describes the
cycles. The templates are named with abbreviated forms of the common cell cycle names:
"Dip" is Diploid, "Ane" is Aneuploid, "Tet" is Tetraploid, "Hyp" is Hyper-Diploid, and "NearDip"
is Near-Diploid.

Notice that the model template name determines the relative positions of the cycles in the
model. For example, with 3 cycles, there is a template for "Dip Ane Tet" and another for "Dip
Tet Ane". The "Dip Ane Tet" template is designed for the Aneuploid appearing between the
Diploid and the Tetraploid populations; the "Dip Tet Ane" is designed for the Aneuploid
appearing above (to the right of) the Tetraploid population.

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The available model template will change depending on the number of cell cycles you have
selected to model.

Range positions

This option allows you to determine whether or not the program will re-position ranges upon
choosing OK. If you have already selected a model and are making some adjustments in this
dialog, choose "Use current range positions". If you are selecting a new model, choose
"Compute range positions" to determine the initial locations for the ranges.

OK

Click this button to create a model matching your selections and display the ranges for the
model. After clicking OK, position the ranges and click the Fit button on the toolbar.

The selected model will be used until 1) another model is selected using Choose Model, 2) an
automatic analysis is performed, 3) a new report document is loaded, or 4) you close the
program.

Cancel

Click this button to close the dialog without creating a model.

Reset

Click this button to restore the dialog to a default model.

See Also

Edit Properties for Cycle

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Edit Properties for Cycle

These options appear when you chose a Cycle in the tree on the left side of the Edit
Properties for Manual Analysis dialog box. The options allow you to fine-tune the properties
for a set of components used to model one cell cycle in the model.

Ploidy

This option allows you to select a different ploidy label for the cell cycle. The model template
you select in the Edit Properties for Manual analysis dialog determines the default value for
this option.

Range labels and results reported on the report are based on the ploidy names assigned to
cell cycles.

G2M

There are three options available for the way G2M is modeled: Disabled, Visible peak, and
Indistinct peak. When Disabled is selected, no G2M will be modeled for the cycle. Choosing
Visible peak allows the program to adjust the mean position of the G2M component for the
best fit, the lowest reduced chi-square (RCS). If the peak for G2M is not clearly defined,
choose Indistinct peak. With this setting, the position of the G2M is based on the G1 position
multiplied by the linearity factor.

S-Phase

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This checkbox allows you to enable or disable the S-Phase component for the cycle.
Normally, you should disable an S-Phase if another more important cycle is significantly
overlapping this cycle. For example, in the near-Diploid model, the Diploid S-Phase is
disabled because the DNA Index (DI) for the Aneuploid cycle is less than 1.3 and therefore
the S-Phases are virtually superimposed on one another.

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Scale command

Use this command to manually adjust the scaling of the loaded histogram data.

A rectangle encompassing the data is displayed. Each corner has a solid square in it. Position
the mouse over a corner square, click and hold the button down, and drag to position a new
boundary.

Data below the left boundary and above the right boundary will be excluded from the
analysis. Adjusting the top of the scaling rectangle re-scales the data on the Y-axis
graphically, but has no effect on the analysis.

Normally when each new data file is opened, the program will automatically scale the data.
See the section on the Options and Configuration dialog box to learn how to disable the auto-
scaling feature.

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Range command

Use this command to manually adjust the ranges for the model.

ModFit LT uses specific ranges to identify important parts of a histogram, usually peaks.
Automatic Analysis positions ranges for you. However, if you need to correct a range position
or if you manually select a model for analysis, you will need to position ranges yourself.

The rule of thumb for positioning ranges is to make sure each range identifies one, unique
peak. Ranges should be positioned so that they do not overlap one another. When a range is
positioned correctly, you will see the associated portion of the histogram filled in with a color
indicating the range estimate.

The exception to the rule for placing ranges is shown in near-Diploid histogram. In these
histograms, you may need to place ranges so that they are not centered on the peaks.
Instead, move the ranges slightly apart to help differentiate the two, overlapping peaks.

Only move or resize ranges that need to be adjusted. By leaving correctly positioned ranges
alone, you help improve user-to-user consistency.

The vertical position of ranges has no impact on the range estimate. You can arrange ranges
vertically so that they are easy to read and access.

Ranges and their uses

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Debris This range provides an estimate of debris. The left side

should be placed at the highest portion of the beginning of
debris. The right side should extend approximately two-
thirds of the histogram scale. The vertical position has no
impact on the estimate.

Stnd1 This range is used to identify the first internal standard, if

there is one. If you have named the internal standard, the
name of the range is changed to the name of the standard.
Center the range over the first standard peak.

Stnd2 This range is used to identify the second internal standard,

if there is one. If you have named the internal standard, the
name of the range is changed to the name of the standard.
Center the range over the second standard peak.

Apop This range identifies the apoptosis peak, if you have

enabled the apoptosis component. The range should be
centered over the peak.

Dip This range identifies Diploid G0-G1 peak. The range should
G1 be centered over the G1peak.

Dip This range identifies Diploid G2-M peak. The range should
G2 be centered over the G2 peak.

An1 This range identifies an Aneuploid G0-G1 peak. The first

G1 Aneuploid cycle will use An1, the second An2, and so forth.
The range should be centered over the G1 peak.

An1 This range identifies an Aneuploid G2-M peak. The first

G2 Aneuploid cycle will use An1, the second An2, and so forth.
The range should be centered over the G2 peak.

G1 This range is used by the Sync Wizard to identify the Diploid

G0-G1 peak. The range should be centered over the peak.

Parent This range is used by the Cell Tracking Wizard to identify

the Parent population.

Gen1 This range is used by the Cell Tracking Wizard to identify a

daughter generation population, if the Floating option is

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selected on the Generations tab of the wizard dialog. Gen1

is the generation closest to the parent, Gen2 is to the left of
Gen1, etc.

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Fit Diagnostics command

Use this command to add a report object that displays a summary of the Fit Diagnostics
dialog box on the report. The object shows a legend of the elements found in the Fit
Diagnostics dialog box. The object can be moved to any convenient location on the report.

To edit the properties of the object, double click on the object after it has been inserted into
the report.

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Fit Diagnostics dialog

This dialog displays information about the current analysis, comparing key aspects with DNA
Consensus Conference recommendations and user-defined cutoffs.

If the S-Phase Assessment system is active, the S-Phase Error Analysis for diploid, aneuploid,
and total (average) S-Phase computations are shown in the middle portion of the dialog. In
DNA Diploid models, the standard deviation for diploid S-Phase is computed. In DNA
Aneuploid models, the standard deviations for aneuploid and total (average) S-Phases are
computed.

Probability of the S-Phase value falling into a user-defined cutoff category is shown in the
listbox at the bottom of the dialog.

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When pressed, %CV, Cell #, Avg Cell #, % Aneuploid, %B.A.D, and RCS buttons
display reference text explaining the generally accepted guidelines for each criterion. The
status boxes display green for results in the Good range, yellow for results in the Fair range,
and red for Poor results.

In the S-Phase Error Analysis section, you can view error distribution plots by clicking the %
Diploid S, % Aneuploid S, or % Total S buttons. Next to the buttons, the computed S-
Phase percentage appears with its standard deviation. Values will only be computed if the S-
Phase assessment system is active. The S-Phase cutoffs dialog controls whether the system
is active or inactive.

See also:

Histogram and S-Phase Assessment

S-Phase cutoffs command (Edit menu)

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Sync Wizard
Sync Wizard->Create or edit model

The Synchronization Wizard guides you through the process of creating or editing a model to
analyze synchronized cell lines. You can initiate the wizard by selecting Sync Wizard-
>Create or edit model from Home tab of the ribbon bar. A dialog will appear containing
several tabs, each presenting you with choices relative to a particular aspect of the model.
The wizard provides full functionality for creating and editing a synchronization model while
maintaining a simple interface.

When starting a Sync Wizard session, you should select each tab to adjust any settings to
your data. When you press the Analyze button, the Wizard dialog will close, the model will
be constructed, and the data file analyzed. In addition, the Wizard toolbar will appear at the
bottom of the main window, allowing you to open files, return to the Wizard, or exit the Sync
Wizard.

If you need to adjust a setting in the model, re-enter the Sync Wizard dialog by clicking the
Wizard button on the Wizard toolbar, or selecting Sync Wizard->Create or edit model
from the Analysis menu. When editing a model, you only need to review the tabs containing
options you want to change. For example, you might view the G0-G1 tab to change the
model from "Adjust automatically" to "Lock the position", then choose Analyze to close the
dialog.

ModFit LT remains in Sync Wizard mode until you quit the Wizard, either by clicking the X on
the Wizard toolbar, by selecting Sync Wizard->Quit.

You can save an active Sync Wizard session in a report to preserve your settings for the
synchronization model you create. The report must be saved with the Wizard toolbar visible
to be able to re-enter the Wizard dialog when the report is reopened.

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Sync Wizard: Start tab

You decide whether to create a model, or edit the one you already created on this tab. The
option to edit is disabled if the Sync Wizard is not already running. Once a model has been
created, you can re-enter the Wizard to edit the model's settings.

Use the Open button to select a data file, if you haven't already selected the file you want to
start with.

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Sync Wizard: G0G1 tab

The purpose of this tab is to allow you to identify the G0-G1 peak. A graphic of the currently
loaded data file is displayed with a cursor used to identify the position of the G0-G1.

The program will position the cursor automatically over the first peak in the sample. If this is
not the correct peak for your analysis, position the G0-G1 peak cursor by clicking on the red
triangle at the top of the graphic and dragging it over the peak. Alternately, you may type a
channel value into the G0-G1 channel edit box.

When you close the Wizard dialog by choosing Analyze and you are creating a model from
scratch, ModFit LT will position the G0G1 range around the channel position you indicate.
The range is placed so that it encompasses 8 channels to the left and 8 channels to the right
of the peak position.

When editing a model, the Sync Wizard does not adjust the range position on the data. You
can manually adjust the range by clicking the Range button on the toolbar and moving or
resizing the G0G1 range.

The Sync Wizard allows you to "lock" the channel position, or to let the program adjust the
position within the G0G1 range. When you choose Lock the position, ModFit LT will not try
to find a better peak position. This setting is useful in case where the G0-G1 peak is indistinct
or not present. You must also specify a standard deviation for the peak with this option.

Choosing Adjust automatically allows the program to look for a peak within the range of
channels encompassed by the G0G1 range, initially plus or minus 8 channels from the
identified peak position. If the program cannot locate a peak within the G0G1 range, the
Sync Wizard Adjustment dialog is displayed with options on how to adjust the model.

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Sync Wizard: G2M tab

The purpose of this tab is to allow you to identify the G2/G1 ratio. This ratio is used to
determine the position of the G2-M peak relative to the G0-G1 peak. A graphic of the
currently loaded data file is displayed with a cursor used to identify the position of the G2M
peak.

You may adjust the G2/G1 ratio three ways: by entering a new ratio in the G2/G1 edit box,
by entering a channel position in the G2-M channel edit box, or by dragging the red triangle
cursor over the G2-M position on the graphic.

The model created by the Sync Wizard will multiply the G0-G1 peak position and standard
deviation by the G2/G1 ratio appearing on this tab to compute the G2-M mean position and
standard deviation. The position of the G2-M peak is always, therefore, dependent on the
position of the G0G1 peak. Similarly, the standard deviation of the G2-M peak is dependent
on the standard deviation of the G0G1 peak.

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Sync Wizard: S-Phase tab

The purpose of this tab is to allow you to control the aspects of modeling S-Phase.

You may adjust the shape used for S-Phase by selecting either Rectangle or Trapezoid.

The Number of Compartments section allows you to pick how many trapezoids or
rectangles you want to use. By choosing more compartments you allow the S-Phase portion
of the model more freedom to conform to your data. However, giving too much freedom may
allow the S-Phase portion of the model to take over data that truly belong to G0-G1 or G2-M.

If you use more than one compartment in S-Phase, you can choose the spacing of
compartments. Choosing Equal spacing distributes the compartments evenly between G0-
G1 and G2-M populations. Choosing Exponential spacing causes compartments to increase
exponentially in size.

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Sync Wizard: Other tab

The purpose of this tab is to allow you select miscellaneous options related to the
synchronization model you are creating.

If you need to perform debris subtraction, enable the Debris checkbox. The Sync Wizard will
add a debris component appropriate for fresh and frozen samples.

If your samples contain aggregates that you want to model, enable the Aggregates
checkbox. The Sync Wizard will add an aggregate compensation component to the model.

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Sync Wizard: Report tab

The purpose of this tab is to allow you to control what information is reported by the Sync
Wizard model.

For Percent report options, you may choose to report percentages for all components and
sub-compartments, or only the primary components.

For Mean report options, you may choose whether or not to report means.

You can include information derived from the data file keywords and other demographic
information by selecting "Show demographics".

If you would like to display the %CV for the G0-G1 peak, enable the Report %CV check
box.

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Sync Wizard->Open Data and Analyze

Use this command to open a data file into ModFit LT and analyze the file with the model
created by the Synchronization Wizard. The standard File Open dialog is displayed for
selection of a file. After selecting a file, the program loads and analyzes the sample in one
step.

If you select more than one file, the program will display a prompt indicating that you should
use the File Batch toolbar to process the files. This technique provides many useful options,
such as automatically saving or printing reports. See File Batch Settings and File Batch
Toolbar for more.

Alternately, you may use the standard Open FCS command to load the next file in a batch.
If you use this option, you will need to click the Fit button to apply the model to the sample.

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Sync Wizard->Quit

Use this command to exit the Synchronization Wizard. The Wizard toolbar will be removed
and normal program operations, such as automatic analysis and opening reports, are
restored.

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Sync Wizard Adjustment dialog

This dialog is displayed when the Sync Wizard model is set to adjust the position of the G0-
G1 peak automatically but there is no peak found under the G0-G1 range.

The Wizard offers four options: to move the G0-G1 range over the first peak in the sample,
to change the model so that the position of G0-G1 is "locked" in its last location, to analyze
without changing the model, or to open the wizard to make your own changes.

If the sample has a visible G0-G1 peak (identified by the peak finder with a black triangle) on
the graphic, you can choose to let the Sync Wizard move the range. It is likely that the
position has shifted from one sample to the next, and it is safe to keep the model in "Adjust
automatically" mode.

If the peak suggested by the dialog is not the G0-G1 population, you should choose to lock
the G0-G1position in its last location. In many synchronized cell lines, the G0-G1 population
disappears. Locking the position to a previously identified value is the best way to handle
these samples.

You can choose to analyze with no changes if you are confident that the model is still
appropriate for the data. Alternatively, you can open the wizard to make adjustments
yourself.

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Cell Tracking Wizard

Cell Tracking Wizard->Create or Edit Model

The Cell Tracking Wizard guides you through the process of creating or editing a model to
analyze cell tracking dye samples. You can initiate the wizard by selecting Cell Tracking
Wizard->Create or edit model from the Home tab of the ribbon bar or from the
Analysis menu. A dialog will appear containing tabs, each presenting you with choices
relative to a particular aspect of the model. The wizard provides full functionality for creating
and editing a Cell Tracking model while maintaining a simple interface.

When starting a Cell Tracking Wizard session, you should select each tab to adjust any
settings to your data. When you press the Analyze button, the Wizard dialog will close, the
model will be constructed, and the data file analyzed. In addition, the Wizard toolbar will
appear at the bottom of the main window, allowing you to open files, return to the Wizard, or
exit the Cell Tracking Wizard.

If you need to adjust a setting in the model, re-enter the Cell Tracking Wizard dialog by
clicking the Wizard button on the Wizard toolbar. When editing a model, you only need to
review the tabs containing options you want to change.

ModFit LT remains in Cell Tracking Wizard mode until you quit the Wizard, either by clicking
the X on the Wizard toolbar.

You can save an active Cell Tracking Wizard session in a report to preserve your settings for
the Cell Tracking model you create. The report must be saved with the Wizard toolbar visible
to be able to re-enter the Wizard dialog when the report is reopened.

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Cell Tracking Wizard: Start tab

You decide whether to create a model, or edit the one you already created on this tab. The
option to edit is disabled if the Cell Tracking Wizard is not already running. Once a model has
been created, you can re-enter the Wizard to edit the model's settings.

Use the Open button to select a data file, if you haven't already selected the file you want to
start with.

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Cell Tracking Wizard: Parent tab

This tab allows you to locate the peak that represents the parent population in the sample.
The program automatically positions the cursor over the right-most peak in the histogram. If
this is not the correct peak for your analysis, position the parent peak cursor by clicking on
the red triangle at the top of the graphic and dragging it over the peak.

Alternately, you may type a channel value into the Parent channel edit box.

This tab also allows you to "lock" the standard deviation (SD) of the Gaussian component
used to model the parent population. The Lock SD option is most useful when analyzing
samples in which there is a great deal of proliferation. In these cases, the parent population
is often difficult for the model to resolve, and the modeling process may broaden the SD of
the parent too much. When this option is enabled, the SD value can be edited.

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Cell Tracking Wizard: Generations tab

This tab displays the information about how the generations will be modeled.

There is an editor for the number of generations to model. The number of generations
should be based on the range between the parent population and the background of the
measurement. For example, if the parent has a mean of 1000 and an unstained population
has a mean of 10, the maximum number of discernible generation is 5 with mean s at 500,
250, 125, 62.5, 31.25, and 15.625. The variability of the populations also has an impact on
when the last generation will no longer be discernible from background.

A dropdown box is provided to select between the Standard and Floating option for
modeling generations.

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In the Standard mode, the mean position of each generation is dependent on the position of
the parent generation. Each generation is positioned at half the intensity of the previous
generation.

The floating option allows the mean of each generation to be computed independently of the
other populations. This option provides much greater flexibility to the model and works well
for samples with visually distinct generations. The floating option is not recommended for
samples where generational boundaries are not obvious to the eye.

The graphic displays a red line where the parent generation is positioned and dotted lines for
each generation.

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Cell Tracking Wizard: Other tab

The purpose of this tab is to allow you select miscellaneous options related to the cell
tracking model you are creating.

The Cell Tracking Options group allows you to identify the generation that is considered
the start of true proliferation. This value is used in computing the Precursor Frequency and
the Upper Generation Proliferation Index (UGPI). For a description of the use of Precursor
Frequency, see "A flow cytometric method to estimate the precursor frequencies of cells
proliferating in response to specific antigens", Givan, A., et al, Journal Of Immunological
Methods Vol. 230, 1999 Elsevier Science B.V.

