Roche uPath RUO User manual

Brand: Roche

Model: uPath RUO

Type: User manual

Roche uPath RUO is an end-to-end digital pathology software solution that allows pathology laboratories to acquire, manage, view, analyze, share, and report on digital images of pathology specimens.

Using the uPath RUO enterprise software, the pathologist can:

View digital images at various magnifications.
Add annotations.
Make tissue section measurements.
Perform image analysis.
Generate reports.

Using the uPath RUO enterprise software, the pathologist can:

View digital images at various magnifications.
Add annotations.
Make tissue section measurements.
Perform image analysis.
Generate reports.

uPath HER2 Dual ISH image analysis

for Breast Algorithm Guide (RUO)

Table of Contents

Introduction 1

Algorithm Summary and Explanation 2

Intended Use 3

Intended Use of product 3

Purpose of Algorithm Guide 3

Test Principles 4

Limitations 5

Data Security 6

Workﬂow for using the uPath HER2 Dual ISH image analysis for Breast algorithm 7

Staining Characteristics 18

Troubleshooting 28

Introduction

The Roche uPath RUO enterprise software (uPath RUO

enterprise software) with the uPath HER2 Dual ISH image

analysis for Breast algorithm is a software system designed to

assist in the quantitative assessment of HER2 gene status using

two-color chromogenic in situ hybridization (ISH) in formalin-

ﬁxed, parafﬁn-embedded (FFPE) normal and neoplastic tissues.

The uPath RUO enterprise software is an end-to-end digital

pathology software solution that allows pathology laboratories

to acquire, manage, view, analyze, share, and report on

digital images of pathology specimens. Using the uPath RUO

enterprise software, the pathologist can view digital images at

various magniﬁcations, add annotations, make tissue section

measurements, perform image analysis, and generate reports.

NOTE: The uPath HER2 Dual ISH image analysis for Breast

algorithm is an adjunctive computer-assisted methodology to aid

in the acquisition and measurement of images from microscope

slides of tissue specimens ISH-stained to determine HER2 gene

status. To assure the validity of image analysis scores, it is the

responsibility of the pathologist to verify agreement by employing

appropriate controls as speciﬁed in the VENTANA HER2 Dual

ISH DNA Probe Cocktail method sheet (available at

www.ventana.com).

For Research Use Only. Not for use in diagnostic procedures.

uPath HER2 Dual ISH image analysis for Breast Algorithm Guide 1

Algorithm Summary and Explanation

For image analysis applications, the pathologist uses the uPath

RUO enterprise software to select and outline one or two (if

necessary) regions of interest (ROIs), using the Quick ROI tool.

Each ROI may be viewed at various magniﬁcations and then

analyzed by the uPath HER2 Dual ISH image analysis for Breast

algorithm. The heat map guides pathologists to the areas the

algorithm identiﬁed as having high ampliﬁcation of HER2 signals.

A count of the total number of HER2 and Chr17 signals for 20

representative cells is generated.

When the pathologist draws an ROI using the Quick ROI tool

(must use Quick ROI to run analysis) the uPath HER2 Dual

ISH image analysis for Breast algorithm ﬁrst identiﬁes the

20 best representative cells. Cells are ranked based on: cell

size, presence of at least one HER2 and one Ch17 signals and

segmentation conﬁdence. The total number of HER2 and Chr17

signals for the 20 representative cells is computed. Through

the quantiﬁcation of HER2 and Chr17 signals the uPath HER2

Dual ISH image analysis for Breast algorithm generates a HER2/

Ch17 ratio. The pathologist can accept the cells selected and the

score provided by the uPath Dual ISH image analysis for Breast

algorithm, or may manually replace cells and override the score

with a different score. The pathologist can also delete the initial

ROI and replace it with a new ROI on a more suitable region of

tissue.

The uPath HER2 Dual ISH image analysis for Breast algorithm

makes no independent interpretations of the data and therefore

should only be used by a qualiﬁed pathologist in conjunction with

histological examination and proper controls. It is designed as an

aid to the pathologist for the assessment of HER2 gene status.

