Rare Cell Analysis Software

Rare Cell Analysis Software

Introduction

The Bio-Rad Rare Cell Analysis Software is a platform used in tandem with the Celselect Slides™
Enumeration Direct Stain Kits and Enumeration Indirect Stain Kits. It is designed to enable analysis
of target cell populations using images and is compatible with multiple microscope models. The
software allows users to create a custom algorithm to identify target cells with up to six fluorescentchannels or five fluorescent channels and brightfield. These settings can be saved for reuse with
future datasets or to share among users. Results can also be revisited and augmented using updated
analysis algorithms.
The software also has the capability to generate a PDF report that includes information about
and images of verified target cells. This report information can then be used for presentations andpublications.
Setup
System Requirements

Installation
Installation on Windows OS

Rare Cell Analysis Software is supported on Windows operating
system 10 (minimum 1903) and 11 versions.

Note: Remove any previous versions, as older versions willinterfere with installation.

Microscopes with Supported Image Analysis Compatibility

1.

Type “Add or Remove Programs” in the Windows search bar.

2.

Click Add or Remove Programs and then type Rare Cell
Analysis Software under the Apps and Features section.

ModelsAgilent Lionheart
Keyence BZ-X
Leica DMi8

a. If an older version of Rare Cell Analysis Software is notinstalled, continue to step 3a.

Nikon Eclipse
Olympus VS200

b. If an older version of Rare Cell Analysis Software is
installed, uninstall the program before continuing to step
3a.

Zeiss Observer■■■

The software is compatible with a Windows platform that at
minimum supports .NET Standard 2.0
The recommended minimum RAM configuration is 2 GB
Anti-virus or anti-malware software can be runningsimultaneously with the software

3.

Certificate Installationa. Download or obtain the installer file, dependency files, andcertificate files.
b. Open the directory from the Windows Start menu bysearching for the filename or by accessing the folder where
the files are stored: .../RareCellAnalysis.UWP_X.X.X.X_x64.
c. Click the directory to open the location of the .cer file.
d. Double click the .cer file and click Install Certificate.

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Rare Cell Analysis Softwaree. Select Local Machine as the store location in theCertificate Import Wizard and click Next.

7.

f. A prompt will appear; click Yes to allow the certificate to
be installed.

File Menus

a. After reading the EULA, click Accept.

g. Select Place all certificates in the following store.

Click File in the top left corner to access file options at any time.
Options include:h. Click the Browse… button.

■

i.
j.

In the pop-up window, select Trusted Root Certification
Authorities and click OK.

■

Click Next.

k. Click Finish. A window notification will appear with: “Theimport was successful.”
4.

End User License Agreement (EULA)

Install Dependencies■

a. Install the following dependencies by double clicking thefollowing files:■

1) Microsoft.NET.CoreFramework.Debug.2.2.appx
2) Microsoft.NET.CoreRuntime.2.2.appx■

If an internet connection is not available, you will need to installthese two dependencies manually:

Save Template File: Saves selected parameters entered in the
Template File Configuration screen
Save Results File: Saves data on selected experiment.
Parameters and reference images entered in the Document
Information and Template File Configuration screen will be savedwith the results. An asterisk at the top of the screen next to the
Results File name indicates unsaved results
Close Results File: Exits current experiment and relocates to
the Start Menu
New Results File: Begins a new image analysis setup with nosaved parameters
Open Results File: Opens existing file that already has analysis
parameters set3) Microsoft.UI.Xaml.2.44) Microsoft.VCLibs.x64.14.00
5.

Application Installationa. Double click the .../RareCellAnalysis.UWP_X.X.X.X_x64.
msixbundle file.
b. Click Install.

6.

Enable File Accessa. By default, this application does not have permission toaccess the active file system. If this setting is not enabled,
the user will be prompted to enable this setting when thesoftware is launched.

