uPath ER (SP1) image analysis,
Breast Algorithm Guide RUO
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
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Publication information
Material number 09411046001
Copyright © 2021, Ventana Medical Systems, Inc. All rights
reserved.
Trademarks The following trademarks are acknowledged:
VENTANA, BENCHMARK, CONFIRM, ULTRAVIEW, and
UPATH are trademarks of Roche.
Other product names and trademarks are the property of
their respective owners.
Manufacturer address
eLabDoc Electronic user documentation can be downloaded using
the eLabDoc e-service on DiaLog:
dialog.roche.com
For more information, contact your local affiliate or Roche
Service representative.
Template version Software version Revision date Change description
1.0 1.0 June 2021 Initial Algorithm Guide
y Revision history
Ventana Medical Systems, Inc.
1910 E. Innovation Park Drive
Tucson, AZ 85755
USA
+1 520 887 2155
+1 800 227 2155 (USA)
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
3
Table of contents
Intended use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Symbols and abbreviations . . . . . . . . . . . . . . . . . . . . . . 4
About the system limitations. . . . . . . . . . . . . . . . . . . . . 6
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Overview of uPath RUO enterprise software. . . . . 8
Description of the algorithm test principles . . . . . 9
Using the algorithm workflow. . . . . . . . . . . . . . . . . 9
Opening a case. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Drawing whole tumor ROI. . . . . . . . . . . . . . . . . . . . . . . 13
Selecting tumor area . . . . . . . . . . . . . . . . . . . . . . . . 13
Excluding an area from a ROI. . . . . . . . . . . . . . . . . 14
Deleting a ROI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Image analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Description of color overlays. . . . . . . . . . . . . . . . . . 17
Performing image analysis . . . . . . . . . . . . . . . . . . . 17
Overriding the slide score manually. . . . . . . . . . . . 18
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Retriggering failed analysis. . . . . . . . . . . . . . . . . . . 19
Understanding areas to exclude from analysis . . 20
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
4 Intended use
Intended use
The uPath ER (SP1) image analysis, Breast RUO algorithm
is an adjunctive computer-assisted tool that identifies ER
negative and positive tumor cell nuclei within a
pathologist annotated viable tumor region in images of
formalin-fixed, paraffin-embedded neoplastic breast
tissue captured on a VENTANA DP 200 slide scanner.
For Research Use Only. Not for use in diagnostic
procedures.
Symbols and abbreviations
Product names Products and name variants for the products mentioned
in this publication.
Symbols used in the publication
Product names Product name variant
uPath ER (SP1) image
analysis, Breast RUO
uPath ER (SP1) algorithm RUO
CONFIRM anti-Estrogen
Receptor (ER) (SP1) Rabbit
Monoclonal Primary Antibody
CONFIRM anti-ER (SP1)
antibody
uPath RUO enterprise
software version 1.1.1
uPath RUO enterprise
software
VENTANA DP 200 slide
scanner
n/a
Benchmark ULTRA instrument n/a
ultraView Universal DAB
Detection Kit
n/a
y Product names
Symbol Explanation
o List item
u Related topics containing further information
q
Tip. Extra information on correct use or useful
hints.
r Start of a task
I Extra information within a task
f Result of an action within a task.
c Frequency of a task.
n Duration of a task.
d Materials that are required for a task.
j Prerequisites of a task.
u Topic. Used in cross-references to topics.
y Symbols used in the publication
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
Symbols and abbreviations 5
Abbreviations The following abbreviations are used.
p Task. Used in cross-references to tasks.
w
Figure. Used in figure titles and cross-
references to figures.
y
Table. Used in table titles and cross-references
to tables.
Symbol Explanation
y Symbols used in the publication
Abbreviation Definition
ER Estrogen Reception
FFPE Formalin-fixed, paraffin-embedded
IHC Immunohistochemistry
ROI Region of interest
RUO Research use only
WSA Whole slide analysis
y Abbreviations
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
6 About the system limitations
About the system limitations
When working with uPath ER (SP1) image analysis, Breast
RUO, cited in this document as uPath ER (SP1) algorithm
RUO, be aware of the following limitations.
The uPath ER (SP1) algorithm RUO is designed to work
with the CONFIRM anti-Estrogen Receptor (ER) (SP1)
Rabbit Monoclonal Primary Antibody, cited in this
document as CONFIRM anti-ER (SP1) antibody. The test
results are only as good as the quality and accuracy of
the immunohistochemistry (IHC) slide that is imaged and
the subsequent image that is analyzed.
q The uPath ER (SP1) algorithm RUO has not been
tested, nor its safety and effectiveness validated when
used with a personal computer from home.
You must validate the staining run by examination of the
control slides to verify that the expected results have
been obtained before images from sample slides are
acquired by the uPath RUO enterprise software.