2 uPath HER2 Dual ISH image analysis for Breast Algorithm Guide

Intended Use of product

The uPath HER2 Dual ISH image analysis for Breast algorithm is

an adjunctive computer-assisted methodology that supports the

detection and enumeration of the HER2 gene in formalin-ﬁxed,

parafﬁn-embedded normal and neoplastic breast tissue when

used with the VENTANA HER2 Dual ISH DNA Probe Cocktail

Assay.

For Research Use Only. Not for use in diagnostic procedures.

Intended Use

Purpose of Algorithm Guide

This uPath HER2 Dual ISH image analysis for Breast algorithm

Guide (Algorithm Guide) is intended to:

• Provide background information on the intended use of the

uPath HER2 Dual ISH image analysis for Breast algorithm, test

principles and limitations.

• Deﬁne the necessary materials, IT, data security and network

requirements.

• Show step-wise directions for running the uPath HER2 Dual ISH

image analysis for Breast algorithm.

• Provide photographic images that illustrate how the uPath HER2

Dual ISH image analysis for Breast algorithm should be used.

• Provide pathologists with a tool to facilitate the use of the uPath

HER2 Dual ISH image analysis for Breast algorithm on FFPE

breast sections stained with the VENTANA HER2 Dual ISH DNA

Probe Cocktail.

• Provide example images of challenging cases to provide

guidance on how to use the uPath HER2 Dual ISH image

analysis for Breast algorithm in their evaluation.

uPath HER2 Dual ISH image analysis for Breast Algorithm Guide 3

Test Principles

Quantitative Scoring Algorithm for the VENTANA HER2 Dual

ISH DNA Probe Cocktail:

The VENTANA HER2 Dual ISH DNA Probe Cocktail uses

quantitative scoring algorithm to determine HER2/Chr17 ratio.

The uPath HER2 Dual ISH image analysis for Breast algorithm

was designed to adhere to the scoring algorithm and workﬂow

detailed in the VENTANA HER2 Dual ISH DNA Probe Cocktail

method sheet.

The uPath RUO enterprise software with the uPath HER2 Dual

ISH image analysis for Breast algorithm employs image analysis

techniques to determine HER2 gene status.

The uPath HER2 Dual ISH image analysis for Breast algorithm

uses pre-deﬁned parameters to score images of tissue stained

with the VENTANA HER2 Dual ISH DNA Probe Cocktail.

Steps involved in image analysis:

• Detection of cells across the entire image.

• Identiﬁcation of scoreable cells and selection of 20

representative cells based on: cell size, number of red and black

signals and segmentation conﬁdence.

• Computation of the HER gene status by dividing the total HER2

signals by the total Chr 17 signals according to the VENTANA

HER2 Dual ISH DNA Probe Cocktail method sheet.

How uPath HER2 Dual ISH image analysis for Breast

algorithm identiﬁes tumor cells and how the score is

calculated:

• The uPath HER2 Dual ISH image analysis for Breast

algorithm identiﬁes tumor cells using color, intensity, size and

morphological features.

• Identiﬁed tumor cells stratiﬁed using pre-set thresholds that are

in line with the VENTANA HER2 Dual ISH DNA Probe Cocktail

method sheet.

• To calculate the HER2 gene status, the uPath HER2 Dual ISH

image analysis for Breast algorithm quantiﬁes the identiﬁed

HER2 (black) and Chr 17 (Red) signals and generates a HER2/

Chr17 ratio according to the VENTANA HER2 Dual ISH DNA

Probe Cocktail method sheet.

4 uPath HER2 Dual ISH image analysis for Breast Algorithm Guide

The uPath HER2 Dual ISH image analysis for Breast algorithm is

designed to work for the VENTANA HER2 Dual ISH DNA Probe

Cocktail. The test results are only as good as the quality and

accuracy of the ISH slide that is imaged, and the subsequent

image that is analyzed.

The pathologist must validate the VENTANA HER2 Dual ISH DNA

Probe Cocktail staining by examining the slide on uPath RUO

enterprise software and using internal controls to verify that the

expected results have been obtained before images of slides are

analyzed.

The manufacturer’s recommendations must be followed for the

VENTANA HER2 Dual ISH DNA Probe Cocktail including using all

positive and negative quality control materials for each staining

run. If the internal controls not acceptable, the tissues need to be

re-stained with acceptable results.