If you need to perform background subtraction, enable the Background checkbox. The Cell
Tracking Wizard will add a rectangle to model background.

When you enable Background subtraction, another option, Asymmetric cytokinesis,

becomes available. With this option checked, the standard deviations of the generations are

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broadened by interpolation along the background rectangle. If this option is not enabled, all
generations have the same standard deviation as the parent population.

With the Asymmetric cytokinesis option enabled, the program sets the name of the model to
"Cell Tracking Wizard Cytokinesis Model". If the option is not checked, the model name is
"Cell Tracking Wizard Basic Model".

Two export options can be enabled for the Cell Tracking Wizard. When Export partition
table is enabled, the program will save a text file containing the "boundaries" of the
generations. These are the points of intersection of the adjacent generations, illustrated
below.

The export file is created each time the wizard analyzes a data file. By default, the export file
name is ProlifWizardPartitions.txt. If you enable the Prompt for file name option, the
wizard will display a File Save dialog before storing the partition file, allowing you to choose a
file name and location.

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The partition file can be imported into other applications, like WinList, for further analysis. It
is a simple, text format file that is readable by spreadsheets, databases, and word
processors.

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Cell Tracking Wizard->Open Data and Analyze

Use this command to open a data file into ModFit LT and analyze the file with the model
created by the Cell Tracking Wizard. The standard File Open dialog is displayed for selection
of a file. After selecting a file, the program loads and analyzes the sample in one step.

If you select more than one file, the program will display a prompt indicating that you should
use the File Batch toolbar to process the files. This technique provides many useful options,
such as automatically saving or printing reports. See File Batch Settings and File Batch
Toolbar for more.

Alternately, you may use the standard Open FCS command to load the next file in a batch.
If you use this option, you will need to click the Fit button to apply the model to the sample.

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Cell Tracking Wizard->Quit

Use this command to exit the Cell Tracking Wizard. The Wizard toolbar will be removed and
normal program operations, such as automatic analysis and opening reports, are restored.

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Cell Tracking Example

Here is an example of a cell-tracking study that helps to explain the results that the Cell
Tracking Wizard displays.

Suppose that we take samples at three time points: T=0, T=3h, and T=6h. For this simple
cell tracking example, we will start at time T=0 with four cells stained for cell tracking.

For the population of cells at T=0, here's what we might see, and some example calculations
for cell tracking equations.

We can now look at the sample at time T=3 and compute the same equations on the current
population of cells. Note that the filled in circles above represent cells that currently exist and
the unfilled, dashed circles are cells that have divided into two daughter cells.

* Note, the circles containing an asterisk are those that went on to “true” proliferation. True
proliferation is defined by g, the index of the user defined generation where true proliferation
began. In this example, “true” proliferation begins at generation 3. Cells that remain in the
parent or generation 2 did not truly proliferate.

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Here is our final time point T=6. Note that the filled in circles above represent cells that
currently exist and the unfilled, dashed circles are cells that have divided into two daughter
cells.

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The number of Modeled Cells is dependent on the number of cells in the original culture and
the amount of cell division. The number of modeled cells will increase with proliferation. This
value is used mostly for computing other cell tracking statistics.

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Non-parent Cells are modeled cells not in the parent generation. The number of Non-parent
cells will be zero in the initial culture and will increase with proliferation. This value will
always be less than Modeled Cells. This value is used mostly for computing other cell tracking
statistics.

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Non-parent Calculated Original Cells are the cells in the original culture that divided and gave
rise to the modeled Non-parent Cells. The number of Non-parent Calculated Original Cells will
be zero in the initial culture and will always be less than the Calculated Original Cells. This
value is used mostly for computing other cell tracking statistics.

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Calculated Original Cells are the cells in the original culture, both those that divided and
those that did not. The number of Calculated Original Cells will be the same as Modeled Cells
in the initial culture. This value is used mostly for computing other cell tracking statistics.

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Percent Divided is the percent of cells in the final culture that divided at least once. The
Percent Divided will be zero in the initial culture and will increase to at most 100%.

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Precursor Frequency is the number of cells in the original culture that divided at least enough
to get to the specified generation of “true” proliferation. In this example the specified
generation is three. The Precursor Frequency will be zero in the initial culture and will
increase with “true” proliferation to a maximum of 1.0.

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Proliferation Index is the number of modeled cells divided by the cells in the original culture.
This is the average number of cells that one original cell became. The Proliferation Index will
be one in the initial culture and will increase with proliferation to a maximum of 2^(maxGen-
1), where maxGen is the highest generation modeled.

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Replication Index is the number of daughter cells divided by the number of cells in the
original culture that divided. The Replication Index will be undefined (0/0) in the initial
culture and will increase with proliferation to a maximum of 2^(maxGen-1), where maxGen is
the highest generation modeled.

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Proliferating Average Number of Divisions is the sum of the number of divisions that resulted
in the cells in each generation divided by the Non-parent Calculated Original Cells. The
Proliferating Average Number of Divisions will be undefined in the initial culture and will
increase with proliferation to a maximum of maxGen-1 where maxGen is the highest
generation modeled.

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Average Number of Divisions is the sum of the number of divisions that resulted in the cells
in each generation divided by the Calculated Original Cells. The Average Number of Divisions
will be zero in the initial culture and will increase with proliferation to a maximum of maxGen-
1 where maxGen is the highest generation modeled.

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Number of Divisions is the total number of divisions that resulted in the cells in each
generation. The Number of Divisions will be zero in the initial culture and will increase with
proliferation.

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Non-proliferative Fraction is the number of modeled cells divided by the cells in the original
culture. The Non-proliferative Fraction will be one in the initial culture and will decrease with
proliferation to a minimum of zero.

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Upper Generation Proliferation Index is the number of modeled cells divided by the cells in
the original culture for generations greater than g, which is 3 in this example. This is the
average number of cells that one original cell became, excluding those in generations less
than g. The Upper Generation Proliferation Index will be undefined in the initial culture and
will increase with proliferation to a maximum of 2^(maxGen-1), where maxGen is the highest
generation modeled.

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Reports
Reports

The documents created by ModFit LT are called reports. Each reports contains the analysis
information for one data sample, and can be saved to disk and re-opened by the program.

When you start ModFit LT, a simple report is created for you automatically. It contains
several objects which are common to all reports: a graphical element which displays the
histogram data, a textual element which displays analysis results, and the gates that have
been created. The histogram is not displayed until there is a data file loaded. The text objects
remain hidden until you have analyzed the data.

To see these elements, load a data file into ModFit LT. The histogram will be displayed. In
addition, if you load listmode data, you can create gates on the data. The gate graphics will
appear on the report, allowing you to manipulate the gates without reloading the sample.

Report Tools

ModFit LT reports can include additional text and graphics that you add to the report with
report tools. These tools are found on the Insert tab of the ribbon bar. In toolbar mode, the
Report toolbar is displayed.

The table below shows the report tools and their functional descriptions. Click a tool for
additional information.

Tool Description

Adds free-form text objects.

Adds keyword information extracted from

the data file you analyze.

Adds a solid line.

Adds an arrow.

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Adds a filled rectangle.

Adds a filled ellipse.

Adds a graph zoomed on the X-axis to

magnify the Diploid G1 component.

Adds a graph object zoomed on Y-axis to

magnify S-Phase and aggregates.

Adds the fit diagnostics graph to the

report.

Moving and resizing report objects

All of the objects on the report can be moved, and most objects can be resized. This allows
you to place objects where you want them on the report page, and adjust the size of
graphics elements.

To move an object, click and hold the mouse button down on the object, then move the
object. Release the mouse to set the new position.

To resize an object, click the object to select it. Handles will appear on the object. Click
and hold the mouse button down on one of the handles. As you move the handle, the object
will resize. Release the mouse to set the new size.

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New command

Use this command to create a new document in ModFit LT.

Any existing objects on a report, including analysis results, are discarded, and new objects
are created in their default locations.

Shortcuts

Keys: CTRL + N on PC; Command + N on Mac

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Open Reports command

Use this command to open one or more existing ModFit LT report documents. Choosing this
option will display the File Open dialog to select reports to open.

A Report document contains an analysis of data and additional report objects that you add to
the document.

A batch of reports may be selected at one time. When more than one report is selected, the
Report Batch Settings dialog is displayed to allow you to set options for batch processing.
Then, the Report batch toolbar will be displayed at the bottom of the application window.
This toolbar will allow you to navigate through the selected reports, and provides additional
batch processing options.

To select a batch of reports, click the mouse on the first item you want to select and drag the
mouse over sequential files in the list. Alternately, you can click the first item, hold down the
Shift key and click the last item to select a group of files. Hold down the CTRL key to select
non-adjacent files in the list.

Shortcuts

Keys: CTRL + O on PC; Command + O on Mac

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Report command

Use this command to display, lay out, and edit the report document.

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Save command

Use this command to save the document with its current name and folder.

When you save a document for the first time, ModFit LT displays the Save As dialog so you
can name your document. The name for the report is based on the data file currently loaded
into it. ModFit LT takes the name of the data file and adds ".mfl" to create the report name.

Once a report document has been saved, this command will not display the Save As dialog; it
will simply resave the report.

If you want to change the name and directory of an existing document before you save it,
choose the Save As command.

The document contains the model, data, gates, and any additional elements that you may
have added to the page.

Shortcuts

Keys: CTRL + S on PC; Command + S on Mac

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Save As command

Use this command to save and name the ModFit LT report document. ModFit LT displays the
Save As dialog so you can name your document.

To save a document with its existing name and folder, use the Save command.

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File Save As dialog box

This dialog is used to save reports in ModFit LT. This is the standard File Save dialog box for
the operating system, so its appearance and features are different on Windows and Mac
versions.

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Print command

Use this command to print a ModFit LT report to the selected printer. The Print dialog box is
displayed allowing you to select printing options.

Shortcuts

Keys: CTRL + P on PC; Command + P on Mac

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Page Setup command

Use this command to set up characteristics of printed pages, including paper size,
orientation, and margins.

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Zoom command

Use this command to change the magnification of the report document as viewed on the
screen. Increase the zoom to magnify the image, or decrease it to reduce the image size.

This has no effect on printed output.

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Recent Reports list

This menu is a convenient list that shows the most recent report files that have been opened
in ModFit LT. Select a file from the list to open the report.

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Edit 1P Graphics

This dialog is used to change the graphic properties for 1P histograms.

Smooth

This value determines the amount of smoothing for the histogram. Set to 0 for no smoothing.

Axis Line Color

Choose the color of the axis line.

Axis Line Weight

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Choose the weight of the axis line.

Background Color

Choose the color of the background page.

Background Transparency

Sets the transparency of the background.

Y Zoom Factor

Select a zoom factor for the Y direction.

Show Peak Locations

Enable this option to see where ModFit found peaks in the file. The peaks are shown as
triangles on the axis.

Data Line Color

Choose the color of the data line.

Data Line Weight

Choose the weight of the data line.

X-axis and Y-axis properties

Edit X (or Y) Parameter

Displays a dialog to Edit Properties for Parameter, to edit transformations, the name, etc.

Color

Choose a color of the axis label.

Font

Select the font of the axis label.

Number Color

Choose the color of the axis tic numbers.

Number Font

Select the font of the axis tic numbers.

Auto Minimum

Enable this option to automatically set the minimum of the axis.

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Minimum Value

When Auto Minimum is not enabled, this value sets the minimum value displayed by the axis.

Auto Maximum

Enable this option to automatically set the maximum of the axis.

Maximum Value

When Auto Maximum is not enabled, this value sets the maximum value displayed by the
axis.

Auto Calculate Major Tics

Enable this option to automatically determine the major tic unit for the axis

Number of Major Tics

When Auto Calculate is not enabled, this value sets the major tic unit of the axis.

Auto Calculate Minor Tics

Enable this option to automatically determine the minor tic unit for the axis.

Number of Minor Tics

When Auto Calculate is not enabled, this value sets the number of minor tics per major tic on
the axis.

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.

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Edit 2P Graphics

This dialog is used to change the graphic properties for 2P plots.

Smooth

This value determines the amount of smoothing for the histogram. Set to 0 for no smoothing.

Axis Line Color

Choose the color of the axis line.

Axis Line Weight

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Choose the weight of the axis line.

Background

Choose the color of the background page.

Background Transparency

Sets the transparency of the background.

Enhance Pops

Enable this option if there are small populations you need to visualize in the graph.

Color Mode

Choose between Rainbow and Gradient color density graphics.

Start Color

For Gradient color mode, this is the color of low frequency events on the axis.

End Color

For Gradient color mode, this is the color for the highest frequency events..

Show All Dots

When enabled, all events in the plot are displayed. If unchecked, the number of dots is
controlled by the Number of Dots edit field.

Number of Dots

When Show All Dots is unchecked, this field determines the number of dots to display on the
plot. Fewer dots may appear if there are fewer events in the plot.

Dot Size

Enter a size for dots. A value of 0 draws a dot 1 pixel in size.

X-axis and Y-axis properties

Edit X (or Y) Parameter

Displays a dialog to Edit Properties for Parameter, to edit transformations, the name, etc.

Color

Choose a color of the axis label.

Font

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Select the font of the axis label.

Number Color

Choose the color of the axis tic numbers.

Number Font

Select the font of the axis tic numbers.

Auto Minimum

Enable this option to automatically set the minimum of the axis.

Minimum Value

When Auto Minimum is not enabled, this value sets the minimum value displayed by the axis.

Auto Maximum

Enable this option to automatically set the maximum of the axis.

Maximum Value

When Auto Maximum is not enabled, this value sets the maximum value displayed by the
axis.

Auto Calculate Major Tics

Enable this option to automatically determine the major tic unit for the axis

Number of Major Tics

When Auto Calculate is not enabled, this value sets the major tic unit of the axis.

Auto Calculate Minor Tics

Enable this option to automatically determine the minor tic unit for the axis.

Number of Minor Tics

When Auto Calculate is not enabled, this value sets the number of minor tics per major tic on
the axis.

Actions

OK

Click this button to accept the changes and close the dialog box.

Cancel

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Click this button to discard any changes and close the dialog box.

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Report tools
Text tool

Use this command to add editable text to the report document.

A dialog will appear allowing you to enter text. After typing the text, click OK on the dialog.
The text will appear on the document. It can be dragged anywhere on the page. Its
bounding rectangle can also be resized.

To edit the text, double click anywhere on the text block.

To drag text, click the text and hold down the mouse button. With the button depressed,
move the mouse. Release the button to set the new position.

To add text to the Flash Comment list, choose the Save button in the Edit Text dialog. The
standard File Save dialog is displayed. Type in a name for the comment file and click OK.
Once the comment has been stored on disk, you will be prompted to type a description for
the comment. Enter the description and click OK. The Edit Text dialog will now display the
description in the Flash Comment list.

To add a stored comment to the report, choose the Text report tool. The Edit Text dialog
appears. From the Flash Comment list, click the comment you want to add. The text of the
comment will appear in the Edit Text dialog. Click OK to add the comment.

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Keywords tool

Use this command to add keywords from your data file and analysis results to the report
document. Some keywords are stored in the file at the time it is created by the acquisition
software. ModFit LT allows you to access these keywords and place them in the report. In
addition, you can access the analysis results as keywords to place on the report. A list of the
keywords appears in the Keywords section.

To move the object after it is inserted, click on it and hold down the mouse button. With the
button depressed, move the mouse. Release the button to set the new position.

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Keywords
This section lists and describes the keywords that are created by ModFit LT. These keywords
can be selected for display on reports using the Keywords command in the Tools menu, and
can be stored in a database as well.

Some keywords are available only when the conditions they relate to exist. For example,
keywords related to a second DNA Aneuploid population will only be available if the model
that is used contains components for a second Aneuploid cycle.

NOTE: The keywords for second, third, and fourth aneuploid cycles are identical to those for
the first, except that they are prefixed with Z_An2, Z_An3, and Z_An4 instead of Z_An1.

Standard Model Comment

Keywords
Z_Aggregates % aggregates
Z_AllCycleEvents Sum of events in cycles
Z_An1 % events in first aneuploid cycle
Z_An1_BAD % BAD in first aneuploid cycle
Z_An1_CV % CV for first aneuploid cycle G1
Z_An1_DI DNA index of G1 in first aneuploid cycle
Z_An1_G1 % of first aneuploid cycle in G1
Z_An1_G1_Mean Mean of G1 in first aneuploid cycle
Z_An1_G2 % of first aneuploid cycle in G2
Z_An1_G2_Mean Mean of G2 in first aneuploid cycle
Z_An1_G2G1Ratio G2/G1 ratio for first aneuploid cycle
Z_An1_Ploidy Ploidy of first aneuploid cycle
Z_An1_S % of first aneuploid cycle in S-Phase
Z_AneuploidS_Bnd S-Phase assessment cut-off value
Z_AneuploidS_Class S-Phase assessment classification
Z_AneuploidS_PValue S-Phase P-value
Z_AneuploidS_Std S-Phase assessment standard deviation
Z_CycleEventsPerChannel Average number of events per channel in
cycles.
Z_Debris % debris
Z_Dip % events in diploid cycle
Z_Dip_BAD % BAD in diploid cycle
Z_Dip_CV % CV for diploid cycle G1
Z_Dip_G1 % of diploid cycle in G1
Z_Dip_G1_Mean Mean of G1 in diploid cycle
Z_Dip_G2 % of diploid cycle in G2
Z_Dip_G2_Mean Mean of G2 in diploid cycle

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Z_Dip_G2G1Ratio G2/G1 ratio for diploid cycle

Z_Dip_Ploidy Ploidy of diploid cycle
Z_Dip_S % of diploid cycle in S-Phase
Z_DiploidS_Bnd S-Phase assessment cut-off value
Z_DiploidS_Class S-Phase assessment classification
Z_DiploidS_PValue S-Phase P-value
Z_DiploidS_Std S-Phase assessment standard deviation
Z_LinearityFactor Linearity value used in the model
Z_ModeledEvents Number of events accounted for by the
model
Z_PloidyStatus S-Phase assessment ploidy
Z_RCS Reduced chi-square
Z_Stnd1 % of events in first internal standard
Z_Stnd1_DI DNA Index of first standard
Z_Stnd1_DipG1Ratio DipG1 mean/First Std mean
Z_Stnd1_Mean Mean of first standard
Z_Stnd2 % of events in second internal standard
Z_Stnd2_DI DNA Index of second standard
Z_Stnd2_DipG1Ratio DipG1 mean/Second Std mean
Z_Stnd2_Mean Mean of second standard
Z_TotalAneuploidFraction Percentage of cells in aneuploid cycles
Z_TotalAneuploidS (Sum all aneuploid S-Phase
areas)*100/(Sum all aneuploid areas). For
simple Diploid models, the keyword reports
the value of Diploid S-Phase. You may
think of this keyword as "tumor S-Phase."
Z_TotalBAD % BAD for histogram
Z_TotalS Average or total S-Phase
Z_TotalS_Bnd S-Phase assessment cut-off value
Z_TotalS_Class S-Phase assessment classification
Z_TotalS_PValue S-Phase P-value
Z_TotalS_Std S-Phase assessment standard deviation

The following keywords store configuration values used by the program. The Auto Analysis
Settings, Peak Finder Settings, and Options and Configuration dialog boxes set these values.