Follow the manufacturer’s recommendations for the
uPath ER (SP1) algorithm RUO, including using all the
positive and negative quality control materials for each
staining run.
Description of the CONFIRM anti-ER (SP1)
antibody
Use the manufacturer’s recommendations for the
CONFIRM anti-ER (SP1) antibody, including all positive
and negative quality control materials for each staining
run. If the control slides are not acceptable, restain the
tissue until acceptable results are obtained. Follow the
interpretation guidelines in the uPath ER (SP1) algorithm
RUO method sheet.
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
About the system limitations 7
Considerations for the uPath ER (SP1)
algorithm RUO
The uPath ER (SP1) algorithm RUO is not designed as a
standalone tool and must only be used by a qualified and
trained pathologist in conjunction with histological
examination and proper controls.
• The uPath ER (SP1) algorithm RUO may generate
incorrect scores if the slide images exhibit abnormal
staining or are improperly scanned.
• The uPath ER (SP1) algorithm RUO was validated on
invasive ductal, lobular, mucinous, and micropapillary
carcinomas. Samples covered grades 1 to 3, scores of
0 to 100%, and tissue biopsies and resections.
• The uPath ER (SP1) algorithm RUO is a tool that
identifies ER negative and ER positive tumor cell
nuclei.
• The uPath ER (SP1) Algorithm RUO was trained to
determine stain intensity using a robust and
representative data set derived from pathologist
assessments.
The uPath ER (SP1) algorithm RUO may misinterpret
some non-tumor cells as tumor cells, especially larger
cells. In most cases there are many more tumor cells than
non-tumor in the annotated area, so this type of error
should not significantly impact the count.
The uPath ER (SP1) algorithm RUO may identify
unstained stromal cells and lymphocytes as negative
tumor cells, which may incorrectly decrease the reported
percent ER positivity. This is more common in high
positive cases and cases with a large lymphocyte
infiltrate, such as medullary carcinomas.
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
8 Overview
Overview
In this section
Overview of uPath RUO enterprise software (8)
Description of the algorithm test principles (9)
Using the algorithm workflow (9)
Overview of uPath RUO enterprise software
The uPath RUO enterprise software version 1.1.1, cited in
this document as uPath RUO enterprise software, is an
end-to-end digital pathology software that consists of
image analysis algorithms and a user interface.
The uPath RUO enterprise software with the uPath ER
(SP1) algorithm RUO provides consistent quantitative
assessment of neoplastic breast tissue.
Multiple functions are available for ease of use while
handling a case:
• Viewing of ROIs at various magnifications.
• Adding annotations to cases.
• Taking tissue section measurements.
• Performing image analysis.
• Generating reports.
uPath ER (SP1) algorithm RUO scoring The uPath RUO enterprise software with the uPath ER
(SP1) algorithm RUO assists in the quantitative
assessment of ER protein expression in IHC stained
histologic sections from FFPE neoplastic breast tissue.
The CONFIRM anti-ER (SP1) antibody counts the total
number of target tumor cell nuclei and stratifies them by
whether they are stained or unstained. Stained cells are
also stratified by stain intensity (weak, medium, or
strong). The uPath ER (SP1) algorithm RUO then divides
the number of ER positive tumor cell nuclei by the total
number of tumor cell nuclei to generate the percent ER
positivity.
The uPath ER (SP1) algorithm RUO reports the percent ER
positivity for each ROI and an aggregate score for all
selected ROIs on that slide. You may either accept the
scores provided by the algorithm, or manually override
them.
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
Overview 9
uPath ER (SP1) algorithm RUO intensity Stained tumor cells are stratified by stain intensity and
categorized as weak, medium, or strong intensity. The
output parameter is displayed as “Weak/Medium/Strong
Intensity Cell Count” in the ROI and Slide Score Panels
within uPath. The reported numbers are cell counts that
are normalized to 100, effectively making the reported
value the percentage of stained cells that fall into each
intensity bin. This is a deviation from other cell counts in
the panels, where the cell count is the absolute cell count.
Description of the algorithm test principles
The uPath ER (SP1) algorithm RUO employs deep
learning and image analysis techniques to obtain a
percent ER positivity.
The uPath ER (SP1) algorithm RUO uses probability maps
to score images of tissue stained with the CONFIRM anti-
ER (SP1) antibody.
Steps in image analysis • Split image into tiles for processing by the
convolutional neural network.
• Detect cells across the entire image.
• Classify cell nucleus as tumor or other types.
• Classify tumor cell nucleus as positive or negative.
• Perform color un-mixing on positively classified tumor
cells to quantify nuclear stain intensity.
• Categorize intensity into weak, medium, or strong.