The pathologist must follow the recommendations for VENTANA

HER2 Dual ISH DNA Probe Cocktail interpretation.

Refer to the VENTANA HER2 Dual ISH DNA Probe Cocktail

method sheet (P/N 1018383EN ) and interpretation guide

(P/N 1018386EN) (available at www.ventana.com).

The uPath HER2 Dual ISH image analysis for Breast algorithm

is designed to be used by a qualiﬁed pathologist in conjunction

with histological examination and proper controls. It is not

designed to be a standalone tool, and requires competent human

intervention throughout the analysis process.

The uPath HER2 Dual ISH image analysis for Breast algorithm

may generate incorrect scores if the captured images have:

tissue folds, non-target staining, overstaining of counter stain,

out-of-focus, etc.

The uPath HER2 Dual ISH image analysis for Breast algorithm

may reject tumor nuclei that are elongated regardless of the

overall shape of the cell. For this reason, tumors containing large

numbers of cells with elongated tumor nuclei may need to be

evaluated manually.

The uPath HER2 Dual ISH image analysis for Breast algorithm

may mischaracterize lymphocytes as a tumor cell. The

pathologist should visually inspect the tissue carefully and, if

possible, avoid regions of tissue where immune cells compose

more than 30% of all cells.

The uPath HER2 Dual ISH image analysis for Breast algorithm has

been trained, developed, and validated on: invasive carcinoma.

The uPath HER2 Dual ISH image analysis for Breast algorithm has

not been tested, or its safety and effectiveness validated, when

used with a personal computer (PC) from home.

Limitations

uPath HER2 Dual ISH image analysis for Breast Algorithm Guide 5

Malicious software or unauthorized instrument access can result

in data loss or instrument unavailability.

To avoid infection by malicious software or unauthorized access

and misuse of the instrument, the following recommendations

are essential:

• Do not install or run any other software on the instrument.

• Make sure that other computers and services on the network

are properly secured and protected against malicious software

and unauthorized access. For example, the laboratory

information system (LIS), archiving share, backup share, or

service.

• Customers are responsible for the security of their local area

network, especially in protecting it against malicious software

and attacks. This protection might include measures, such as

a ﬁrewall, to separate the device from uncontrolled networks.

This protection might also include measures that ensure that the

connected network is free of malicious code.

• Restrict physical access to the instrument and all attached IT

infrastructure (computers, cables, network equipment, and so

on).

• Make sure that instrument backup and archive ﬁles are

protected from any unauthorized access and disaster. This list

includes the remote storage location, disaster discovery sites,

and the secure transfer of backup ﬁles.

• If possible, use a ﬁrewall to restrict network trafﬁc.

• USB ﬂash drives can be used for several kinds of backups and

restores. Wrong handling of a USB ﬂash drive may result in data

loss or malfunction of the instrument.

• Use only USB ﬂash drives that are tested and installed by your

local Roche service representative.

• At any one time only one USB device can be in use. Before

inserting a USB ﬂash drive, check that no other USB device is

inserted.

• Before removing a USB ﬂash drive, choose the Eject button in

Windows.

• The default operating system (OS) conﬁguration provided with

the server should not be altered as this has implications on the

hardened OS conﬁgurations.

• To prevent a virus from infecting the uPath RUO enterprise

software, use the USB ﬂash drive exclusively on the instrument.

Do not store other data on this USB ﬂash drive.

Data Security

6 uPath HER2 Dual ISH image analysis for Breast Algorithm Guide

Workﬂow for using the uPath HER2 Dual ISH image analysis for Breast algorithm

Materials needed

• uPath RUO enterprise software

• uPath HER2 Dual ISH image analysis for Breast algorithm

• Breast tissue slides stained with the VENTANA HER2 Dual ISH DNA Probe Cocktail (using the VENTANA Silver ISH DNP and

VENTANA Red ISH DIG Detection Kits) stained on the BenchMark ULTRA instrument

• VENTANA DP 200 slide scanner

Workﬂow

1. The breast tissue on a glass slide is stained with the VENTANA HER2 Dual ISH DNA Probe Cocktail using a BenchMark ULTRA

instrument.