This screen also contains buttons to progress through the twomain workflows. Select one of the following buttons based on
intended operations:■■b. Click Launch Settings and Exit to open the Windows File
Access Settings menu. To manually open this window, go
to Windows Settings > Privacy > File System. Turn on fileaccess for Rare Cell Analysis Software.

© 2024 Bio-Rad Laboratories, Inc.

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New Results File: For analyzing new images with an existing
template file or from scratch. Selecting this will open theDocument Information screen to enter experiment details
Open Previous Results File: For revisiting or adjusting analysis
on an experiment analyzed before. Selecting this will take theuser to File Explorer, where a previous session may be reopened

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Rare Cell Analysis Softwaree. Template: Allows the user to select a general set of
established parameters saved as a file locally (optional). If
a Template File is not selected, the parameters will need to
be entered or optimized by the user. Click the file icon tonavigate to a file of choice through a File Explorer window.

Process Step Menu (Navigation Menu)

To navigate between analysis parameter screens, select Proceedor Back in the top menu. To choose specific steps for analysis,
access the Process Step Menu by clicking the green tab with threecircles on the left side of the screen. Click the page title to return tothat step in the analysis process.

New Results File1.

To begin analyzing a new set of images, open the Rare Cell
Analysis Software.

2.

On the Start menu, click New Results File.

3.

In Document Information, enter the appropriate information.

Click Proceed to continue.

5.

In most cases, the settings for an image can be initiatedfrom a Template file. This file can be loaded in the Document
Information Page and may be imported or created by theuser. Once these settings are established for a particular
microscope, the image information should not need to be
recreated. If this is the first Template file for a particular
microscope, follow the steps below based on whether the
microscope produces a mosaic of images (stacks) or one
image for the entire slide (stitched):a. Stacked Images:

a. Username: Open text field that autofills based on thecurrent user directory (required).

1) Sample Volume: To calculate the concentration oftarget cells, enter the undiluted volume of sample thatwas loaded into the Inlet Funnel (mL, range 0.1–99).

b. Sample ID: Open text field that assigns the name of thesample being analyzed (optional).

2) Microns per Pixel: To calculate multiple metrics aboutcell size, enter the ratio of microns per pixel (range
0.2–2.0).

c. Slide ID: Open text field for the user to enter the serial
number of the Celselect Slides being analyzed (optional).
d. Date: Field for user to define a date, normally when an
image set was collected or analyzed (required). The default
setting will fill in the date on the computer being accessed.
Select the calendar icon and select the date by clicking a
day from the calendar navigation pane.

© 2024 Bio-Rad Laboratories, Inc.

4.

3) Tiles per Column: In image stacks, to correctly identify
cells on the borders of multiple images, enter thenumber of columns that make up the entire scannedarea (range 1–52).

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Rare Cell Analysis Software

Click the Source button and select one of the image
files from your dataset.

4) Tiles per Row: In image stacks, to correctly identify
cells on the borders of multiple images, enter thenumber of rows that make up the entire scanned area
(range 1–52).
5) Scan Direction: In image stacks, to correctly identify
cells on the borders of multiple images, enter the
direction in which the image index increases. It can beleft to right row after row (Horizontal Line), it can be left
to right for one row and then right to left for the row
underneath (Horizontal Zigzag), or it can be the sametwo fundamental patterns in the vertical direction: top
to bottom for every column (Vertical Line) or alternating
between top to bottom and bottom to top (Vertical
Zigzag).
6) Anchor Point: In image stacks, in addition to knowing
how many tiles there are and how they’re arranged, thesoftware needs to know where the images start. The
user must select the corner where the first image of theseries is positioned. The options are top left, top right,
bottom left, and bottom right.

Confirm the detected file pattern matches what is
expected. For example, A1_01_[Channel]_[Tile]_
DAPI_001.tif should be displayed for European Visitor
datasets with the positions of [Channel] and [Tile]
correctly identified.
7) File Wizard: The File Wizard is used for determining thefile pattern name associated with stacked images andcan be used to automatically select and map stacked
images to channel folders. Note that all of the stacked
images should be placed in the same directory. Subdirectories are not included in the search.