• Calculate the percent ER positivity by dividing the
number of ER positive tumor cell nuclei by the total
number of tumor cell nuclei.
Tumor cell identification and scoring Cell locations and classifications are identified by parsing
probability maps generated by the deep learning network.
The classification with the highest probability determines
whether cell nuclei are classified as tumor positive, tumor
negative, or other cell types. The overlay only identifies
negative and positive tumor cell nuclei.
Using the algorithm workflow
The workflow for the uPath ER (SP1) algorithm RUO
includes the following:
• Staining with the CONFIRM anti-ER (SP1) antibody.
• Scanning with the VENTANA DP 200 slide scanner.
• Scoring with the uPath ER (SP1) algorithm RUO.
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
10 Overview
Antibody Staining The CONFIRM anti-ER (SP1) antibody identifies ER
positive tumor cell nuclei in neoplastic breast tissue.
Tissue samples are stained with the ultraView Universal
DAB Detection Kit on a Benchmark ULTRA instrument.
Follow the recommendations provided in the CONFIRM
anti-ER (SP1) antibody method sheet for tissue
preparation, staining, and review of controls.
Restain the slides if staining does not meet the guidelines
outlined in the CONFIRM anti-ER (SP1) antibody method
sheet.
Scanning The VENTANA DP 200 slide scanner is an imaging device
used to scan, digitize, compress, store, retrieve, and view
digitized images of slide specimens.
u For more information, refer to the VENTANA DP 200
slide scanner User Guide.
Software The uPath RUO enterprise software is customizable to
meet both site and pathologist needs.
u For more information, refer to the uPath RUO
enterprise software User Guide
d Make sure that the following supplies are ready before
starting this procedure:
m uPath ER (SP1) algorithm RUO
m uPath RUO enterprise software
m VENTANA DP 200 slide scanner
m Neoplastic breast tissue slides stained with the
CONFIRM anti-ER (SP1) antibody on Benchmark
ULTRA instrument with the ultraView Universal DAB
Detection Kit.
r To create and analyze slide images
1
Use a Benchmark ULTRA instrument using the
ultraView Universal DAB Detection Kit to stain a
neoplastic breast tissue sample with the CONFIRM
anti-ER (SP1) antibody on a glass slide.
2
Scan the slide with the VENTANA DP 200 slide
scanner in BIF file format at 20X magnification and at
one z-plane.
f The VENTANA DP 200 slide scanner transfers the
slide image to the image management system.
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
Overview 11
3
Create a case in the uPath RUO enterprise software
either manually in data management or automatically
through communication with the laboratory
information system.
I The uPath RUO enterprise software triggers WSA,
which automatically analyzes the entire scanned
image, and indicates in the viewer when analysis is
complete.
4
Use the ROI and Exclusion tools to annotate any viable
tumor region.
5
Review the results, and then either accept the score or
manually override it.
u Related topics
• Retriggering failed analysis (19)
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
12 Opening a case
Opening a case
Manage slide images for uPath ER (SP1) algorithm RUO
in the CASES screen.
j Make sure the following prerequisites are completed
before starting the procedure.
m You must have the pathologist role or the correct
permissions assigned before you can perform this
task.
r To open a case
1
In the CASES screen, view the cases assigned to you.
2
Choose a case by doing one of the following:
• Double-click on the desired case.
• Highlight the case and choose the VIEWER tab.
f The VIEWER screen displays the case with all the
images.
u Related topics
• Selecting tumor area (13)
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
Drawing whole tumor ROI 13
Drawing whole tumor ROI
In this section
Selecting tumor area (13)
Excluding an area from a ROI (14)
Deleting a ROI (16)
Selecting tumor area
Each ROI that you create and its analysis status is
displayed in the SLIDE PANEL. If you start to create an
annotation and decide not to continue, you can clear the
selection.
c A slide evaluation or analysis is needed.
j Make sure the following prerequisites are completed
before starting the procedure:
m The VIEWER screen is open.
m You must have the pathologist role or the correct
permissions assigned before you can perform this
task.
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
14 Drawing whole tumor ROI
r To select a tumor area
1
Choose a slide, and then magnify the image.
I The pathologist may view each ROI at various
magnifications and analyze it with the uPath ER
(SP1) algorithm RUO.
f The buttons in the ANNOTATION area of the
Quick Action menu bar become available (change
to bright white font).
2
Choose the ROI button, and then choose an option
from the tool bar.
f The hand cursor changes to a crosshair cursor.
3
Move the cursor over the tissue section where you
want to create the ROI, and then choose and drag the
cursor to produce the ROI.
f The ROI is created, and an annotation label above
the ROI displays the ROI name and number.
q All annotation types except a text box are
assigned a name that includes a number, such as
ROI#1, or A#1 for an arrow.