Configuration Keywords Comment

CF_APOPTOSIS Auto Analysis state for apoptosis
CF_AUTOAGGREGATE Auto Analysis state for AutoAggregates
CF_AUTODEBRIS Auto Analysis state for AutoDebris

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CF_MOD_S_COMPARTMENTS Number of S-Phase compartments in this

model
CF_MOD_S_SHAPE Shape of S-Phase in this model
CF_G2G1RATIO Auto Analysis state for linearity or G2/G1
ratio
CF_PLOIDY_DIP_STD_RATIO Auto Analysis state for diploid-to-
standard ratio
CF_PLOIDY_MODE Auto Analysis state for ploidy detection
mode
CF_SPHASE_COMPARTMENTS Auto Analysis state for number of S-
Phase compartments
CF_SPHASE_SHAPE Auto Analysis state for shape of S-Phase
CF_SPHASE_DI_THRESHOLD Auto Analysis state for DI for S-Phase
CF_TET_RANGE Auto Analysis state for Tetraploid range
CF_PF_STATE State for Peak Finder
CF_PF_RISE Peak Finder setting for rise
CF_PF_LOWCV Peak Finder setting for low CV
CF_PF_HIGHCV Peak Finder setting for high CV
CF_PF_CC Peak Finder setting for correlation
coefficient
CF_PF_PCNTSTART Peak Finder setting for starting threshold

MF keywords

Internal keywords are prefixed with "MF_" and store data computed by the program. They
are listed in the table below.
Internal Keywords Comment
MF_ANALYSIS Type of analysis performed
MF_AUTODEBRIS_FIRSTCUT Setting for AutoDebris first cut
MF_AUTODEBRIS_RESOLUTION Setting for AutoDebris resolution
MF_AUTODEBRIS_SECONDCUT Setting for AutoDebris second cut
MF_AUTODEBRIS_THIRDCUT Setting for AutoDebris third cut
MF_DATE Date of analysis
MF_FILE Full path name of the data file
MF_FILEPATH Pathway to the data file
MF_GATE1 Parameters for gate
MF_GATE1_PERCENT Percent of events in gate
MF_GATE2 Parameters for gate
MF_GATE2_PERCENT Percent of events in gate
MF_GATE3 Parameters for gate
MF_GATE3_PERCENT Percent of events in gate

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MF_MODEL Name of the model used in analysis

MF_PLOIDYINDEX 0=Diploid, 1=Tetraploid, 2= One
Aneuploid, 3=Two Aneuploid, etc.
MF_RCS Reduced chi-square
MF_REPORT Name of the report
MF_REPORTPATH Pathway to the report
MF_TIME Time of the analysis
MF_VERSION Version of the program

USER keywords

These keywords show the results of user-entered equations found in Options and
Configuration dialog. The name for the keyword is prefixed with "USER_". This allows you to
display and database results of user-entered equations.

C keywords

Keywords that begin with "C_" display the final value for a detail of a model component. The
naming convention starts with "C_", followed by the name of the component, an underscore,
and the name of the detail. For example, "C_An1 G1_Area" displays the area of the An1 G1
component.

Sync Wizard keywords

When using the Sync Wizard, several additional keywords are made available. These allow
you to extract the percentage of each compartment used in modeling S-Phase. If, for
example, you use 3 rectangles to model S-Phase, you will find three additional keywords:
Z_DIPS_C1, Z_DIPS_C2, Z_DIPS_C2.

Proliferation Wizard keywords

When using the Proliferation Wizard, additional keywords are available to allow you to display
and database all of the information generated by the wizard. These keywords are prefixed
with "Z_PROLIF", and are named to indicate the value that it stores.

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Line tool

Use this command to add lines to the report document. Lines can be dragged into position
anywhere on the report.

To drag a line, click on the object and hold down the mouse button. With the button
depressed, move the mouse. Release the button to set the new position.

To change the size or angle of a line, click on the line to make it the active object. Handles
will appear on a rectangle that contains the line. Click and hold one of the handles to
reposition that corner of the bounding rectangle. The line will reorient as you move the
handle. Each of the handles can be moved in the same manner.

To change the properties of a line, double click on the line.

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Arrow tool

Use this command to add lines with arrowheads to the report document. Arrows can be
dragged into position anywhere on the report.

To drag an arrow, click on the object and hold down the mouse button. With the button
depressed, move the mouse. Release the button to set the new position.

To change the size or angle of an arrow, click on the arrow to make it the active object.
Handles will appear on a rectangle that contains the arrow. Click and hold one of the handles
to reposition that corner of the bounding rectangle. The arrow will reorient as you move the
handle. Each of the handles can be moved in the same manner.

To change the properties of an arrow, double click on the arrow.

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Rectangle tool

Use this command to add rectangles to the report document. Rectangles can be dragged into
position anywhere on the report, and have selectable colors.

To drag a rectangle, click on the object and hold down the mouse button. With the button
depressed, move the mouse. Release the button to set the new position.

To change the size of a rectangle, first click on it to make it the active object. Click and hold
one of the handles to reposition that corner of the rectangle. Each of the handles can be
moved in the same manner.

To change the properties of a rectangle, double click on the rectangle.

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Ellipse tool

Use this command to add ellipses and circles to the report document. Ellipses can be dragged
into position anywhere on the report.

To drag an ellipse, click on the object and hold down the mouse button. With the button
depressed, move the mouse. Release the button to set the new position.

To change the size of an ellipse, first click on it to make it the active object. Click and hold
one of the handles to redefine the size and shape of the ellipse. Each of the handles can be
moved in the same manner.

To change the properties of an ellipse, double click on the ellipse.

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X Zoom Graph

Use this command to add a graph object to the report that shows a zoomed view of the
histogram. The inserted graph can be moved and resized on the report.

The X Zoom graph object is especially useful for visually identifying near Diploid populations.
It is designed to crop the X-axis so that only the Diploid G1 peak is on scale.

To edit the object zoom settings, double click on the object after it has been inserted into the
report. Then, adjust the X-Axis minimum and maximum values to include the portion of the
histogram you want to view.

You can set the default values for this object using the Options and Configuration dialog box.

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Y Zoom Graph

Use this command to add a graph object to the report that shows a zoomed view of the
histogram. The inserted graph can be moved and resized on the report.

The Y Zoom graph object is designed to enhance low-level histogram data. It provides a
detailed view of S-Phase and small G2M and aggregate forms. Use the graph to verify the fit
of the model and ensure the correct model is selected.

To edit the object zoom settings, double click on the object after it has been inserted into the
report. Select a new Y Zoom factor to increase or decrease the magnification of the
histogram.

You can set the default values for this object using the Options and Configuration dialog box.

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Fit Diagnostics command

Use this command to add a report object that displays a summary of the Fit Diagnostics
dialog box on the report. The object shows a legend of the elements found in the Fit
Diagnostics dialog box. The object can be moved to any convenient location on the report.

To edit the properties of the object, double click on the object after it has been inserted into
the report.

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Batch Processing
Batch Processing

ModFit LT provides powerful batch processing of data files and reports. Batch processing of
data files supports automatic analysis, saving and printing of reports, and databasing of
results, in addition to other features. When processing batches of reports, you can re-analyze
data, perform AutoLinearity adjustments, re-save and print reports, and store results in
databases.

Batch Processing Settings

The Options tab contains entries for File Batch Settings and Report Batch Settings. These
options allow you to select what you want to do as you process data files and reports,
respectively. Refer to these sections for specific details.

Batch Selection

If you select more than one file in either the Open FCS or Open reports dialogs, the program
automatically enters batch-processing mode. A toolbar appears at the bottom of the
application window when more than one file is selected. The toolbar allows you to navigate
through the files, performing the selected operations. Refer to sections on File Batch Toolbar
and Report Batch Toolbar for details.

Example 1

Suppose that you want to select a set of data files, auto-analyze them, and save reports for
each. In addition, you do not what the program to prompt you to store a record in the
database, nor for any listmode gating changes.

1. From the Options tab, choose File Batch Settings.

2. In the dialog, choose Auto-analyze and Save report. Choose a location for the reports
using the Save Report Path button. Clear the checkboxes for Database prompt and File
prompt. Click OK to apply the settings and close the dialog.

3. Next, choose the Open FCS command. Select the files that you want to analyze and click
Open. The File Batch toolbar will appear at the bottom of the application window, the first file
will be opened and auto-analyzed, and a report will be saved for the analysis. Click the Auto-
advance button on the File Batch toolbar to automatically process the rest of the files.

Example 2

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Suppose you want to review a set of reports, one at a time, and print them after making
changes to them. You can approach this either of two ways: A) review, correct, and print
each report, or B) review, correct and re-save each report, then re-run the entire batch and
automatically print each report.

1. From the Options tab, choose Report Batch Settings. Clear all of the checkboxes and click
OK.

2. Next, choose Open to open reports. Select the reports you want to review and choose
Open. The Report Batch toolbar will appear at the lower right of the application window, and
the first report will be displayed.

3. Review the report, making changes as needed.

4. When you are ready to proceed, you have 2 choices. If you prefer the first method
described above, you would click the Print button on the main toolbar to print the report, and
click the Save button if you made any changes. For the second method, just click the Save
button to resave the report.

5. To move to the next report, click the Next button on the Report Batch toolbar and repeat
the process above until you have reviewed all of the reports.

6. If you want to batch print all of the saved reports, you would return to the Report Batch
Settings dialog. This time, you would check the option to Print reports and click OK. Click the
button on the Report Batch toolbar that moves you to the first report. The report will be re-
opened and automatically printed. Now click the Auto-advance button to open and print each
of the remaining reports.

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File Batch Settings

This option displays the Edit Properties for File processor dialog box, allowing you to choose
what happens during batch processing of data files. The dialog is displayed automatically
when you select more than one data file for analysis.

Batch processing options apply when you select more than one data file in the Open FCS
dialog box. When more than one file is selected, the File Batch toolbar is displayed at the
bottom of the application window. You can navigate through the batch one file at a time, or
auto-advance through the batch.

Analysis

Choose what analysis option you want to occur when a new data file is opened. The options
available are listed below:
No analysis: No analysis is performed.
Auto-analyze: Automatic Analysis is performed.
Auto-analyze and Auto Linearity: Automatic Analysis is performed, followed by Auto
Linearity.
Fit with current model: The Fit command is performed with the current model.

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Fit and Auto Linearity: The Fit command is followed by Auto Linearity with the current
model.

Save Report

Enable this option to automatically save a report for each data file. The report is
automatically named using the Auto Save Report command. Saving occurs after any analysis
has been performed, and again when moving to another batch item.

Save Report Path

If the Save report option is enabled, reports will be saved to the path shown here. Click the
Folder button to select a new location for reports.

Print Report

Enable this option to automatically print a report to the current printer. Printing occurs when
moving to another batch item.

Save PDF

Enable this option to automatically generate a PDF version of the report. The PDF is created
in the same location as the Save Report Path.

Save Graphics

This option allows you to save graphics for the histogram or report. Graphics are
automatically named based on the data file that is loaded, as described in Export Histogram
Graphic and Export Report Graphic. The save operation occurs when moving to another
batch item. The options available are listed below:
No: No graphics are saved to disk.
Histogram: Histogram graphics are saved.
Report: Report graphics are saved.
Histogram and Report: Both Histogram and Report graphics are saved.

Database prompt

Enable this option if you want to be prompted to store the analysis in any active databases.
If you clear this checkbox and databases are active, records will be stored without prompting.
If databases are inactive, this option is ignored.

File prompt

Enable this option if you are opening listmode files and you want the program to allow you to
change the selected parameter or gates, or if you are opening histogram files with multiple
histograms in each and need to select the histogram of interest.

Multi Dataset prompt

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Enable this option if you are opening files that contain more than one FCS dataset. For
example, Beckman Coulter FC-500 files are often stored with 2 datasets: one containing FCS
2 data, and the second containing FCS 3 data. In order to select a dataset, this option must
be enabled.

Once you have selected a dataset, you can disable this option and process an entire batch of
files without having the prompting dialog appear.

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File Batch Toolbar

This toolbar is displayed at the bottom of the ModFit LT application window when you select
more than one file in the Open FCS dialog.

Most of the buttons on the toolbar are used to navigate through the batch. The left-most
button is used to edit the properties of the batch processor. See File Batch Settings for more
on file batch properties.

As you navigate through the batch, the actions and options in the File Batch Settings dialog
are performed on each file that is opened. This allows you to perform a series of automatic
operations just by moving to another batch item, such as automatic analysis, saving and
printing reports. The function of each button is described below.

Edit the properties of the batch.

Display the Choose Batch dialog to select a

batch item to open.

Go to the first file in the batch.

Go to the previous file in the batch.

Redo the current file in the batch.

Go to the next file in the batch.

Go to the last file in the batch.

Auto-advance through the batch.

Stop the auto-advance process.

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Choose Batch Dialog

This dialog is displayed when the Choose button on the File Batch or Report Batch toolbar is
clicked. It allows you to choose a batch item to open.

The listbox displays all items in the batch. To select an item, click on it in the list and click
OK. The item will be opened.

Click Cancel to close the dialog without changing the batch item.

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Report Batch Settings

This option displays the Edit Properties for Report processor dialog box, allowing you to
choose what happens during batch processing of reports. The dialog is displayed
automatically when you select more than one report for analysis.

Batch processing options apply when you select more than one report in the Open Reports
dialog box. When more than one report is selected, the Report Batch toolbar is displayed at
the bottom of the application window. You can navigate through the batch one report at a
time, or auto-advance through the batch of reports.

Analysis

Choose what analysis option you want to occur when a new data file is opened. The options
available are listed below:
No analysis: No analysis is performed.
Auto-analyze: Automatic Analysis is run the data in the report.
Auto-analyze and Auto Linearity: Automatic Analysis and Auto Linearity are performed
on the report data.
Fit with current model: The Execute Model command is executed on each data file.
Fit and AutoLinearity: An AutoLinearity adjustment is performed for each report, and
the model is finally fit with the best linearity setting. See Using AutoLinearity for more
on this option.

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Add Zoom Objects

Enable this option to automatically add an X-Zoom and Y-Zoom graph to each report that is
processed. These objects allow close examination of the low level events such as debris,
aggregates, and S-Phase, and provide a magnified view of the area around the Diploid G1
peak.

Save Report

This drop-down provides three report saving options described below. Saving occurs after
any analysis has been performed, and again when moving to another batch item.
Don't re-save report: The report is not resaved.
Re-save report: The report is resaved, using the save report name.
Auto-name and save report: The report is resaved using the Auto Save Report
command. The name of the report may change from the original name with this
option.

Print Report

Enable this option to automatically print the report to the current printer. Printing occurs
when moving to another batch item.

Save PDF

Enable this option to automatically generate a PDF version of the report. The PDF is created
in the same location as the Save Report Path.

Save Graphics

This option allows you to save graphics for the histogram or report. Graphics are
automatically named based on the data file that is loaded, as described in Export Histogram
Graphic and Export Report Graphic. The save operation occurs when moving to another
batch item. The options available are listed below:
No: No graphics are saved to disk.
Histogram: Histogram graphics are saved.
Report: Report graphics are saved.
Histogram and Report: Both Histogram and Report graphics are saved.

Database prompt

Enable this option if you want to be prompted to store the analysis in any active databases.
If you clear this checkbox and databases are active, records will be stored without prompting.
If databases are inactive, this option is ignored.

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Report Batch Toolbar

This toolbar is displayed at the lower right of the ModFit LT application window when you
select more than one file in the Open Reports dialog.

Most of the buttons on the toolbar are used to navigate through the batch. The leftmost
button is used to edit the properties of the batch processor. See Report Batch Settings for
more on report batch properties.

As you navigate through the batch, the actions and options in the Report Batch Settings
dialog are performed on each file that is opened. This allows you to perform a series of
automatic operations just by moving to another batch item, such as rerunning the analysis
and printing reports. The function of each button is described below.

Edit the properties of the batch.

Display the Choose Batch dialog to select a

batch item to open.

Go to the first file in the batch.

Go to the previous file in the batch.

Redo the current file in the batch.

Go to the next file in the batch.

Go to the last file in the batch.

Auto-advance through the batch.

Stop the auto-advance process.

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Clipboard
Copy command

Use this command to copy the report or a single object to the clipboard. This command is
unavailable if there is no data currently loaded into the program.

Copying to the clipboard replaces the contents previously stored there.

To copy a single object, click the object to select it, and then choose the Copy command. The
object will be placed in the clipboard.

To copy the entire report, make sure no object is selected in the report, and then choose the
Copy command. The report will be copied as a graphic image to the clipboard.

Shortcuts

Keys: Ctrl + C on PC; Command + C on Mac

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Paste command

Use this command to insert a copy of the clipboard contents onto the report. This command
is unavailable if the clipboard is empty or if ModFit LT does not support the format of the
data.

When the clipboard contains more than one format at a time, ModFit LT prompts you to
select the format you want. Select the format you want from the list and click OK to paste
the object.

After it is pasted, the object may be selected and dragged to a new location on the screen.

Supported formats include the following:

PC version: Text, Bitmap, PNG

Mac version: Text, Bitmap, PNG

Shortcuts

Keys: Ctrl + V on PC; Command + V on Mac

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Databases
Databases

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins and ModFitEditors have permission to
perform them.
Purpose: To add, delete or edit one or more databases. See Databasing with ModFit LT for
more on databases.