Select a mapping for the images. By default, thedisplayed image will map image files with filenames
indicating channel 1 to the first enabled channel,
channel 2 to the second enabled channel, etc. Both thechannel as indicated in the filename and the enabledchannel to map to may be changed as necessary
toward the expected mapping. Select Do Not Map
from the dropdown list to not map any images files
indicating that channel number.

After filling out the Image Information and Channel
sections of the Template Configuration page, run theFile Wizard by clicking the green File Wizard button.

Click Apply.
b. Stitched Images:
1) Sample Volume: To calculate the concentration oftarget cells, enter the undiluted volume of sample thatwas loaded into the Inlet Funnel (mL, range 0.1–99).
2) Microns per Pixel Ratio: To calculate multiple metrics
about cell size, enter the ratio of microns to pixels(range 0.2–2.0).
3) When using a stitched image, adjust the tile and row
count to optimize processing. For best practices,use the same numbers as you would for the
stacked images.

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6.

Scroll down to the Channel configuration.

7.

Select the number of channels necessary for the assay
by clicking the checkbox next to the channel number.
(Note: limited to six channel fluorescent markers.)

8.

Select the dropdown box to choose the fluorescent marker
used for each channel. If a custom marker is being used,
select Custom and enter the name of the fluorescent label.

9.

Click the Source folder to upload the corresponding images
for each channel.

c. Fluorescence Intensity: Using the intensity of an object
in a specific channel, the software will identify objects forfurther analysis. For optimal analysis, select a potential
range of fluorescence levels. Enter a value for the range
minimum and maximum (maximum range 1–65535).

Note: If using image stacks, all scan files affiliated with thechannel must be entered at the same time by selecting theimage files while holding the Ctrl and/or Shift keys.
2.

Click Proceed when ready.

3.

A loading screen will appear with the estimated time to analyze
images and identify potential cells.

Note: The maximum cell count that can be detected is 100,000
potential cells. This prevents the user from using incorrect settings,
which would result in a runaway indefinite processing time.

10. An optional text box is available to enter any sample
information that may be helpful for reference later. For
example, a user may want to associate a specific channel witha protein or other label of interest.
Total Cell Identification

The initial analysis begins with identification of all objects that may
be cells. This is done by selecting one universal channel and theparameters of interest that will define potential cells.
1.

Scroll down to the Cell Identification Parameters box, selectthe desired channel, and enter in the desired values for thefollowing cell identification features:

4.

After the analysis time has completed, a new screen willappear. Click OK to continue.

a. Diameter: Using the Microns per Pixel Ratio entered in the
Image Information section, the software will identify objects
based on their outer diameters. For optimal analysis, selecta potential range of cell diameters. Enter a value for therange minimum and maximum (maximum range 1–50 μm).
b. Aspect Ratio: Using the long and short axis of each object,
the software will identify objects based on the aspect ratioof their long-to-short dimensions. For optimal analysis,
select a potential range of aspect ratios. Enter a value forthe range minimum and maximum (maximum range 1–20).

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Rare Cell Analysis Software

Target Cell Identification

In the Target Cell Identification Lists & Graphs page, information
can be viewed on the cells identified previously. Additional
analysis tools in the Charts menu can be used to narrow searchparameters to specific target cell characteristics in one or more of
the selected channels.
1.

Click the Charts tab to bring up options for:a. Area: Using the Microns per Pixel Ratio entered in the
Image Information section, the software will identify objects
based on their areas. The range may have a minimum, amaximum, both, or neither (maximum range 1–99999 μm2).

4.

b. Diameter: Using the Microns per Pixel Ratio entered in the
Image Information section, the software will identify objects
based on their outer diameters. For optimal analysis, selecta potential range of cell diameters. The range may have aminimum, a maximum, both, or neither (maximum range
1–50 μm).