2. Image acquisition (whole slide scanning) is performed with the VENTANA DP 200 slide scanner at 40x magniﬁcation at one z-plane.

3. Once digital images are acquired, these images are transferred from the computer associated with the VENTANA DP 200 slide

scanner to the image management system (IMS) on a centralized server.

4. Following transfer to the server, a case will be created in the uPath RUO enterprise software. Case creation can occur automatically

through communication with the laboratory information system (LIS) using identiﬁcation information (i.e., tissue type and assay)

contained in the barcode label of the glass slide or entered manually into the uPath RUO enterprise software (refer to uPath RUO

enterprise software User Guide (1019288EN)).

5. If the uPath HER2 Dual ISH image analysis for Breast algorithm is installed (must be installed on a separate server from the uPath

RUO enterprise software and the IMS) and a 40x image is accessioned with the appropriate stain and tissue type, the uPath RUO

enterprise software then automatically triggers Whole Slide Analysis (WSA).

6. WSA automatically analyzes the entire scanned image.

7. Once WSA is completed, the pathologist is notiﬁed within the software that “analysis is complete.”

8. When the pathologist accesses the image they will have the ability to toggle a heat map that will highlight areas suitable for scoring

based on the presence of HER2 signals. (need heat map values)

9. Once a suitable area is identiﬁed the pathologist can select an ROI using the Quick ROI tool, which will automatically generate a

0.4 mm x 0.4 mm square to score 20 representative cells selected by the uPath HER2 Dual ISH image analysis for Breast algorithm

based on: cell size, number of HER2 and Chr17 signals and segmentation conﬁdence. If the pathologist does not agree with the cell

selection, they can remove and add cells by selecting cells on the ROI Details panel or within the ROI. The pathologist can also delete

the initial ROI and replace it with a new ROI on a more suitable region of tissue.

10. If the HER2/Chr17 ratio is between 1.8 and 2.2 the pathologist will be able to use the Quick ROI to draw a second ROI and analyze a

second set of 20 cells according to the the VENTANA HER2 Dual ISH DNA Probe Cocktail method sheet. However, if the score for the

ﬁrst ROI is >1.8 or <2.2 the quick ROI tool will be disabled and the HER2 status will be based only on the ﬁrst ROI. If the pathologist

does not agree with the score, they can manually override the algorithm.

Staining

• Tissue preparation and staining should follow the recommendations provided in the VENTANA HER2 Dual ISH DNA Probe Cocktail

method sheet.

• All proper controls should be reviewed and slides should be re-stained if the staining does not meet the guidelines outlined in the

VENTANA HER2 Dual ISH DNA Probe Cocktail method sheet.

• The uPath HER2 Dual ISH image analysis for Breast algorithm requires use of the VENTANA HER2 Dual ISH DNA Probe Cocktail,

and any additional material or supplies listed in the VENTANA HER2 Dual ISH DNA Probe Cocktail method sheet, to stain tissues

prior to analysis. The VENTANA HER2 Dual ISH DNA Probe Cocktail determines HER2 gene status in FFPE breast tissue stained

with the VENTANA Silver ISH DNP and VENTANA Red ISH DIG Detection Kits on a BenchMark ULTRA instrument.

uPath HER2 Dual ISH image analysis for Breast Algorithm Guide 7

Image Capture

The VENTANA DP 200 slide scanner is required for scanning of the slides. Images are required to be scanned at 40x magniﬁcation. It is

recommended that the tissue be free of folds and ink. If large sections of the image are out of focus, it is recommended that the slides

be rescanned. For further information on scanning, please refer to the VENTANA DP 200 slide scanner User Guide (PN 1016906EN).

General Navigation: uPath enterprise software

The uPath RUO enterprise software is meant to be customizable to individual and site needs including but not limited to report

conﬁguration and user interface. This Algorithm Guide will focus on the tools necessary for using the uPath HER2 Dual ISH image

analysis for Breast algorithm only. For further information on the uPath RUO enterprise software please refer to the uPath RUO

enterprise software User Guide.