The Edit All Databases dialog can be displayed by choosing Edit Databases from the Edit
tab.

Overview

ModFit LT can have one or more databases defined at any time. The database format is
ASCII-formatted, tab-delimited database. Any FCS keyword or analysis result can be mapped
to a database field. A complete description of the functionality of the database system can be
found in the Databasing with ModFit LT discussion.

Edit

If a database is selected, this option shows the Edit Keyword Map dialog box. You can also
double-click the database item to activate this option.

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Properties for the selected item

This button appears in the group of buttons below the listbox. It allows you to edit properties
for the selected item in the list.

If a database is selected, the Edit Properties for Databasen dialog will be displayed.

New

This command creates a new, empty database.

Delete

This option will delete the selected database from the list.

OK

Put away the Edit All Databases dialog box.

Properties

This button edits properties for the containing object.

The Edit Properties for All Databases dialog box will be displayed.

Help

Show this help topic.

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Edit Properties for All Databases

Purpose: To perform actions that will apply to all databases in the Edit All Databases.

Choosing Edit Databases from the Edit tab, then clicking the Properties button on the right of
the dialog will display the Edit Properties for All Databases dialog.

Overview

The dialog is displayed when the Properties button is clicked in the Edit All Databases dialog,
and no databases are selected in the list. It allows you to perform action on all of the defined
databases at once.

Activate all

Activates all databases. When a database is active, it can store records of information as you
analyze files in WinList.

De-activate all

Disables all databases. No database will store information until it is reactivated.

Delete files

Removes all database files on disks for databases in the Edit All Databases dialog. This is a
convenient file management button, but should be used with great caution. Make sure you
have adequate backup copies of important databases before using this option.

OK

Close the dialog and accept changes.

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Cancel

Since the options in this dialog box are immediate, Cancel is the same as clicking OK.

Help

Show this help topic.

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Edit Properties for Database

Purpose: To activate, display, and set other attributes of a database. See Databasing with
ModFit LT for more on databases.

The Edit Properties for Database dialog can be displayed by choosing Edit Databases from
the Edit tab, selecting the database you want, then clicking the Properties button. If the
Database Viewer is displayed, clicking a button on the viewer's toolbar can also access this
dialog.

Overview

Each database can be activated independently. Additionally, the database can be set for
prompting or automatic mode, and a viewer window can be displayed. This dialog also allows
you to set the path and other properties for the database.

Active

Check this option to activate the database. When the database is active, it will attempt to
store a record each time an analysis is performed. Uncheck this option to disable a database;
records will not be saved in databases that are not active.

Prompting

Check this option if you want ModFit LT to prompt you each time it is ready to save a record.
The prompt will allow you to confirm or cancel the saving of the record into each active
database. Uncheck this option for automatic operation where ModFit LT stores the
information without prompting.

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Viewer

Check this option to display a Database Viewer for the database. The viewer displays the
fields in columns and each row represents a record in the database. Additional options are
available on the viewer window.

Viewer Font

Select a font to use for the database viewer.

Choose database

This option allows you to choose an existing database on disk. Click the "Link to" button. A
file selection dialog box will appear, allowing you to navigate to the existing database and
select it. Linking to an existing database will rename the database in memory to match the
one selected on disk, and the associated database map on disk will be loaded as well.

Change location

This option allows you to change the path for the defined database. Click the "Path" button
to display a Choose database location dialog. Navigate to the location in which you want to
store the database.

If a database of the same name already exists in that folder, you will be offered three
choices. First, you can choose to overwrite the database with the one you have defined.
Secondly, you can choose to use the database that exists in the new location, replacing the
structure you have defined in memory. This is convenient if you have a database already
defined that you want to use. Or you can choose to cancel the action.

OK

Save property settings and put away the Edit Properties for Databasen dialog box.

Cancel

Close the dialog and discard any changes you have made.

Help

Show this help topic.

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Edit Keyword Map

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins and ModFitEditors have permission to
perform them.
Purpose: Select the information you want to save in a database. See Databasing with ModFit
LT for more on databases.

The Edit Keyword Map dialog can be displayed by choosing Edit Databases from the Edit
tab, selecting the database you want to edit, then clicking the Edit button. If the Database
Viewer is displayed, clicking a button on the viewer's toolbar can also access this dialog.

Overview

Each database in ModFit LT has its own structure, made up of a set of keywords to database
and a matching set of fields in a database. The Edit Keyword Map dialog determines how the
keywords are "mapped" to fields in the database.

Results Tree

This tree expands to display all of the available groups of keywords and the keywords in each
group. A branch of the tree can be expanded by double clicking the mouse on it. Select a
branch of the tree by clicking once on it.

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This tree is used to select the keywords you want to store in the database. It allows multiple
selection. See the discussion in Databasing with ModFit LT for more on proper selection of
information to database.

Map Definition

This list displays the field names and the keywords they relate to. The first column showing
the field name can be edited; other columns are for display only. To edit a field name, click
once on the field to select it, then click again after a moment to enter edit mode.

Add

Click this button to add the keywords selected in the Results Tree to the database definition
shown in the Map Definition list. Fields are named automatically to show the source of the
information. You can also double-click on a keyword to add it to the definition.

Replace

The option is available when you select an item in the tree and an item in the Map Definition
list. It allows you to map a new keyword to an existing field in the database.

Delete

Click this button to delete the fields selected in the Map Definition list from the database.

Up/Down

These buttons allow you to change the order of fields in the Map Definition list. Select one or
more fields in the list, and then use the Up and Down buttons to move the item(s).

OK

Save changes to the database. If records already exist in the database, the structure of the
records will be revised to match the new structure you have defined.

Cancel

Close the dialog and discard any changes you have made.

Help

Show this help topic.

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Database Viewer

Purpose: Display the contents of a database.

The Database Viewer can be displayed by choosing to show the viewer in the database
properties. To do so, choose Edit Databases from the Edit tab, select the database you
want, and then click the Properties button. In the Edit Properties for Database dialog,
enable the viewer and click OK.

Overview

This window can be displayed for any database you have created. The viewer displays the
fields defined in the database as column headings. Each record in the database is shown as a
row of information. The window can be scrolled horizontally and vertically if there is
information out of view. Each column can be resized by dragging the partition between the
column headings with the mouse.

At the top, a toolbar provides buttons to manually add, delete, and replace records.

Click the copy button to copy the database as text to the clipboard.

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Click the "+" (add) button to add the current analysis information to the database.

Click the "-" (delete) button to delete the selected records from the database; if the button is
grayed, there are no records selected.

Click the replace button to store the current analysis into an existing record in the database.

Additional buttons activate and deactivate the database, display the Edit Keyword Map
dialog, display the Edit Properties for Database dialog, and backup the database.

See Databasing with ModFit LT for more on databases.

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Add Record

This button appears on the Edit tab of the ribbon bar. It can be used to manually trigger the
addition of a record to active databases.

See also:

Databases

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Database Wizard

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins and ModFitEditors have permission to
perform them.
The Database Wizard is a tool that makes it easy to set up a database for the ModFit analysis
results.

To use the wizard, start by opening a data file and analyzing it so that you have a set of
analysis results on the report. Then, start the database wizard by clicking this button, found
on the Edit tab of the ribbon bar.

The wizard's greeting page will display.

Click Next to proceed. The Database page of the wizard will display.

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Select an existing database from the list or use the New button to create a new one. Once
you have made your selection, click the Finish button to complete the wizard.

The wizard will add fields to the selected database for all of the analysis results that are
shown on the report. If a field is already in the database, it will not be added again.

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Exporting
Export: Save PDF

Use this command to export the report to PDF format on disk.

When you select this option, the File Save dialog is displayed. Navigate to the location where
you want to store the file, and then enter a name for the file. Click the Save button to store
the file on disk.

Exporting is also available in batch processing. See File Batch Settings and Report Batch
Settings for more.

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Export: Save Histogram Graphic

Use this command to export the graphics for the histogram to a file on disk. The default
format of the graphic can be selected using the Options and Configuration dialog. Supported
formats currently include bitmap (bmp), JPeg (jpg), Tiff (tif), Portable Network Graphics
(png), Portable Pixmap (ppm), X11 Bitmap (xbm), X11 Pixmap (xpm), and Scalable Vector
Graphics (svg).

When you select this option, the File Save dialog is displayed. Navigate to the location where
you want to store the graphic file, and then enter a name for the file. Click the Save button
to store the file on disk.

Exporting is also available in batch processing. See File Batch Settings and Report Batch
Settings for more.

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Export: Save Report Graphic

Use this command to export the graphics for the entire report to a file on disk. The format of
the graphic can be selected using the Options and Configuration dialog. Supported formats
currently include bitmap (bmp), JPeg (jpg), Tiff (tif), Portable Network Graphics (png),
Portable Pixmap (ppm), X11 Bitmap (xbm), X11 Pixmap (xpm), and Scalable Vector Graphics
(svg).

When you select this option, the File Save dialog is displayed. Navigate to the location where
you want to store the graphic file, and then enter a name for the file. Click the Save button
to store the file on disk.

Exporting is also available in batch processing. See File Batch Settings and Report Batch
Settings for more.

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Logging
Edit Log

Research and Compliance Mode note:

In RUO mode, users can turn logging on and off, and set properties.
In Compliance mode, logging is always enabled.

ModFit LT allows you to create a log of operations that you perform. This is useful if you
have any difficulties with the program and need to interact with the Technical Support
team. Logging is always enabled when the program is running in Compliance mode.

To view the current log, click the Logging button on the Edit tab of the ribbon bar.

To adjust properties of the log, click the Properties button. See Edit Properties for Log for
details on the log settings.

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Edit Properties for Log

Research and Compliance Mode note:

In RUO mode, users can turn logging on and off, and set properties.
In Compliance mode, logging is always enabled.

This dialog allows you to activate and adjust the program's logging features, useful in
isolating problems that you may have in working with the program.

Active

To enable logging of program operations, check this option. This file is a plain text file, with
fields separated by tabs. It can be opened in most text editors, or attached to email
messages.

To disable logging, clear the check from this checkbox.

Log File

This read-only field shows the name of the log file on disk.

Logging Level

This field selects the level of detail stored in the log. Choose from "Basic" and
"Comprehensive". Comprehensive logging is typically only used when working with a support
technician at Verity Software House to determine the cause of a problem.

Maximum Log Size

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Select the maximum size for the log file. If this is set for Unlimited, the log will grow without
limitation (not recommended).

Full Error Report

Generally, this option should not be enabled. However, Technical Support make ask you to
enable this option to determine the cause of a problem.

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Preferences
Auto Analysis Settings

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins have permission to perform them.

This dialog is used to configure several properties of the Auto Analysis command.

AutoDebris

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When this option is checked, the program will enable the AutoDebris component to model
debris in the data file during Auto Analysis. Debris is often created in the sample preparation
process. If the option is not checked, the AutoDebris component will be disabled.

AutoAggregates

When this option is checked, the program will enable the AutoAggregate component to
model aggregation in the data file during Auto Analysis. Aggregation in the sample can
produce a continuum of particles that are stuck together, significantly affecting S-Phase
estimates. If the option is not checked, the AutoAggregate component will be disabled.

Apoptosis

NOTE: We strongly recommend that this option should be disabled in your configuration. To
get more robust and accurate results, use additional markers for apoptosis detection.

If this option is checked, the first peak detected after any internal standards will be
considered an apoptotic population and modeled with a Gaussian.

Linearity Settings

Linearity

This value is used in the computation of some G2M positions, and in the AutoAggregate
model component. The default value is 2.0, as G2M peaks are expected to appear at two
times the associated G1 peak. Adjust this value to match the known linearity setting for your
instrument.

When Auto Analysis does not detect a distinct G2M peak for a cycle, it assumes the location
of the G2M peak to be at the G1 position times this linearity setting. This is called a
dependent G2M, because its position depends on the G1.
The linearity value is used by the AutoAggregate model component to determine the
expected locations of peaks created by aggregation.

Standards and References

Internal Standards

Select the number of internal standards that your data files contain.

Most DNA internal standards are designed to appear to the left of the first cycle's G1
population. By default, the program will assume that the first peaks it finds should be
assigned to the number of standards you select here. Use the Properties of Standards button
below to give the program additional information about the internal standards in your
samples.

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Properties of standards

Click this button to display the Standards dialog. This dialog allows you to edit the names and
expected locations of internal standards in your samples. By entering expected locations, the
program can more accurately determine which peaks are standards, and where the Diploid
population occurs. See the description of Diploid determination below for more on using this
value to assign ploidy.

External reference standard

If you enter a value of an external standard location, you can make use of this value in
assigning ploidy labels to the cycles that AutoAnalysis finds. For example, if you run a normal
Diploid control at a particular channel, you can enter that channel position in this edit box.
See the description of Diploid determination below for more on using this value to assign
ploidy.

Ploidy Determination

Maximum cell cycles

Select the maximum number of cell cycles that Auto Analysis can model. By default, this is
set to "Unlimited". If the program detects numerous false peaks in your data files, you may
want to specify a maximum of 3 or 4 cycles. See the Peak Finder Settings for more
information about finding peaks.

Diploid determination

This option determines the method that Auto Analysis will use to identify the Diploid cycle in
your data files.

First cycle is Diploid

This is the default selection. Use this option if you want the first (leftmost) cell cycle always
to be classified as the Diploid cycle.

Based on external reference

Use this option if you have entered an expected location for an external reference above, and
you want to use that position as the basis for identifying the Diploid cycle. The expected
Diploid location will be computed as the External reference standard location multiplied by
the Diploid-to-Standard ratio.

Based on Standard1 and Based on Standard2

Use this option if you have entered expected location(s) for internal standards above in
Properties of standards, and you want to use one of the standards to compute the expected
location of the Diploid cycle. The expected Diploid location will be computed as the Expected
position of the standard multiplied by the Diploid-to-Standard ratio.

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Examples:

1. Suppose you have one internal standard in your samples that is normally found at channel
38 in the DNA histogram, and that the normal Diploid G1 location is 1.3 times the standard's
position. You would enter the expected location of the standard using Properties of
standards, select Based on Standard1 for the Diploid determination option, and enter 1.3 in
the Diploid-to-standard edit box.

2. Suppose you run a normal Diploid control that is positioned at channel 55 everyday, and
that you expect this to be the location of Diploid cycles in your test samples as well. You
would enter 55 in the External reference standard field, select Based on external reference
for the Diploid determination, and enter 1.0 for the Diploid-to-Standard ratio.

Diploid-to-Standard ratio

This value is used if you have the Diploid determination option selected for Based on external
reference, Based on Standard1, or Based on Standard2. The expected location for the Diploid
G1 is computed by multiplying this value times the expected location of the reference or
standard.

When Diploid determination is set for First cycle is Diploid, this value is not used.

G1 threshold

This value sets the minimum relative height for a peak to be considered a G1, after adjusting
the height for aggregates. The relative height is a value relative to the largest peak, where
the largest peak is 100%. The default setting for G1 threshold is 5%. To allow smaller peaks
to be considered G1 peaks, lower the value. To make the setting more restrictive, increase
the setting.

Peak location range

This value controls how much variation the program allows in looking for a peak at an
expected location. It is a percentage of the expected location. If, for example, the expected
location of the Diploid G1 is channel 50 and this value is set for 10%, Auto Analysis will look
for a peak between channel 50 plus and minus 5 channels, or 45 to 55.

Dependent G2M

With this option enabled, Means and Standard Deviations for all G2M components will be
dependent on the associated G1 components in models created by Auto Analysis. This setting
creates more consistent analysis results from one operator to the next, and it is
recommended. With the option disabled, any G2M component that has a well-defined Peak
Finder peak will have a floating Mean and Standard Deviation. Those without well-defined
peaks will be dependent on associated G1 components.

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S-Phase

S-Phase shape

This option controls the mathematical function that is used to model S-Phase.

Rectangle
This is the default selection. It provides the most conservative estimate of S-Phase and is the
recommended option.

Trapezoid
This option provides one additional degree of freedom over the Rectangle.

Polynomial
This option adds two degrees of freedom over a rectangle.

Number of compartments

Select the number of compartments to use in modeling S-Phase. By default, this option is 1,
the recommended value.

DI for S-Phase

Enter a DNA index value (DI) below which the program will disable S-Phase modeling for the
lower of two overlapping cycles. The default and recommended value is 1.3.

When two cell cycles are significantly overlapped, the S-Phases can be arbitrarily inflated or
deflated in the modeling process. In this situation, it is better to disable the S-Phase for the
lower-positioned cycle. The DI for S-Phase setting controls the threshold for this decision.

Tetraploid

G2M Threshold

This value determines when a peak at a G2M location is considered to be too large for a G2M
peak, and is classified as a G1 peak for another cycle. The value used in making this decision
is a simple statistical fit of the peak, not the modeled result for the peak.

The value is a percentage of the estimated area of the associated G1 peak. By default, the
G2M can be up to 15% of its associated G1 before it is reclassified as a G1 of another cycle.

Tetraploid location range

This value controls how much variation the program allows in looking for a potential
Tetraploid peak. It is a percentage of the expected location.

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Open

If you have previously saved Auto Analysis settings to disk, you can use the Open button to
load those settings into the program.

Save

Use this option if you want to store the current Auto Analysis settings to a file on disk. The
standard file save dialog box will be displayed, allowing you to navigate to a location and
name the file to save.

Reset

Click this button to restore the Auto Analysis values to the factory default settings.

OK

Click this button to accept the changes you have made and close the dialog.

Cancel

This option closes the dialog and discards any changes you might have made.

Help

This option displays the on-line help for this dialog.

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Standards
Research and Compliance Mode note:
In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins have permission to perform them.

This dialog allows you to edit how ModFit LT interprets the internal standards in your data
files.

If your files contain internal standards, you should fill in the expected location for each
standard in this dialog. Doing so will assist the program in identifying standard peaks, as well
as to correctly classify cell cycles. After you enter location values in this dialog, you can
choose a ploidy determination method in the Auto Analysis Settings dialog that makes use of
the location.

Select the standard that you want to edit in the list on the left and the right hand list will
show the properties that you can adjust for the standard.

See also: Properties for Standards

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Properties for Standards

Name

Enter a name for the standard. This name will be assigned to a range for the standard peak.
The range can be viewed and adjusted by clicking the Range button on the toolbar.