In the Target Cell List tab, select the single cell or groupof cells by clicking the row to view the fluorescence andmorphological data. Selected cells will be highlighted in theCharts tab and in Graphs. Cells can also be selected on
the graph, and their corresponding rows in the table will behighlighted.

c. Aspect Ratio: Using the long and short axes of eachobject, the software will identify objects based on the
aspect ratio of their long-to-short dimensions. For optimal
analysis, select a potential range of aspect ratios. The
range may have a minimum, a maximum, both, or neither(maximum range 1–20).
d. Fluorescence Intensity: Using the intensity of an object
in a specific channel, the software will identify objects forfurther analysis. For optimal analysis, select a potential
range of fluorescence levels. The range may have aminimum, a maximum, both, or neither (maximum range
0–65535).
e. Perimeter: Using the Microns per Pixel Ratio entered in the
Image Information section, the software will identify objects
based on their outer perimeters. For optimal analysis,
select a potential range of cell perimeters. The range may
have a minimum, a maximum, both, or neither (maximum
range 1–99999 μm).
Note: Fluorescence settings are unavailable for brightfield.
2.

Click Refresh Filtering next to the Graphs heading to update
the targeted cell selection in the Identified Target Cell Count.

3.

In the Graphs tab click the dropdown menu to select whichaxes will be shown for the target cells, given the ranges of theparameters set earlier.

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5.

Clicking the gear in the Graphs section opens additional
options.

Generate Report

When analysis is completed, a PDF report of the selected target
cells can be generated.

a. Copy Graph to Clipboard: Copies the graph as an image
for use in presentations and publications.

Click the gear toolbox to open Report File Settings to set up:

b. Open in Expanded Window: Enlarges the selected graphin a separate window for additional manipulation.
c. Edit Graph Axes: Allows the user to alter the range of theparamters shown on the graph without changing the filter
parameters.
d. Add New Graph: Creates an additional graph in thesame workspace. Up to four graphs can occupy theworkspace at one time. These all showcase the same
selected data but different axes can be showcased in eachseparate graph.
After the parameter ranges are finalized, click Proceed toadvance to the Target Cell Confirmation page.

7.

In the Target Cell Confirmation page, after reviewing the celldata, users can select or deselect individual cells or groupsof cells that do not match desired cell identification features.
Information about each potential cell is listed, as well imagesof the cell in all channels selected and the cell’s locationtranslated onto a representative grid of the stitched image orstack montage. Click the checkbox next to the target cell to
confirm or cancel the item.

8.

This table of cell data can be exported into a .csv file for
additional statistical analysis or other forms of manipulation inthird-party software.

© 2024 Bio-Rad Laboratories, Inc.

Location: The folder where the report will be saved.

2.

Report Logo: A user-defined image that will be in the headerof the report.

3.

Header Configuration: Contains fields in the report that willbe specific to the user, including the company/lab name, its
website, and a potential report title.

After the PDF report is complete, a confirmation message willappear. Click Home to return to the Welcome page. Click Save
Results File to save all the settings and image associations (justthe thumbnails, not the entire files) as a single file. This can bedone at any time while setting parameters. In addition, saving thesettings by clicking Save Template File will allow the same metrics
to be used to analyze other datasets.

e. Remove Graph: Deletes the currently selected graph.
6.

1.

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Open Previous Results File Option

1.

In the Welcome page, click Open Previous Results File toopen past experiments to edit.

2.

Locate the desired file and click Open.

3.

Users will be transferred to the Document Information page.

4.

Click Proceed or use the Navigation bar to be transferred to
the Template File Configuration page to make any necessarychanges to the image source, color, channel, or cell
identification parameters. After adjusting these parameters,
the settings may be saved as a Template File or the modified
findings can be saved as a Results File.

5.

Click Proceed or use the Navigation bar to be transferred to
the Target Cell Identification Lists & Graphs page to review
data or generate a PDF report.

BIO-RAD and CELSELECT SLIDES are trademarks of Bio-Rad Laboratories, Inc. in
certain jurisdictions. All trademarks used herein are the property of their respective owner.
© 2024 Bio-Rad Laboratories, Inc.

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