Excluding an area from a ROI
You can draw an area on a slide image to exclude it from
the ROI.
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
Drawing whole tumor ROI 15
j Make sure the following prerequisites are completed
before starting the procedure.
m The VIEWER screen is open.
m You must have the pathologist role or the correct
permissions assigned before you can perform this
task.
r To exclude an area from a ROI
1
Choose a slide and then magnify the image.
I All images in the case are displayed. Choose an
image by magnifying it or clicking on it.
f The buttons in the ANNOTATION area of the
Quick Action menu bar are activated.
2
Choose the ROI on the slide image.
I Any existing text, arrow, measurement, and
exclusions are displayed in the SLIDE PANEL.
f The selected ROI is highlighted in blue in the
SLIDE PANEL.
3
Choose the Exclusion button, and then choose an
option from the list.
4
Draw the region to be excluded on the image, and
then choose CONFIRM.
u Related topics
• Understanding areas to exclude from analysis (20)
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
16 Drawing whole tumor ROI
Deleting a ROI
You can change the ROI on a slide image.
j Make sure the following prerequisites are completed
before starting this procedure.
m The VIEWER screen is open.
m You must have the pathologist role or the correct
permissions assigned before you can perform this
task.
r To delete a ROI
1
Choose on the ROI.
f The ROI annotation label pop-up is displayed.
2
In the annotation label pop-up or the STATUS PANEL
functions area, choose the icon.
I You can also delete the ROI from the ROI
DETAILS panel.
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
Image analysis 17
Image analysis
In this section
Description of color overlays (17)
Performing image analysis (17)
Overriding the slide score manually (18)
Description of color overlays
A color overlay displays after ROI Analysis is completed.
The overlay disappears while moving the image. It returns
when the image is no longer selected.
Performing image analysis
The uPath RUO enterprise software allows for the
pathologist to select the ROI for each slide to be analyzed.
j Make sure the following prerequisites are completed
before starting the procedure.
m The VIEWER screen is open.
m You must have the appropriate privileges to perform
this task.
Color Overlay result
Yellow Tumor cell nucleus is positive for ER
Black Tumor cell nucleus is negative for ER
y Color overlay
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
18 Image analysis
r To perform image analysis
1
Choose the whole tumor ROI by doing one of the
following:
• Choose the center of the ROI to be analyzed.
• Choose the ROI in the SLIDE PANEL.
2
For each ROI, choose the Image Analysis button
either in the SLIDE PANEL or next to the ROI.
f Results are displayed in the SLIDE PANEL: under
Slide Score and next to the ROIs.
Overriding the slide score manually
The uPath RUO enterprise software allows for the
pathologist to review all cases analyzed and change
scores if necessary.
j Make sure the following prerequisites are completed
before starting this procedure.
m The VIEWER screen is open.
m You must have the pathologist role or the correct
permissions assigned before you can perform this
task.
r To override the slide score manually
1
Choose a slide for the score override, and then from
the SLIDE PANEL.
f The SLIDE SCORE secondary panel is displayed.
2
Choose the button in the Slide Score area.
3
In the Percent Positivity field, enter the score, and then
choose the CONFIRM button to save the score.
• (Optional) Enter comments in the comment field.
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
Troubleshooting 19
Troubleshooting
In this section
Retriggering failed analysis (19)
Understanding areas to exclude from analysis (20)
Retriggering failed analysis
If an error occurs while performing WSA, an analysis error
message is displayed beneath the slide image.
c As needed.
j Make sure the following prerequisites are completed
before starting the procedure.
m You must have Administrator privileges to perform
this task.
r To retrigger failed analysis
1
Login to the uPath RUO enterprise software.
2
Navigate to Administrator Settings and choose Job
Queue.
I The administrator settings is only available if you
have administrator privileges.
3
Choose the Failed tab.
f All slides with failed analyses are displayed.
4
Choose the slide to be analyzed then choose the Start
Job button.
f Slide moves to the Canceled/Completed tab when
done.
Roche Diagnostics
uPath ER (SP1) RUO algorithm v1.0 · 1020567EN · uPath RUO enterprise software · v1.0
20 Troubleshooting
Understanding areas to exclude from analysis
Follow best practices in selecting ROIs. Exclude areas
from ROIs that are typically avoided when scoring
manually. If you disagree with the score given by the
uPath ER (SP1) algorithm RUO, you have the option to
override it.
Areas to exclude from analysis No overlay With overlay
Necrosis
Exclude area from analysis. It can affect
the slide score.
.
Tissue folds
Exclude area from analysis. It can affect
the slide score.
Off-target staining
Exclude area from analysis. It can affect
the slide score.
Ink
Exclude area from analysis. It can affect
the slide score.
y Areas to exclude from analysis
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