Expected position

Enter the channel location on the histogram where the standards peak is typically found. You
should ensure that your protocols for running samples on your flow cytometer consistently
maintain this position for the standard.

Generate debris

When checked, internal standard components are considered to be involved in the generation
of debris in the histogram.

Note: Models that are created by AutoAnalysis ignore internal standards from debris
generation.

Generate aggregates:

When checked, internal standard components are considered to be involved in the generation
of aggregates in the histogram.

Note: Models that are created by AutoAnalysis ignore internal standards from aggregate
generation.

See also: Standards

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Peak Finder Settings

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins have permission to perform them.

The Peak Finder dialog adjusts the settings used by the system to determine how peaks will
be identified in your samples. The peak finder statistically determines what populations are
peaks in the histogram data, and then applies filters to remove unwanted peaks from
consideration.

The peak finder is of fundamental importance in Automatic Analysis. Therefore, it is very

important to approach changes to the peak finder with care and thoughtfulness.

Changes to these settings will affect how the program defines a peak, which in turn will alter
ploidy detection, model selection, and analysis results in Automatic Analysis. Test any
changes you make on a reasonable sampling of your data files. Make changes to improve the
overall effectiveness of the peak finder, not to handle an exceptional sample. Exceptional
samples should be handled using manual analysis.

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The dialog displays the analysis histogram. The graphic indicates the currently identified
peaks with black triangles at the base of the histogram. In addition, there are controls that
allow you to adjust the peak finder settings and see the effect immediately.

As you move your mouse pointer over the histogram, boundaries of peaks are drawn over
the data, illustrated in the graphic above. The status of the peak for each criterion is shown
next to the control for the filter. Using this information, you can determine why the peak
finder excludes peaks, and make adjustments to the filters accordingly.

Start at

The value controls the point at which ModFit LT starts looking for peaks. It allows you to skip
over channels where your data has debris that may be misinterpreted as a peak. The value is
a percentage of the histogram scale, and higher values move the starting point to higher
channel location.

Low %CV

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This value controls the low %CV cutoff for data to be considered a peak. %CV reflects the
broadness of the peak; those peaks which have %CV lower than this filter are excluded. To
remove more narrow peaks, increase the value. To allow more peaks, lower the value.

High %CV

This value controls the high %CV cutoff for data to be considered a peak. %CV reflects the
broadness of the peak; those peaks which have %CV higher than this filter are excluded. To
allow more peaks to pass the filter, increase the value. To reduce the number of peaks that
pass the filter, reduce the value.

Rise Threshold

This value controls how far a peak must rise above adjacent data to be considered a real
peak. Peaks that extend above adjacent data less than this value are excluded from the peak
finder. To increase the minimum rise for a peak and reduce the number of passing peaks,
increase this value. To reduce the filter and allow more peaks to pass, reduce the value.

Correlation

This value controls the minimum correlation coefficient allowed for a peak to pass this
"shape" filter. Each potential peak is fit with a Gaussian curve; the resulting correlation
coefficient is compared to this filter. To increase the minimum allowable correlation
coefficient and reduce the number of passing peaks, increase the value. To increase the
number of peaks that pass the filter, reduce the value.

Open

If you have previously saved peak finder settings to disk, you can use the Open button to
load those settings into the program. This is useful if you analyze sets of data that have
different peak characteristics.

Save

Use this option if you want to store the current peak finder settings to a file on disk. The
standard file save dialog box will be displayed, allowing you to navigate to a location and
name the file to save.

Reset

Click this button to restore the peak finder values to the factory default settings.

OK

Click this button to accept the changes you have made and close the dialog.

Cancel

This option closes the dialog and discards any changes you might have made.

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Help

This option displays the on-line help for this dialog.

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Options and Configuration

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins have permission to perform them.

This dialog is used to set program configuration options.

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Data Processing and Display

Apply Dither

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When enabled, a small random value is added to listmode events to make the distributions
continuous. This is enabled by default.

Apply Compensation Keywords

When enabled, the program will read compensation keywords from the FCS file and apply
compensation defined in those keywords.

Desired resolution

Select the desired resolution for the analysis histogram. It is strongly recommended that you
use the default setting of 256 channels to improve performance and consistency. The
histogram will be displayed and analyzed at the desired resolution, provided that the
parameter's resolution is greater than or equal to the desired resolution.

Log Decade Option

This option determines how many decades will be displayed for listmode data. Choose
between "Computed Decades" and "Force Four Decades". Computed decades are based on
the ADC resolutions stored in the FCS file keywords.

Axis Units

Choose between "Histogram channels" and "Data (ADC) values". Historically and by default,
ModFit displayed the axis with histogram channel tic marks, and results were reported on
that scale. To display the axis with the true resolution of the FCS data, choose "Data (ADC)
values"; this will also change results to be on the ADC scale.

Auto-scale X axis

Auto-scale Y axis

These options control the auto-scaling of the main histogram graphic. When checked, each
data file is automatically scaled on the X-axis and/or Y-axis as it is opened. The Y scaling is
set to the height of the highest peak. The low X scale is set to 0. The high X scale is set to
the highest channel that contains data, to a maximum of resolution minus 1.

Uncheck one or both of these options to leave the scaling unchanged as new data files are
opened. This is convenient for studies where it is useful to see all histograms on a fixed
scale.

Parameter Auto-zoom

Zoom Type

Choose how this parameter should be zoomed when ModFit reads it.

Off: no zooming is performed.

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To highest used channel: zooms to the highest channel that has events in it.

To fixed upper boundary: zooms to the channel defined in the Fixed Upper Boundary edit box
below.

To Percentile: zooms to a percentile location in the data.

Percentile

This field is used when To Percentile is selected for Zoom Type. It defines the percentile of
data to include in the zoomed area.

Zoom Graphs

These settings determine the default values for X and Y Zoom graph objects. These objects
can be placed on the report to show the histogram zoomed in the X or Y scale, respectively.

Y Zoom Graph Factor

Enter the initial value for Y Zoom graph objects. The value is the number of times to magnify
the scale, e.g. 25 means multiply data points on the Y scale by 25.

X Zoom Graph Width

Enter the initial width as a number of standard deviations to display in the X Zoom graph
object.

Update X Zoom Window

The X Zoom window object on the report can adjust to the position of peaks in each file, or
only when created. Select the appropriate option.

Modeling

Dip G2M Fraction

This value is used to compute the relative area of the Diploid G2M in Tetraploid models.
Because the Diploid G2M in a Tetraploid model is in the same location as the Tetraploid G1
peak, the program disables modeling of the Diploid G2M. After the model fit is completed,
the program adjusts the Tetraploid G1 by subtracting a portion of its area and assigning it to
the Diploid G2M. The adjustment is computed by multiplying the Diploid G1 area by the Dip
G2M Fraction. The default value is 0.08, or 8%, which means that the Diploid G2M area will
be 8% of the Diploid G1, and the Tetraploid G1 will be reduced by the same area.

AutoLinearity

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These settings control the AutoLinearity function of the program. This feature is available
when batch processing reports, and in the Quick Model Editor. See Using AutoLinearity for
more on this feature.

Low linearity

This field sets the lowest value used by the AutoLinearity operation. Values for this field must
be between 1.85 and 1.94, inclusive.

High linearity

This field sets the highest value used by the AutoLinearity operation. Values for this field
must be between 1.95 and 2.2, inclusive.

Miscellaneous

Default Graphic output

Choose the desired graphic format to create when exporting histogram and report graphics.
Export commands are found in the File menu and in the File and Report Batch Settings
dialog.

Supported formats currently include bitmap (bmp), JPeg (jpg), Tiff (tif), Portable Network
Graphics (png), Portable Pixmap (ppm), X11 Bitmap (xbm), X11 Pixmap (xpm), and Scalable
Vector Graphics (svg).

Parameter Database

ModFit LT keeps track of which parameters from the FCS file you have used for gating and
analysis. This command allows you to view and edit settings for these parameters, including
whether they are linear or log. See Edit Parameter Database for more information.

PDF Output Quality

This option controls the quality of the PDF files created by the program. Generally, higher
quality PDF files are larger than lower quality files.

Save Recovery Backup

ModFit can save your work periodically so that if there is a problem you can recover. Choose
the time interval for automatic backups.

Open

If you have previously saved configuration settings to disk, you can use the Open button to
load those settings into the program.

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Save

Use this option if you want to store the current configuration settings to a file on disk. The
standard file save dialog box will be displayed, allowing you to navigate to a location and
name the file to save.

Reset

Click this button to restore the configuration values to the factory default settings.

OK

Click this button to accept the changes you have made and close the dialog.

Cancel

This option closes the dialog and discards any changes you might have made.

Help

This option displays the on-line help for this dialog.

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Edit Parameter Database

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins have permission to perform them.

This dialog displays the parameters that have been customized by the user.

FCS Keyword Name($PnN_$PnS)

This field identifies the entry and cannot be edited. The label is composed of two keywords for a
specific parameter in the FCS file: the $PnN and $PnS keywords. These keywords allow ModFit
to distinguish your preferences for each uniquely-identified parameter.

Parameter Alias

This field can be edited. It is displayed on graphs that display this parameter.

Transform

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Select the transformation that you want to use for this parameter. Choices include:

Linear
Log
HyperLog
Hyperbolic Sine
VLog

Zoom Type

This field allows you to set up automatic parameter "zoom". It is useful for parameters where
only a small portion of the dynamic range is used. For example, with DNA samples acquired on
an Accuri instrument, only a small portion of the channels are used to store data.

The default setting is for zooming to be "Off". Other options include:

"Highest used channel": zooms the data to exclude any empty channels on the high end of the
parameter's range.

"Fixed upper boundary": zooms the data to exclude channels above a specified channel.

"Percentile": zooms the data to exclude channels above the specified percentile of the data area.

Fixed Upper Boundary

This field is used if Zoom Type is set for "Fixed upper boundary". It defines the channel to
which you want to zoom. Channels above this channel will be excluded.

Percentile

This field is used if Zoom Type is set for "Percentile". Enter the percentile of data you want to
include.

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.

Open

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If you have previously saved parameter preferences to disk, you can use the Open button to load
those preferences into the program.

Save

Use this option if you want to store the current parameter preferences to a file on disk. The
standard file save dialog box will be displayed, allowing you to navigate to a location and name
the file to save.

Delete All

Click this button to clear out all entries from the parameter preferences. It is recommended
that you Save your parameter preference to a file prior to deleting all, in case you want to
restore settings to a previous state.

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Edit Properties for Parameter

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins and ModFitEditors have permission to
perform them.

FCS Keyword Name

This is the name from the FCS file for the parameter.

Alias

This field contains the name to display when this parameter is used in ModFit. It can be
edited, and it appears on the axis of the graphs that display the parameter.

Transform

Choose either a Log or Linear transform to use for this parameter.

Parameter auto-zoom

Zoom Type

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Choose how this parameter should be zoomed when ModFit reads it.

Off: no zooming is performed.

To highest used channel: zooms to the highest channel that has events in it.

To fixed upper boundary: zooms to the channel defined in the Fixed Upper Boundary edit box
below.

To Percentile: zooms to a percentile location in the data.

Fixed Upper Boundary

This field is used when To fixed upper boundary is selected for Zoom Type. It defines the
channel location to zoom to.

Percentile

This field is used when To Percentile is selected for Zoom Type. It defines the percentile of
data to include in the zoomed area.

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.

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Edit User Equations

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins and ModFitEditors have permission to
perform them.

ModFit LT allows you to create additional equations to compute when the model is executed,
either with Auto Analysis or Execute Model. Equations that you enter here are remembered
when the program closes. They can be added to databases and displayed on the ModFit LT
report page as keywords.

These equations must follow the syntax described in the section on Equation syntax.

To add a new equation, click New and enter the equation (see Equation syntax for details).

To change an equation, select the equation in the list and click the Properties button, or
edit the equation directly. See Edit Properties for Equation for details.

To display an equation on the report, check the Add to Report Results checkbox.

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Use the Model Type drop-down list to determine which model types will compute and
display you equation.

To save the current equations to disk so that they can be opened on another computer, click
the Save button.

To open a previously saved set of equations from disk, click the Open button.

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Edit Properties for Equation

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins and ModFitEditors have permission to
perform them.

ModFit LT allows you to create additional equations to compute when the model is executed,
either with Auto Analysis or Execute Model. These equations must follow the syntax
described in the section on Equation syntax.

You can enter a name for each equation and the equation expression. The name will be used
as a keyword name, prefixed with "USER_", and can be selected for databasing and display
on the report.

For example, if you name your equation, "SumOfAneuploidAreas", you can select the
keyword "USER_SumOfAneuploidAreas" to display on the report using the Tools menu,
Keywords command. You can also select this keyword to be databased using the Edit menu,
Databases command.

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S-Phase Cutoffs command

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins have permission to perform them.

Use this command to review, edit, and create cutoff ranges for different tissue types. The
cutoff ranges you enter are used by the Fit Diagnostics routine to assign S-Phase
percentages into low, intermediate, or high classifications.

Refer to the DNA Consensus Conference report (Cytometry 14, p471-500, 1993 (c) Wiley-
Liss, Inc.) for information on establishing cutoffs in your laboratory.

To enable the S-Phase assessment system, check the Activate Assessment System
checkbox.

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To disable the S-Phase assessment system, clear the check from the Activate Assessment
System checkbox.

To add a tissue cutoff, enter a tissue type in the Tissue edit box. Then enter the thresholds
for low-to-intermediate and intermediate-to-high for each ploidy type. Click the Add button
to add the new type to the list of choices.

To set a tissue type as the Current Tissue, double click the entry in the listbox. The current
tissue is displayed at the top of the dialog and on the Toolbar in the Tissue type drop-down
listbox.

To edit a tissue cutoff, click the listbox entry for the tissue you want to edit. Make changes to
the ranges in the edit boxes, and then click Replace to update the entry.

To delete a tissue cutoff, click the listbox entry you want to delete. Then click the Delete
button.

To accept changes to the dialog, click OK.

To discard changes to the dialog, click Cancel.

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Settings Manager

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins have permission to perform them.

This dialog allows you to manage the program settings. You can back-up settings so that
they can be transferred to other computers or for archival purposes. Similarly, you can select
settings from previous back-ups and load them as current settings.

Export Settings

Click this button to export the current settings. The standard file save dialog will be
displayed. Navigate to the location where you want to save the settings, type a file name,
and click Save.

Import Settings

Click this button to import the settings. The standard file open dialog will be
displayed. Navigate to the settings file that you want to open, select it, and click Open.

Reset Settings

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Click to clear the current settings and reset to defaults. The program will confirm that you
want to perform this operation. If you proceed, it will offer to restart the program for you.

OK

Click this button to close the dialog box when finished importing or exporting settings.

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Research and Compliance Modes

Research and Compliance Modes

ModFit LT can be set up to run in either a Research-Use-Only mode, or in Compliance mode.

There are some differences in how the program operates in each mode.

 Compliance mode depends on operating system user groups to control the level of
access to program features. Research mode allows any user to perform any operation.
 Compliance mode displays a login dialog when the ModFit LT is starting up to verify the
user and determine the group to which the user belongs.
 Compliance mode enables logging so that there is a record of the activities of each user.
Research mode can be run with logging enabled or disabled.
 Compliance mode is designed to support clinical labs and can be part of a compliance
plan for the US-FDA's 21 CFR Part 11 regulations. Research mode does not provide
compliance with 21 CFR Part 11.
 Both modes use the same intuitive interface and ModFit LT engine.

The program displays which mode it is operating in. In the Ribbon bar interface, the mode is
displayed in the upper right corner of the ribbon:

or

In the Toolbar interface, the mode is displayed on the main toolbar next to the Help button.

Setting Up Research Use Only Mode

After initial installation, ModFit LT will automatically be in Research mode. There is no further
configuration required.

To revert to Research mode after setting up Compliance mode, the user groups that were
created for Compliance mode must be removed. Once the user groups are removed, the
program will revert to Research mode.

Setting Up Compliance Mode

Setting up Compliance mode is something that should be performed by IT personnel.
While it is not a complicated task, it requires administrative access rights and a clear
understanding of user groups and permissions. It is therefore something that we strongly
recommend be performed by the IT team at your institution. The task involves creating
several required ModFit LT user groups for your computer or domain, and then adding users

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to those groups. Once the groups are defined, the program automatically operates in
Compliance mode. Click here for details.

21 CFR Part 11 Compliance Matrix

This matrix provides a simple summary of how ModFit LT in Compliance mode helps you with
21 CFR Part 11 compliance. Click here for details.

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Setting Up Compliance Mode

Setting up Compliance mode should be performed by your IT team. It requires

administrative rights and a clear understanding of your network operating
system.

The task involves creating several required ModFit LT user groups for your computer or
domain, and then adding users to those groups. Once the groups are defined, the program
automatically operates in Compliance mode.

ModFit LT User Groups

There are three (3) user groups for Compliance mode: ModFitAdmins, ModFitEditors, and
ModFitUsers. Optionally, you can define an additional user group, ModFitResearchers,
for research users within the Compliance setting who are not restricted by Compliance
requirements.

The table below shows the kinds of features that group members can access.

Feature ModFitAdmins ModFitEditors ModFitUsers ModFitResearchers RUO

Mod
Any
user
Set shared
folder Yes No No No No
location
Edit
Preferences Yes No No Yes Yes
Check for
Updates Yes No No Yes Yes
Edit
Logging Yes No No Yes Yes
Create and
Edit Models Yes Yes No Yes Yes
Edit
Databases Yes Yes No Yes Yes
Select
Models Yes Yes Yes Yes Yes
Add files to
batch Yes Yes Yes Yes Yes

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Run a
batch of Yes Yes Yes Yes Yes
files
Log and
Preference Per
Shared Shared Shared Per user
folder user

Setting Shared Folder location

When Compliance mode is enabled, there is an additional command that appears on the
Options tab of the ribbon bar for ModFitAdmins. In Toolbar mode, the command appears in
the Edit menu.

The Select Shared Folder command is used to select a settings folder that is shared by all
users in Compliance mode. The common settings folder ensures that all Compliance-mode
users work with the same configuration.

ModFitAdmins can use this command at any time to set or change the location for shared
settings. Other users will not see this command.

Creating User Groups

The program requires at least one of the ModFit LT user groups to be defined in order to run
in Compliance mode. ModFit LT user groups can be defined for your computer or for your
entire domain using tools provided by the operating system. This is typically done by
information technology personnel because it requires administrative rights.

The advantage of defining the user groups at the domain (network) level is that the groups
are defined for all computers on the domain at the same time. If you define the user groups
on an individual computer, those groups are available only on that computer.

Below we describe how to create user groups on a Windows server or on a Windows

compute as examples. The specific methods you need to use may differ depending on the
version of the operating system you are working with.

After you have defined the user groups and added users to them, ModFit LT will detect the
groups when it starts up and operate in Compliance mode automatically.

Windows Server User Groups

Follow these steps to define user groups for an entire network or domain. Note: if you use
this technique, you do not need to define user groups on each individual
computer.

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 On a domain server, launch the "Active Directory Users and Computers" administrative
tool.
 Right click the "Users" folder in the left panel and choose New->Group. The New Group
dialog is displayed.

 Type ModFitAdmins for the group name and click OK.

 Once the group is created, double-click the group in the list to display the Properties for
the group.
 Click the Members tab and click Add to select members for the ModFitAdmins
group. You should have at least one user in the ModFitAdmins group. Members can be
added by typing user names, or by using the dialog's search features.

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 Repeat this process for the ModFitEditors, ModFitUsers, and optionally

ModFitResearchers groups, adding appropriate users to each group.
 Once the groups are created and users are added to them, the program will
automatically use Compliance mode when it starts up and is connected to the domain.

Windows Computer User Groups

Follow these steps to define user groups for an individual Windows computer. Note, if you
already defined user groups for your domain, you do not need to define the
groups again for each computer.

 On a Windows 7 computer, right click Computer and choose "Manage". For Windows 10
and newer, right click the Windows button (lower left) and choose "Computer
Management". The Computer Management app will display.
 Expand "System Tools" and "Local Users and Groups" in the left panel.

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 Right click the "Groups" folder and choose "New Group". The New Group dialog is
displayed.

 Type ModFitAdmins for the group name and enter a description for the group.
 Click Add to select members for the ModFitAdmins group. You should have at least one
user in the ModFitAdmins group. Members can be added by typing user names, or by
using the dialog's search features.
 Click Create to create the new local user group.
 Repeat this process for the ModFitEditors, ModFitUsers, and optionally
ModFitResearchers groups, adding appropriate users to each group.
 Once the groups are created and users are added to them, the program will
automatically use Compliance mode when it starts up on that computer.

Mac OS X User Groups

Follow these steps to define user groups for an individual Mac OS computer.

 On a Mac OS computer, launch System Preferences. Choose "Accounts" or "Users and

Groups", depending on the version of the OS.
 Click the lock icon to authenticate to make changes. This will require administrative
rights.

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 Click the "+" button under the list of users and groups. The "New Account" dialog is
displayed.
 Change the New Account type to "Group".
 Type ModFitAdmins for the group name and click "Create Group".
 Select the users you want to be members of the ModFitAdmins group. You should
have at least one user in the ModFitAdmins group.
 Repeat this process for the ModFitEditors, ModFitUsers, and ModFitResearchers
groups, adding appropriate users to each group.
 ModFit LT will run in Compliance mode the next time it starts.

See also:

21 CFR Part 11 Compliance

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21 CFR Part 11 Compliance

ModFit LT can be part of your lab's compliance strategy for 21 CFR Part 11 when it is setup
and operated in Compliance mode. It has been developed under a quality management
system and validated with a suite of quality assurance tests. The table below summarizes
how the key elements of Part 11 relate to ModFit LT and its use in Compliance mode
settings.

Section Summary of Rule Compliance Strategy

Controls for closed ModFit LT can be accessed only through the user
11.10
systems interface; it is a closed system.
ModFit LT uses externally-generated data to validate
Validation and
11.10 (a) its results and employs proprietary binary format,
accuracy.
static PDF, and/or CRC methods to ensure valid files.
ModFit LT generates Xml-format model files, CRC-
Record generation
11.10 (b) checked log files, and static PDF that are human-
and readability
readable and machine-readable.
Record protection Record retention and retrieval plan is managed by the
11.10 (c)
and retrieval laboratory’s file management practices.
ModFit LT uses the operating system’s user login
11.10 (d) Authorized access validation, and user group membership determines
permissions within the software.
ModFit LT employs a CRC-checked logging system
11.10 (e) Audit trails with time stamps, and log files are preserved. Log
files cannot be deleted by ordinary means.
Operational system ModFit LT enables and disables portions of the UI to
11.10 (f)
checks enforce permitted sequencing of steps and events.
ModFit LT uses the operating system user groups and
11.10 (g) Authority checks
login validation to assure authorized access.
Device checks and ModFit LT performs validation checks on the FCS data
11.10 (h)
validity files.
ModFit LT provides a User Guide, technical support,
Education, training and tutorial videos to assist with the proper, intended
11.10 (i)
and experience use of ModFit. Specific on-site training may be
arranged.
This is an individual laboratory requirement beyond
11.10 (j) Electronic signatures
the scope of use of ModFit LT.
ModFit LT version releases are accompanied by an
Systems
11.10 (k) updated User Guide. Verity employs document control
documentation
procedures.
Controls for open
11.30 ModFit LT is designed as a closed system.
systems

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Signature
11.50 ModFit does not require or offer electronic signatures.
manifestations
ModFit LT does not require or offer electronic
11.70 Signature linking
signatures.
Unique electronic Your laboratory must ensure that electronic signatures
11.100 (a)
signatures are unique to each user on the network.
Each laboratory must verify its own employment
11.100 (b) Identity verification
records and IDs.
Signature Each laboratory must verify electronic signature
11.100 (c)
verification eligibility with the FDA.
11.200 Two-component ModFit LT does not require or offer electronic
(a)(1) signing signatures.
11.200 Authenticity and ModFit LT does not require or offer electronic
(a)(2,3) falsification signatures.
11.200 (b) Biometric signatures ModFit LT does not utilize biometric IDs.
Controls for ModFit LT uses the operating system user groups and
11.300 (a)
identification login validation to assure unique user identities.
The individual laboratory IT structure must control
11.300 (b) Identification aging
password checks and aging requirements.
Not applicable. ModFit LT does not utilize external
External ID
11.300 (c) tokens, cards, RFID, or other physical identity-
validation
confirming devices.
The individual laboratory IT structure must control
11.300 (d) Unauthorized use login authenticity and manage safeguards to prevent
unauthorized use.
Not applicable. ModFit LT does not utilize external
External device
11.300 (e) tokens, cards, RFID, or other physical identity-
testing
confirming devices.

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Customizing Models
Model Descriptions

This section contains descriptions of the models used by ModFit LT.

Model naming

When Auto-analysis or Choose model commands are used to create models, the program
uses the following logic to create a name for the model.

Position in name Meaning

1 Number of cycles

2 'D' if AutoDebris is enabled; 'n' if not

3 'A' if AutoAggregates is enabled; 'n' if not

4 Number of standards

5 'A' if Apoptosis is enabled; 'n' if not

For each cycle in the model, the name includes the following:
An underscore (_) character;

The first character of the cycle's ploidy, i.e. D=Diploid, A=Aneuploid, T=Tetraploid, H=Hypo-
diploid;

'S' if S-Phase is active, or 'n' if not; and

'F' if G2M is active and floating, 'D' if active and dependent, 'n' if G2 inactive. Floating means
that the mean position and standard deviation are adjusted during the modeling process.
This gives more freedom to the model, but the peak must be unambiguous. Dependent
means that the position and standard deviation of the G2M are computed by multiplying the
G1 position and standard deviation by a factor of 2.0 or your chosen linearity setting. Inactive
means that the G2M is not modeled.

For example, the model name "3DA0n_DSD_AnD_ASF" can be interpreted to mean that there
are 3 cycles, AutoDebris is enabled, AutoAggregates is enabled, zero standards, and
apoptosis is not being modeled. The first cell cycle is Diploid, with S-Phase active and a
Dependent G2M. The second cycle is Aneuploid, S-Phase is turned off and the G2M is
dependent. The third cycle is also Aneuploid, S-Phase is enabled, and the G2M position is
allowed to float.

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Ranges and their uses

The table below shows the ranges used by ModFit LT and the populations they are used to
identify.

Range Usage

Debris Left side placed at start of downward sloping

debris, right side extended through roughly 2/3
of the histogram. Provides estimates for
AutoDebris component.

Stnd 1 Identifies the peak of the first standard. The

range should be positioned to encompass the
standard peak. If you have named your
standards, the range will show the name you
assign. See Auto Analysis Settings for details on
naming standards.

Stnd 2 Identifies the peak of the second standard. The

range should be positioned to encompass the
standard peak. If you have named your
standards, the range will show the name you
assign.

Dip G1 Identifies the G1 of the Diploid cycle. The range

should be positioned to encompass the peak.

Dip G2 Identifies the G2 of the Diploid cycle. The range

should be positioned to encompass the peak.

Cycle G1 and G2 Identify the G1 and G2 for each additional cycle.

The range should be positioned to encompass
the associated peak.

When you use automatic analysis, ModFit LT constructs a model for you based on the peaks
it can identify in the data sample. It looks at how many peaks there are, their standard
deviations, areas, and relative locations, then takes its best shot at deducing the ploidy,
debris, and aggregation aspects of the sample. It builds a model that matches what it finds.
The black triangles at the base of the histogram show the peaks the program has identified.

When you select a model yourself using the Choose Model option, you are presented with
the dialog pictured below. With this dialog, you make the decisions about the sample.

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Colors

ModFit LT allows you to edit the colors used by the model using the Colors command. The
default color assignments are listed in the table below.

Component Color

Diploid G0-G1 red

Diploid G2-M red

Diploid S-Phase reverse-hatched

blue

Aneuploid G0-G1 1st is bright yellow;

others become
darker shades of
yellow

Aneuploid G2-M 1st is bright yellow;

others become
darker shades of
yellow

Aneuploid S-Phase forward-hatched

blue

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AutoAggregates light green

AutoDebris medium blue

Apoptosis sky blue

1st Standard dark gray

2nd Standard light gray

Component Shapes

The parts of ModFit LT's models are comprised of several component shapes. The table
below shows the shapes used by the models, and where they are used.
Component Shape Uses
Gaussian Gaussian
components
are used to
model peaks.
They are
often linked to
ranges.

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Rectangle This
component is
typically used
to model S-
Phase. The
peaks on
either side of
it determine
boundaries
and standard
deviations.

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Trapezoid This
component
can be used
to model S-
Phase. It has
an additional
degree of
freedom over
the rectangle
and generally
yields higher
S-Phase
values. The
peaks on
either side of
it determine
boundaries
and standard
deviations.

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Polynomial This
component
can be used
to model S-
Phase. It has
an additional
degree of
freedom over
the trapezoid
and should be
used with
caution. The
peaks on
either side of
it determine
boundaries
and standard
deviations.

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AutoDebris The
AutoDebris
component is
used to model
debris. Its
starting point
is based on
the position of
the Debris
range.

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AutoAggregate This
component is
used for
aggregate
compensation.
Its shape is
unique to
each
histogram. It
does not use
a range.

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Weibull This
component
can be used
to model
skewed peaks
in custom
models.
Contact Verity
technical
support for
assistance
with this
component.

See also:

Choose Model command (Analysis menu)

Manual analysis with ModFit LT

Analysis Results

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Colors command

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins and ModFitEditors have permission to
perform them.

Use this command to adjust colors and other graphical aspects of the model. You can change
color, fill style, and transparency for colors in the current model. The changes you make can
also be remembered and applied to other models.

Model Definition

With the Model Definition selected in the tree on the left, you can edit characteristics of the
fit line displayed on the modeled histogram. The color, style, and weight of the fit line can
be adjusted.

Components

To choose a new color for a model component, click the color swatch next to the component.
The standard dialog for choosing colors will appear. Select a new color and click OK.

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To choose a new fill style for a model component, click the sample fill for the component. A
drop-down list of choices will appear. Select a new fill style from the list.

Each component can either be transparent or opaque. This determines how overlapping
components are drawn. To make a component transparent, enable the checkbox in the
Transparent column. Uncheck the option to make a component opaque.

OK

When you click OK, you will be asked whether you want to apply the changes to all models,
or just the current model. If you choose to apply to all models, the changes are stored for
future sessions of ModFit LT. If you choose to apply the changes only to the current model,
the changes will be lost when you load a new model, perform an Auto Analysis, or close the
program.

Reset All

Click this button to clear all color selection overrides.

Cancel

Click this button to discard changes to colors.

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Quick Model Editor

Overview

The Quick Model Editor allows you to make changes to the current model. It is available
when the model you are using is created by AutoAnalysis or by using the Choose Model
option.

Changes you make with this dialog are applied to the model when you click OK. In order to
see the changes to the model, you need to re-analyze the data by clicking the Fit button or
choosing Execute Model from the Analysis menu.

AutoDebris

This checkbox determines whether the AutoDebris model component is active or not. When
checked, AutoDebris is activated. The AutoDebris component is used to model debris in data.
It can be left active even when samples display little or no debris, because its shape and
amplitude are based on probability distributions unique to each sample.

AutoAggregates

When this option is checked, the AutoAggregates model component is activated. This
component is used to model the continuum of aggregate forms found in many samples. It
can be left active even when there is little or no aggregation in the sample, because its shape
and amplitude are based on probability distributions unique to each sample

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Linearity

This field modifies the appropriate G2M dependency settings and modifies the aggregate
model component's linearity setting. When the user indicates that a G2M is not visible, the
program makes the position and standard deviation of the G2M model component dependent
on the G1 by this linearity setting. It updates the linearity setting for the aggregate model
component as well.

Internal standards

This option determines the number of peaks attributed to standards. Standard peaks typically
occur below the first G1 peak. However, you can configure the expected locations for
standards in your samples using the Auto Analysis Settings dialog box.
G2M Section

The options in this section of the dialog allow you to control how the G2M for each cycle is
modeled. If the option is checked (Visible), the program will use the associated G2M range to
estimate the position and it will allow that position to be adjusted by the modeling process. If
the option is not checked, the position of the G2M will be computed by multiplying the G1
position by the linearity value.

Generally, if a G2M is a well-defined peak that does not overlap another peak, you can
enable the option and model will provide a more accurate result. However, if the G2M peak is
not clearly defined or if it substantially overlaps another peak, uncheck the option. If the
option is checked when a peak is not well defined, the model will produce less accurate
results.
S-Phase Section

The options in this section control the state of the S-Phase model component for each cell
cycle in the model. If a check appears in the checkbox, the S-Phase component is active and
the program will attempt to model it. Clear the check to disable the S-Phase component for
the model.

In samples such as near Diploid histograms, the S-Phases of two or more cycles can be
significantly overlapped. This can create problems for the modeling process. In these cases,
it is better to disable one of the S-Phase components, normally the Diploid S-Phase.

OK

Apply the settings to the current model and put away the dialog box.

Cancel

Close the dialog and discard any changes you have made.

Reset

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This option resets the the properties in this dialog to their default states.

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Full Model Editor

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins and ModFitEditors have permission to
perform them.

This dialog allows editing of existing models and creation of new models. It can be used to
change any and all aspects of a model, and therefore should be used with great care.

The listbox displays the aspects of the model that can be edited: ranges, components, and
results.

Ranges:

Ranges are used to provide estimates for the model components. They are displayed when
you click the Range button on the ribbon bar. Most ranges are used to identify peaks in the
model, and associated model components use the range position, size, and statistical data as
a starting point in the modeling of the histogram. See Edit Ranges for more on this topic.

Components:

Components of the model define the mathematical pieces and their interdependencies. There
are components to model debris, aggregates, peaks, and broad distributions. Each

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component, in turn, has a set of details that further describe the properties of the
component. See Edit Properties for Component for more on this topic.

Equations:

Equations are expressions used to display the result of the modeling process. They can show
simple information, such as keywords stored in the data file, or sophisticated mathematical
expressions based on details of model components. See Edit Equations for more on this
topic.

OK:

Click this button to close the dialog and apply the changes to the current model.

Open:

This option allows you to open a previously saved model from disk. The File Open dialog box
is displayed, allowing you to navigate to and select the model. Note: Models from ModFit LT
2.0 and earlier cannot be opened with this option.

Save:

This option allows you to save the current model to disk for later use. The File Save dialog is
displayed, allowing you to navigate to the location where you wish to store the model.

Help:

Click this button to display online help for this topic.

Edit:

This button is enabled when one of the list entries is selected. Clicking the button will display
the edit dialog for the selected item.

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Edit Components

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins and ModFitEditors have permission to
perform them.

This dialog allows you to add, delete, and edit components of the model. Each model
component mathematically describes a piece of the model. The collection of components
attempts to fully define the histogram as a mathematical model.

Component names can be edited by clicking slowly once on the name, then clicking again.
The list item will enter edit mode; press the Tab key or click another item to accept the
revision.

Select a component in the list, and then click one of the action buttons to modify it.

OK:

Click this button to close the dialog and accept the changes.

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Help:

Click this button to display online help for this topic.

Properties:

This button is enabled when one of the list entries is selected. Clicking the button will display
the edit properties dialog for the selected item.

Edit:

This button is enabled when one of the list entries is selected. Clicking the button will display
the edit dialog for the selected item.

Copy:

Click this button to copy the selected component. The new copy will be added to the end of
the component list, and can be selected for editing.

Delete:

Click this button to delete the selected component. If other components are dependent on
the deleted component, they will become inactive. Use great care when deleting components
to ensure the remaining model components are not adversely affected.

See also:

Edit Properties for Component

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Edit Properties for Component

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins and ModFitEditors have permission to
perform them.

This dialog allows you to edit properties for a single model component. In addition, you can
select and edit the properties for each detail of the component. Component details vary
depending on the type of component.

A tree showing the component and its details is displayed on the left side of the dialog.
Initially, the component itself is selected, and the list on the right shows the properties of
that component.

When you click one of the details in the tree on the left, the properties for that detail will
appear. See Edit Properties for Detail for more on this topic.

Component State:

This checkbox shows the current state of the component.

Name:

This edit box displays the name of the component. It can be edited by click inside the edit
box and typing new text.

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Type:

This drop-down list displays and selects the type of mathematical expression used for this
component. Changing the type may also change the details, shown in the tree on the left of
the dialog. See Model Descriptions for details on each model component type.

Associated range:

This drop-down list displays and selects the model range, if any, used to provide estimates to
this model component. In most cases, ranges identify peak locations. They appear on the
histogram when you click the Range button on the toolbar, or select Position ranges from the
Analysis menu. If a component is not associated with a range, the selection will display "No
range".

Number of compartments:

This list selects the number of sub compartments for the component. Some components, like
the Polynomial, do not support more than one compartment. Setting this to 0 will disable the
component.

Color:

Click this button to display the standard color selection dialog and choose a new color for the
component.

Fill Style:

This list contains graphical "hatch" options to use when drawing the component. Using a
hatch pattern allows an additional degree of transparency to overlapping components, in
some cases, making it easier to review the model.

Transparency:

This option determines whether the component coloring will be transparent or not.

Generate Debris:

Enable this option if the population associated with the component contributes to debris in
the sample. The debris modeling uses this to determine the expected shape of debris.

Generate Aggregates:

Enable this option if the population associated with the component contributes to aggregates
in the sample. The aggregate modeling uses this to determine the expected shape of
aggregates.

OK:

Click this button to accept the changes and close the dialog.

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Cancel:

Click this button to cancel changes and close the dialog. This button may be disabled if
changes are made that cannot be cancelled.

Help

Click this button to display online help for this topic.

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Edit Properties for Detail

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins and ModFitEditors have permission to
perform them.

This dialog allows you to edit the properties of a model component detail. The details of a
component will depend on the particular component type. The properties of each detail
define how the detail is related to other component details, how it is computed, and the
source of its estimates. See Edit Properties for Component for additional information.

Dependency:

This option determines how the detail is computed. Floating means that the modeling
process will adjust the detail to find the best value. Floating details are commonly used for
means and standard deviations of well-defined peaks modeled with Gaussian component.
Dependent details are computed directly by applying a multiplicative and/or additive factor to
the value of the controlling detail. Fixed details are not adjusted during the modeling
process; their values are determined either by a static value or from a range estimate.
Interpolate can be selected to compute the value of the detail by interpolation. It is only
applicable to standard deviation details. An example best describes the use of this option.
Suppose you have a model that describes a diploid population. Now suppose there is an extra
peak in S-Phase that you believe should be broadened in the same way as the S-Phase is
broadened. You would choose Interpolate for the standard deviation of the extra peak, and

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make its controlling component and detail the S-Phase lower standard deviation. The upper
boundary of the interpolation is automatically the upper standard deviation.

Controlling component:

If Dependent or Interpolate is selected as the Dependency option, the controlling component

determines what model component this detail is dependent upon. You must also select a
Controlling detail (below) to complete the dependency. If the Dependency option is Floating
or Fixed, the controlling component is ignored.

Controlling detail:

If Dependent or Interpolate is selected as the Dependency option, the controlling detail

determines the detail of the Controlling Component on which this detail depends. If the
Dependency option is Floating or Fixed, the controlling component is ignored.

Multiplicative factor:

If Dependent is selected as the Dependency option, the final value of the controlling
component's detail is multiplied by the multiplicative factor. As an example, the G2M
component's mean position is often made dependent on the G1 component's mean by a
factor of 2.

Additive factor:

If Dependent is selected as the Dependency option, this value is added to the final value of
the controlling component's detail, after it has been multiplied by the multiplicative factor.

Estimation:

This drop down provides choices for the source of the estimate for the detail. Outside-in and
Inside-out draw the estimate from simple statistics performed before the modeling process.
Range draws the estimate from a range associated with this component. User-entered
estimate uses the value entered in the Estimated value field as the source of the estimate.
Estimated value:

This field displays the estimate that was used for the detail when the model was last run. If
Estimation is set to User-entered estimate, the value will not be adjusted. In all other cases,
the value may change each time you choose Fit, depending on how the details dependencies
are set.

Final value:

This read-only field displays the result of the last modeling process for this model.

OK:

Click this button to accept the changes and close the dialog.

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Cancel:

Click this button to cancel changes and close the dialog. This button may be disabled if
changes are made that cannot be cancelled.

Help

Click this button to display online help for this topic.

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Edit Ranges

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins and ModFitEditors have permission to
perform them.

This dialog allows you to add, delete, and edit ranges of the model. Ranges are graphical
elements that you can position on the histogram. They are used to supply estimates to model
components.

Range names can be edited by clicking slowly once on the name, then clicking again. The list
item will enter edit mode; press the Tab key or click another item to accept the revision. See
Edit Properties of Range and Edit Properties for Component more on this topic.

OK:

Click this button to close the dialog and accept the changes.

Help:

Click this button to display online help for this topic.

Properties:

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This button is enabled when one of the list entries is selected. Clicking the button will display
the edit properties dialog for the selected item.

Delete:

Click this button to delete the selected range. If components are dependent on the deleted
range, they will become inactive. Use great care when deleting range to ensure model
components are not adversely affected.

Properties:

This button is enabled when one of the list entries is selected. Clicking the button will display
the edit dialog for the selected item.

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Edit Properties of Range

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins and ModFitEditors have permission to
perform them.

This dialog allows you to edit the properties of a model range. Ranges are graphical elements
that you can position on the histogram, used to supply estimates to model components.

Label:

This edit box displays the name of the range. It can be edited by click inside the edit box and
typing new text.

Active:

When checked, the range is displayed on the histogram (in Range view) and it can be used
as an estimation source for model details. When unchecked, the range will not be displayed
and any components that use the range will be deactivated.

Left:

The edit box shows the location of the left side of the range. You can edit the field manually,
or position the range graphically to change the value. To display ranges, click the Range
button on the toolbar, or choose Position Ranges from the Analysis menu.

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Width:

The edit box shows the width of the range. The right side of the range is positioned at Left
plus this value.

Y Position:

This value determines the vertical location of the range label. The value is a percentage of
the Y scale.

Color:

Click this button to display the standard color selection dialog and choose a new color for the
range.

Type of estimate:

There are three types of range estimates: Gaussian, Debris and Aggregate. Most ranges are
Gaussian type, used to identify peaks in the histogram. There is typically only one Debris
estimate range in a model, used by the AutoDebris component for estimation of the debris.
The Aggregate estimate can be associated with the AutoAggregate component, but is
typically not used.

Auto-position to:

In most models, the ranges are positioned and sized by the user for manual
analysis. Automatic analysis positions ranges based on the peaks it finds in the
data. However, you can create your own custom models, too, and for those models you can
tell ranges how to position themselves.

The peaks that are considered by the auto-positioning logic are those that show a black
triangle on the main histogram.

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No peak: This turns off auto-positioning and leaves the range where it is.
Closest peak: Moves the ranges to the closest peak.
Dimmest peak: Moves the ranges to the dimmest peak.
Brightest peak: Moves the ranges to the brightest peak.
Largest peak: Moves the ranges to the largest peak (by area).
Smallest peak: Moves the ranges to the smallest peak (by area).
Tallest peak: Moves the ranges to the tallest peak.
Shortest peak: Moves the ranges to the shortest peak.

OK:

Click this button to accept the changes and close the dialog.

Cancel:

Click this button to cancel changes and close the dialog. This button may be disabled if
changes are made that cannot be cancelled.

Help

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Click this button to display online help for this topic.

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Edit Analysis Results

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins and ModFitEditors have permission to
perform them.

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This dialog displays the list of analysis results that are computed for the current model. Each
item in the list has a Result Name and a Result Equation. The equation uses ModFit LT's
equation syntax to compute the results.

For models that are created by ModFit LT's Auto Analysis, Choose Model, Sync Wizard, and
Cell Tracking Wizard, there is little need to edit the Analysis Results. The main use for this

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dialog is for models that you design yourself from scratch, or models you customize from a
ModFit LT model.

The items in this list are available for databasing as well as being added to the report.

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Edit Properties for Analysis Result

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins and ModFitEditors have permission to
perform them.

This dialog is used to edit an Analysis Result. It allows editing of the Result Name and the
Result Equation.

See Equation Syntax for details on writing equations.

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Edit Report Results

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins and ModFitEditors have permission to
perform them.

This dialog allows you to edit the equations used to display results on the report.

You can edit equations by selecting an item in the list and clicking Properties. Alternately, you
can edit an equation directly by clicking once slowly to select the equation, then clicking
again to enter edit mode. To leave edit mode, press the Tab key or click another item in the
list.

Properties:

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This button is enabled when one of the list entries is selected. Clicking the button will display
the edit properties dialog for the selected item.

New:

Click this button to add a new equation. The new equation will be added to the end of the
list, and can be selected for editing.

Delete:

Click this button to delete the selected equation. Be careful when deleting equations that
contain "T[n]" expressions, where n is an integer. These equations compute temporary
variables used by other equations; the other equations may fail if they make use of a deleted
expression.

OK:

Click this button to close the dialog and accept the changes.

Help:

Click this button to display online help for this topic.

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Edit Properties of Report Result

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins and ModFitEditors have permission to
perform them.

This dialog allows you to edit an equation for a line of results that appears on the report.

Equation:

Type or edit the equation expression. See Equation syntax for details.

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Equation syntax

ModFit equations are expressions that are compiled by the program and evaluated each time
the model is analyzed. Equations can contain literal text, keywords from the FCS file being
analyzed, and symbols that provide access to the computed results of the analysis.
Mathematical operations include addition, subtraction, division, and multiplication, grouping
of operations using parentheses, and use of build-in functions.

Strings

String literals are enclosed in quotation marks.

Example equation: "This is text."

Report output: This is text.

Keywords:

Keywords can be draw from the FCS file and from internally computed values. Keywords are
enclosed in square brackets.

FCS keywords vary from file to file, and are generally created by the software that was used
to acquire the data file.

Internal keywords include the following:

[DATE] displays date of analysis.

[TIME] displays time of analysis.

[FILE] displays name of data file analyzed.

[REPORT] displays name of report.

[AUTONAMEREPORT] displays name generated by

AutoNameReport.

[MODEL] displays the name of the model.

[ANALYSIS] displays the type of analysis performed,

e.g. Automatic, Manual.

[RCS] displays the reduced chi-square value

from the last fit of the model.

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[GATE1] displays name of gate one, or "Off" if no

gate

[GATE2] displays name of gate two, or "Off" if no

gate

[GATE3] displays name of gate three, or "Off" if

no gate

[FILEPATH] displays full path to data file.

[MODELPATH] no longer supported

[REPORTPATH] displays full path to the report

[VERSION] displays the internal version of the

program

[LINEARITYFACTOR] displays the configuration setting for

linearity

[EXTREFMEAN] displays the value for the External

Reference Mean position from the Auto
Analysis Settings dialog.

[DIPTOSTDRATIO] displays the value for the Diploid-to-

Standard Ratio from the Auto Analysis
Settings dialog.

[RANGEnNAME] displays the name of range with ID of n

[RANGEnLEFT] displays the left channel position of range

with ID of n

[RANGEnRIGHT] displays the right channel position of

range with ID of n

Example equation: "Today is ";[DATE]

Report output shows date of analysis (for example): Today is 28-Feb-2005

Example equation: [RANGE4NAME]

Report output shows name of range with ID of 4: "Dip G1"

Internal functions

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Internal functions are used to gain access to component details, areas, and other computed
values. Below is a table describing each function. Note that component and compartment
indexing start at zero (0).

Function Description

A[m, n] Returns the area of component m,

compartment n. To return the area of all
compartments for a component, use an
asterisk (*) for n. To return the area of
all components, use an asterisk for m.

M[m, d] Returns the value of detail d for model

component m.

T[n]=equation Sets the value of temporary variable n

to the expression in equation. After
setting a temporary variable, you can
use the variable without the
"=equation" portion to return the value
of the equation.

T[n] Returns the value stored in temporary

variable n.

P[n] Set the floating-point precision for the

rest of the equation to n decimal places.

Y[m, start, end] Returns the portion of the area of

component m that falls between
channels start and end.

S[start, end] Returns the sum of events between

channels start and end.

U[start, end] Returns the mean between channels

start and end.

H[ ] Returns the mean of the highest active

cycle component. NOTE: There is no
space between the open and close
square brackets.

F[expression] Returns the value of a keyword

converted into a floating-point number.
This allows the keyword to be used in
calculations.

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For example, the $P1B keyword stores

the number of bits used to store a value
for parameter 1 in an FCS file. To
compute the number of bytes used by
parameter 1, the following equation
could be used: "Parameter 1 bytes =
";F[[$P1B]]/8

Mathematical operations

The following operations are supported.

Operation Usage

Addition Use the plus symbol (+) between

numeric values.

Multiplication Use the asterisk (*) between numeric

values.

Subtraction Use the minus symbol (-) between

numeric values.

Division Use the slash (/) between numeric

values.

Grouping Use parentheses to group mathematical

operations.

Concatenation

Equations can contain multiple elements separated by a semi-colon (;).

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Help
Check for Updates

Research and Compliance Mode note:

In RUO mode, all users can perform these tasks.
In Compliance mode, only ModFitAdmins have permission to perform them.

This command launches the VSH Updater application, which checks for program updates. In
order to function correctly, the VSH Updater needs access to the Verity Software House web
site (www.vsh.com) and administrative rights to some areas on your hard drive. You may
need to provide authorization credentials for it to run.

Follow instructions provided by the VSH Updater to perform the check and update your
software.

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Ask Tech Support

This command interacts with your email application to create a new message to Verity
Software House Tech Support. A new message is created to the Tech 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 questions you want to ask. A
member of the Tech Support team will respond to your question as quickly as possible.

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Make a Suggestion

This command interacts with your email application to create a new message to Verity
Software House Tech Support. A new message is created to the Tech 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.

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Report a Problem

This command interacts with your email application to create a new message to Verity
Software House Tech Support. A new message is created to the Tech 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.

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Application Info

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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User Guide

Use this command to display the User Guide in your browser.

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About command

Use this command to display the About dialog box.

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ModFit LT Help Appendices

Appendices
Analysis Results

This section describes the results that are displayed by ModFit LT after an analysis has been
performed. The results are usually displayed on the upper right side of the report, although
you can move the results to anywhere on the report.

There are several types of models available in the program, each with its own set of results.
Standard models are created by Automatic Analysis and by choosing a model for manual
analysis. These models include components and results for debris, standards, aggregates,
and apoptosis, as well as for one or more cycling populations. For each of the cell cycles, a
G0G1, G2M, and S-Phase component is included.

Sync Wizard models are created when using the Sync Wizard. These models are designed for
the special demands of perturbed cell cycle analysis.

The Cell Tracking Wizard creates Cell Tracking Wizard models. These models are used to
study cell proliferation in samples using cell-tracking dyes and proliferation markers.

The program calculates percentages based on the number of events that are actually
modeled. The number of events modeled can be less than the total number of events in a
sample if you apply a gate, scale the data to eliminate events, or if the model itself does not
fit the data exactly. Suppose, for example, that a listmode file contains 10,000 events. If you
gate out 10% of the events, the analysis will be performed on 9,000 events, or 90% of the
total events.
Standard Models
Description
Label
Model This shows the name of the model used for the
analysis. For information on how to decode model names,
see Model Descriptions.
Analysis type: This will show either "Automatic analysis" or "Manual
analysis", depending on how the model was selected and
whether any user-edits were made to the model set up.
Auto Linearity This will show "Yes" if Auto Linearity was performed, or
"No" if it was not.
Ploidy Mode Displays the Auto Analysis Diploid Determination setting.
Diploid Reference If the Diploid Determination setting is External Reference,
Based on Standard 1, or Based on Standard 2, this result
will display the location of the ploidy reference.
If First Cycle is Diploid mode, this result is meaningless and
is not displayed.
% Cycle Percent of all events that are associated with cycling

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populations
%G0G1 G0G1 as a percent of all cells in that cycle
G0G1 mean G0G1 mean channel
%G2M G2M as a percent of all cells in that cycle
G2M mean G2M mean channel
% S-Phase S-Phase as a percent of all cells in that cycle
G2/G1 ratio Ratio of G2 mean channel to G1 mean channel
%CV Coefficient of variation for G0G1 cells
DI DNA Index, defined as the mean channel of G1 cells of an
aneuploid population compared to Diploid G1 mean
channel.
DI based on reference If the Diploid Determination setting is External Reference,
Based on Standard 1, or Based on Standard 2, this result
will display the DNA index based on the computed
reference position for the Diploid G1.

Total Aneuploid S- Sum of all Aneuploid S-Phase areas as a percentage of the

Phase total (G1G1 + S + G2M) area for all Aneuploid populations.
This result is not displayed for Diploid models. However it is
computed as a keyword even for Diploid models. For
Diploid models, the "tumor" S-Phase is considered to be the
Diploid S-Phase, and so the keyword reports the S-Phase of
the Diploid.
Total S-Phase Also called "Average S-Phase". Sum of all S-Phase areas as
a percentage of the total area for cycling cells of all
populations.
Total B.A.D Amount of debris and aggregates found within the region
between the first G1 and the last G2 peak.

Standards
Stnd % Area of standard as a % of all modeled events minus debris
Stnd mean Mean channel position of standard
DI DNA Index of standard as standard mean channel relative
to the Diploid G1 mean channel.
Dip G1 Ratio Diploid G1 mean channel divided by mean channel of the
standard
%CV Coefficient of variation for standard

%Debris Debris area as a % of all modeled events

%Aggregates Aggregate area as a % of all modeled events
%Apoptosis Apoptosis area as a percent of all modeled events minus
debris

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Modeled events Number of events accounted for by all components in the

model
All cycle events Number of modeled events in the G0G1, G2M, and S-Phase
components of all cycling populations
Cycle events per All cycle events divided by the number of channels between
channel the first G0G1 and the last G2M of the model.
RCS Reduced chi-square, a measure of goodness of fit. Values
from 1.0 to 3.0 are good, 3.0 to 5.0 are fair, and greater
than 5.0 are poor.

Tetraploid Model

ModFit LT handles Tetraploid histograms slightly differently than most other ploidy types.
Because the Diploid G2M and the Tetraploid G1 peaks are superimposed, the program
disables the Diploid G2M during the modeling process. After the model has been applied to
the histogram using the Fit with Current Model command or Auto Analysis, the program will
add back a specified amount to the Diploid G2M and reduce the Tetraploid G1 by the same
amount. This amount is computed as a percentage of the Diploid G1 peak, and the
percentage is specified in the Options and Configuration dialog box. The default percentage
is 8% (0.08).

Sync Wizard Model

In addition to many of the results described for standard models, the Sync Wizard also
computes the percentage for sub-compartments of S-Phase
Result Computation
S-Phase Cells in S-Phase as a percent of all the cells in that cycling
population
Compartment n Area of S-Phase sub compartment as a percent of all the
cells in that cycling population

Cell Tracking Wizard Model

See the Cell Tracking Example for an explanation of Cell Tracking results.

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Histogram and S-Phase Assessment

ModFit LT contains two systems to evaluate confidence in the S-Phase estimates it computes.
The first method evaluates several histogram features that have been published as affecting
S-Phase accuracy (Cytometry. Vol. 14. Number 5, 1993.): %CV, Cell Number and %B.A.D.
The Prout's Neck DNA Consensus Conference published a set of guidelines for these
histogram attributes and ModFit LT uses these published guidelines to provide you with a
graphical and quantitative picture of how the histogram satisfies the requirements. By
pushing the button next to each of these features you can examine the actual published
quote.

ModFit LT evaluates an additional feature not covered by the Consensus Conference: RCS.
Reduced Chi-square is a measure of how well the model fitted the data. Based on our
experience with RCS values, we added our own guidelines for this feature.

The second method of S-Phase assessment is unique to ModFit LT and is based on a

statistical approach to the problem rather than a consensus of experiences. By using
established statistical methods, ModFit LT calculates the error probability distribution about
each of its S-Phase estimates (e.g. Total or Average S, Aneuploid S, and Diploid S). The
standard deviation of this probability distribution provides a direct quantifiable way of
evaluating the confidence of a given S-Phase estimate. If you are getting glassy-eyed on this
statistical stuff, let us show you four graphs that will make it crystal clear how this system
works. The first graph demonstrates how cell cycle programs used to work.

Let's say that your old cell cycle program reported a Total (average) S-Phase estimate, also
known as "Average S-Phase", of 20.5%. Since the program did not give you any information
on the variability associated with this estimate, you had to assume that it was correct.

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Another way of saying the same thing is that the probability of the Total S-Phase being
20.5% was assumed to be unity or one. Largely because of this limitation, a number of S-
Phase criteria were developed that normally would be conducive to obtaining accurate S-
Phase estimates (cell number>10,000, %CV<8.0, etc).

ModFit LT has an exclusive statistical method of estimating the probability distribution about
each of its S-Phase estimates. Let's take a look at how that changes our figure.

Since ModFit LT calculates the probability distribution about its estimate, you now have a way
of quantifying the S-Phase estimate's variability with a standard deviation. Rather than
20.5% in this example, you now know that it is really 20.5% +/- 1.2%. However, ModFit LT
takes this S-Phase assessment one step further. By evaluating S-Phase cutoffs against this
probability distribution, the program can calculate the probability of the estimate being in a
particular category (e.g. Low, Intermediate, or High). Let's look at the next graph to see how
this works.

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The areas of the probability distribution between each of the S-Phase cutoffs indicate how
likely the S-Phase estimate is within that category. For example, the area of the High
category is 0.95; thus, there is a 95% chance that the specimen has a high S-Phase with
your laboratory's specific S-Phase cutoffs. Normally in statistics, you express the probability
of the hypothesis not being correct (p-value). In this case, the p-value is simply 1.0 - 0.95, or
0.05, and it is the p-value which ModFit LT displays.

That's just about all there is to it. Not only does ModFit LT indicate the variability of its
estimate, but it also merges your S-Phase cutoffs with the derived probability distribution to
provide you with just the information you need to convey to the physician about the
proliferative activity of the specimen. Is the S-Phase high? How sure are you of this
assessment? You can now answer both questions quantitatively.

We promised you four graphs and have only shown three so far. Suppose the derived S-
Phase estimate was one of those borderline calls. It overlaps one of your S-Phase cutoffs.
Let's modify the above graph to show that situation.

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How do you classify this specimen? If you choose High, then you have a 25% chance of
being wrong. When confronted with this situation it's best to combine two categories. By
calling the specimen: Intermediate to High, the probability of being wrong is back to a
reasonable level. What you are really saying is that the S-Phase is probably not Low.

If ModFit LT determines that the certainty (probability) of a particular S-Phase level is less
than 0.9, it will automatically combine the two most probable categories. If the certainty is
still less than 0.9 with the combined categories, then the program will display a question
mark (?) next to the corresponding S-Phase assessment.

ModFit LT enables the inspection of the error distributions about each of its S-Phase
estimates. If the sample is DNA diploid, only the diploid S-Phase error distribution can be
evaluated. If the sample is DNA aneuploid or tetraploid, only the aneuploid and total S-Phase
error distributions can be evaluated.

See also:

S-Phase cutoffs command (Edit menu)

Fit Diagnostics command (Analysis menu)

S-Phase Assessment Options dialog

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DDE Support

NOTE: This feature is only available on Windows versions of ModFit LT. DDE is not supported
on the Macintosh.

Dynamic Data Exchange (DDE) in ModFit LT allows the program to be driven by and to
provide information to other applications. WinList for Windows can use DDE to connect with
ModFit LT and to share data with it. In most cases, the DDE scripts provided with WinList are
sufficient for this interaction. If you have another application you want to connect with
ModFit LT using DDE, please contact Verity Customer Support.

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ModFit LT Help Appendices

Strengths and Limitations of Automatic Analysis

ModFit LT uses some very sophisticated logic when it attempts to automatically analyze your
data. The system has been designed to work with a great variety of data samples, samples
that vary in almost an unlimited number of ways. When your data samples fit into the
assumptions built into the automatic analysis functions, the program works well in
automatically modeling them. Conversely, as you might expect, the automatic systems do not
work as well when your samples differ from what it has been designed to analyze.

To better understand the strengths and limitations of automatic analysis, it may be helpful to
examine some of the assumptions built into the system.
Peak Finder

When you open a data file with the program, one of the first things the software attempts to
do is locate "peaks" in the sample. This step is the foundation of automatic analysis: if it finds
the important peaks in the sample, the remaining steps in auto-analysis have a good chance
at succeeding. If it cannot detect the important peaks, it will more than likely have difficulty
or may fail to automatically analyze the sample.

While it is relatively easy for our eyes to see peaks in a histogram, the computer algorithms
that perform this function are anything but simple. The process ModFit LT uses works well
with the large library of data files we use to test the system. Here is how it works.

The program performs a statistical analysis of the histogram to identify potential peaks. Then
the peak finder begins to filter out peaks that do not meet certain criteria. There are filters
for high and low %CV, relative peak height, and how well the peak can be fitted with a
Gaussian in a quick analysis. These filters can be viewed in the Peak Finder Settings dialog.

If one or more of the filtering criterion filters out an important peak, the system will likely fail
in a later step. For this reason, you can adjust some of the filters used by the peak finder so
that the system better matches the data samples analyzed in your laboratory.

A very important note on the peak finder settings: adjusting one or more settings to work
with a single sample is a bad idea. The reason is simple: without looking at numerous
samples to test your new setting, you may find you have fixed the peak finder for one
exceptional sample, and broken it for the typical sample. Weigh your adjustments carefully.
Do not make adjustments to accommodate a single sample; use manual analysis to handle
the exception.
Ploidy Detection

The peak finder identifies the peaks it finds by displaying a small, black triangle under the
peaks on the histogram display. The next step in automatic analysis is to look at the pattern
presented by those peaks and attempt to match the pattern to a type of histogram the

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program knows about. This occurs when you click the Auto button on the toolbar, or choose
Automatic Analysis from the Analysis menu.

So just what is an "important" peak?

The most important peaks the program needs to find are peaks for internal standards,
apoptosis, and G0G1 peaks for each cycling population. If the program finds these peaks, it
generally succeeds in auto-analysis.

You need to tell the program how many standard peaks your sample contains. This is done in
the Auto Analysis Settings dialog box. The program assumes that standard peaks, if any, are
the first peaks, followed by an apoptosis peak, if selected, and the remaining peaks are
cycling population peaks.

If you have correctly informed the program as to how many standard peaks the sample
contains and whether or not it has an apoptosis peak, and the peaks found by the peak
finder are the important peaks, the ploidy detection step is off to a good start.

The remaining step is a pattern-matching step, which ignores standard and apoptosis peaks.
Here, the program analyzes relative sizes and positions of the peaks remaining, looking for
cell cycles. ModFit LT is designed to handle any number of cell cycles in a histogram, though
it is rare to find samples with more than 4 cell cycles.

In general, the more complicated the histogram, the more difficult it is to classify. You may
need to manually analyze very complex samples.

Once the sample is classified, a model is created to match the pattern. Then ranges are
assigned to important peaks, and the non-linear least-squares analysis of the sample is
performed.
Debris and Sample Quality

As you might expect, the success of automatic analysis is closely linked to the quality of the
data you want to analyze. Samples with lots of debris or aggregation detract from the
program's ability to find peaks or determine the right model to use. Efforts to gate out debris
and aggregation sometimes work, but they are usually going to work against the program's
built-in functions designed to compensate for debris and aggregates. The better solution is to
perfect the sample preparation and acquisition techniques you use. Starting off with a cleaner
sample greatly simplifies the peak and ploidy detection operations, and helps ensure that the
peaks seen by you and the program are real peaks.

Follow the suggestions of experts to prepare and analyze your samples. Establish standards
that must be met before a sample can be considered for analysis. The DNA Consensus
Conference (Cytometry. Vol. 14. Number 5, 1993) made many specific recommendations on
DNA analysis, many of which relate to sample quality. Your standards should be at least as
restrictive as those presented in that report.

336 © 1994-2017 Verity Software House. All Rights Reserved.

ModFit LT Help Appendices

Be aware of obvious things, as well. If you accidentally try to analyze forward angle light
scatter instead of the DNA parameter, you will end up with nonsensical information when you
perform automatic analysis.
Your Responsibilities

Based on this discussion, it is probably obvious that there are numerous ways automatic
analysis of your samples can fail. The problem is that the program itself doesn't know when it
has failed; only you can make that determination.

You should view automatic analysis with scientific skepticism. You must review the
information on the report to ensure all the model matches your understanding of the
histogram. You should always review the results of an automatic analysis to ensure that the
program has identified the standard peaks, apoptosis peaks, and G0G1 peaks of the cycling
populations. In addition, you should verify that the ploidy classification assigned by the
program concurs with your knowledge of the sample.

See also:

Automatic Analysis command

Manual analysis with ModFit LT

© 1994-2017 Verity Software House. All Rights Reserved 337

 ModFit LT Help Index

Index
Batch processing 203
2
Batch Processing Settings 203
21 CFR Part 11 Compliance 272
Batch settings for Files 205
A Batch settings for Reports 210
About command 322 Bitmap 229
Activate all 217 Bmp 230
Activate Assessment System 260
C
Active 78, 219
CD Key 23
Add Record 225
Cell Tracking Example 155
Analysis 203
Cell Tracking Wizard 52, 145, 146, 147,
Analysis Population 111
148, 150, 153, 154, 155
Analysis Results 305, 308, 323
Generations tab 148
Aneuploid 7, 123, 274
Other tab 150
Apoptosis 234, 323
Start tab 146
Appendix C
Cell Tracking Wizard dialog 59, 145
Histogram and S-Phase Assessment 326
Cell Tracking Wizard->Create or edit
Appendix G
model (Analysis menu) 145
Strengths and Limitations of Automatic
Cell Tracking Wizard->Open data and
Analysis 331
analyze (Analysis menu) 153
Application Info 320
Change Data Set... 114
Arrow 171
Check for Updates 316
Arrow tool 197
Choose Batch Dialog 209
ASCII text 78
Choose Model 37
Ask Tech Support 317
Choose Model command 120
Auto Analysis 72, 86, 116
Colors command 284
Auto Analysis command 116
Compliance 264, 266, 272
Auto Analysis Settings 234
Component 274
Auto Analyze and Auto Linearity 117
Component Shapes 274
Auto Calculate Major Tic 186
Computations 323
Auto Calculate Minor Tics 186
Configuration 86, 234, 242, 246
Auto Linearity 119
Copy command 213
Auto Maximum 186
Creating a report template 94
Auto Minimum 186
cutoffs 326
Auto Save Report 171
Cycle properties 123
AutoAggregates 234, 274
AutoDebris 234, 274 D
AutoLinearity 72 Data 109
automatic analysis 32 Data set 114
Automatic analysis limitations 331 Database 78, 215, 217, 219, 221, 223,
Auto-scale 246 225, 226
Average S-Phase 326 Database files 78
Database keywords 192
B
Database Viewer 223
BAD 323

© 1994-2017 Verity Software House. All Rights Reserved 338

 ModFit LT Help

Database Wizard 226 Export Settings 262

Databasing with ModFit LT 78 External reference 234
DDE Support 330
De-activate all 217 F
Debugging 231 FDA's 21 CFR Part 11 264
Delete files 217 Field Testing ModFit LT 98
deleting 171 fields 78, 221, 226
Detail 296 File 109
Diploid 7, 123, 274 File batch settings 203, 208
Diploid determination 7, 234 File Batch Settings (Edit menu) 205
Diploid G2M Fraction 323 File Batch Toolbar 208
Diploid-to-standard ratio 234 File Open dialog box 110
Division error index 323 File Save As dialog box 178
DNA Consensus Conference 326 Files
listmode 111
E First Time Using ModFit LT 32
Edit 1P Graphics 183 Fit Diagnostics command 129, 202
Edit Components 291 Fit Diagnostics dialog 129, 130, 202
Edit Databases 215 Fit with Current Model command 118
Edit Equations 309 Fixed Upper Boundary 255
Edit Keyword Map 78, 221 Full Model Editor 289
Edit Log 231 Functions 312
Edit model 286, 289
Edit Parameter Preferences 252 G
Edit Properties for 2P Graphics 186 Gate 111, 205
Edit Properties for All Databases 217 graphics 171
Edit Properties for Component 293 I
Edit Properties for Cycle 123 Import Settings 262
Edit Properties for Database 78, 219 Installation and Setup of ModFit LT 21
Edit Properties for Details 296 Internal keywords 192
Edit Properties for Equation 259
Edit Properties for Log 232 J
Edit Properties for Parameter 255 JPeg 229, 230
Edit Properties of Equation 311
Edit Properties of Range 301 K
Edit Ranges 299 Keyword Groups 78, 221
Edit User Equations 257 Keyword-Database Map 78, 221
Ellipse 171 Keywords 78, 192, 221, 312
Ellipse tool 199 Keywords tool 191
Enhanced Cell Tracking Analysis 59 L
Equation syntax 312 Latest Versions 6
Equations 308, 311, 312, 323 License and Warranty 4
Error with database 78 Limitations to database 78
Export Histogram Graphic 229 Line 171
Export Report Graphic 230 Line command (Tools menu) 196
Export Save PDF 228

© 1994-2017 Verity Software House. All Rights Reserved 339

 ModFit LT Help

Linear 255 Portable Network Graphics 229, 230

Linearity 72, 205, 234 Ppm 229
Listmode 111, 205 Precursor frequency 155, 323
Log 255 Preferences 246
Log properties 232 Print command 179
Logging 231 printing
batch 203
M probability 326
Macintosh version 21 Problem 319
Make a Suggestion 318 Proliferation index 155, 323
Manual Analysis 120 Proliferation results 323
Manual analysis with ModFit LT 37 Prompting 78, 219
Memory 20 Properties for Standards 241
Model colors 274 p-value 326
Model components 274, 289
Model descriptions 274 Q
Model names 274 Quick Model Editor 286
ModFit LT Help 3
Moving 171 R
RAM 20
N Range command 126
Naming Convention 7 Range position 126
New command 173 Ranges 126, 274
New Features in ModFit LT 8 RCS 326
New version 6 Recent Files list 115
Nonproliferative fraction 323 Recent Reports list 182
Non-proliferative Fraction 155 Recommended approach 72
Rectangle 171
O Rectangle tool 198
Open FCS 109 Region 111
Open report 32, 174 Registering your new software 23
Open Reports command 174 Registration dialog 23
Optimizing Auto Analysis 86 Replication Index 155
Options and Configuration 246 Report 32, 171
P Report a Problem 319
Page Setup command 180 Report batch settings 203, 210
Parent 147 Report Batch Toolbar 212
Paste command 214 Report command 175
PC versions 21 Report objects 129, 171, 202
Peak Finder 86 Report Page 100, 171
Peak finder limitations 331 Report results 311, 323
Peak Finder Settings 242 Report template 94
PICT 229 report tools 171
Ploidy determination 234 Research and Compliance Modes 264
Png 230 resizing 171
Population 111 Resolution 246

340 © 1994-2017 Verity Software House. All Rights Reserved.

 ModFit LT Help

results 78, 312, 323 Syntax 312

Ribbon Bar 100, 103 System Requirements 20
RUO Mode 266
T
S Temporary variables 312
Save 171, 177, 178 Tetraploid 7, 234, 323
Save As command 177 Text command 190
Save command 176 To Percentile 255
Save graphic 229 Toolbar 100, 107
Save PDF 228 Total S-Phase 326
Save picture 229
Scalable Vector Graphics 229, 230 U
Scale command 125 Unlock Code 23
Scaling 246 Update 6
Serial Number 23 Updates 316
Settings Manager 262 Upper Generation Proliferation Index 52,
Software security 23 150, 155, 323
S-Phase 234 User equations 257, 259
S-Phase assessment 326 User group 272
S-Phase cutoffs 326 User Guide 321
S-Phase Cutoffs command 260 Using AutoLinearity 72
spreadsheet 78 Using the Sync Wizard 43
Standards 234, 240, 241 V
Strings 312 Viewer 78, 219, 223
Subset 111
Suggestion 318 W
Support 6, 319 Web 6
Svg 229, 230 Weibull 274
Sync Wizard 43, 133, 136, 139, 140, 142, Windows 21
144
X
G0G1 tab 134
G2M tab 136 X Zoom Graph 200
Other tab 139 Y
Report tab 140 Y Zoom Graph 201
S-Phase tab 138
Start tab 133 Z
Sync Wizard Adjustment dialog 144 Zoom command 181
Sync Wizard->Create or edit model Zoom Graph 200
(Analysis menu) 132 Zoom Type 255
Sync Wizard->Open and analyze 142
Sync Wizard->Quit 143

© 1994-2017 Verity Software House. All Rights Reserved 341