8 uPath HER2 Dual ISH image analysis for Breast Algorithm Guide

Pathologist Workﬂow

Opening a Case

Images of Breast tissue stained with VENTANA HER2 Dual ISH DNA Probe Cocktail can be accessed by double-clicking on a case

(Figure 1).

Or selecting a case and pressing the viewer tab within the uPath RUO enterprise software (Figure 2).

A screen with all images associated with a case will appear (Figure 3).

Figure 3

Figure 2

Figure 1

uPath HER2 Dual ISH image analysis for Breast Algorithm Guide 9

Once a glass slide stained with the VENTANA HER2 Dual ISH DNA Probe Cocktail is scanned on a VENTANA DP 200 slide scanner

at 40x, the image is imported into the uPath RUO enterprise software and associated with a case. The uPath HER2 Dual ISH image

analysis for Breast algorithm will automatically trigger WSA. Times to complete the WSA precomputing step depend on server

speciﬁcations, image sizes and number of images in the queue. When displayed, “waiting to start auto-analysis” speciﬁes the images

are in queue and yet to be analyzed and “analyzing” is used when WSA is being performed (Figures 4 and 5).

Once the image is completely analyzed via WSA in the uPath RUO enterprise software, “analysis successful” will be displayed

underneath the slide image within the viewer in the uPath RUO enterprise software (Figure 6). Images cannot be scored prior to

successful WSA completion.

Heatmap

The WSA precomputing step allows for the uPath HER2 Dual ISH for Breast algorithm to generate a Heatmap available in the Slide

Navigator panel (Figure 7). The Heatmap can be toggled on and off using the Hide Heatmap button and Show Heatmap button on the

lower right hand corner of the Slide Navigator panel (Figure 7). The Heatmap is intended to identify areas with increased HER2 signals,

deemed ideal for scoring based on the in the VENTANA HER2 Dual ISH DNA Probe Cocktail Interpretation Guide. It ranges from

yellow (low HER2/Chr17 ratio) to red (high HER2/Chr17 ratio). No overlay will appear for areas (and cases) where the algorithm deems

there not to be ampliﬁcation. Despite what the Heatmap shows, it is necessary for the user to review the image as would be done in a

manual read.

Figure 5Figure 4

Figure 6

Figure 7

10 uPath HER2 Dual ISH image analysis for Breast Algorithm Guide

Scoreable Cells Overlay

An overlay outlining scoreable cells in red will appear when the image is at 20x magniﬁcation or greater (Figure 8). The uPath HER2

Dual ISH image analysis for Breast algorithm selects scoreable cells based on the following criteria: size, presence of at least one

HER2 and one Chr17 signal, and segmentation conﬁdence (cell is clearly separated from neighboring cells). If any of the three

criteria are not met, the cell will be deemed not scoreable and no overlay will appear. The overlay identiﬁes scorable tumor cells

allowing the user to determine areas with a sufﬁcient number of scorable tumor cells.

Once the image has been reviewed and an area of interest is identiﬁed (area of tumor cells and appropriate staining internal

controls cells), use the Quick ROI tool to initiate analysis (Figure 9).

Figure 8

Figure 9

uPath HER2 Dual ISH image analysis for Breast Algorithm Guide 11

Upon selecting the Quick ROI tool, the following will occur:

1. A 0.4 mm x 0.4 mm ROI central to the viewer appears (Figure 10).

2. The overlay of all scoreable cells will disappear within the ROI.

3. A bolded red overlay will appear, highlighting the 20 representative cells as selected by the algorithm within this ROI (Figure 10).

4. The HER2 count and Chr17 count for each cell will appear within the ROI Details Flipout Panel (Figure 11).

5. Ratio of HER2/Chr17, Total HER2 Signals and Total Chr17 Signals will be calculated and displayed within the ROI Details Flipout Panel

(Figure 14). The ROI Details can be toggled by pressing the hide/retrieval Button (Figures 11 and 12).

Figure 12Figure 10 Figure 11

12 uPath HER2 Dual ISH image analysis for Breast Algorithm Guide

Pathologists can select any one of the 20 cells with the red overlay to display the corresponding number of HER2 and Chr17 signals

within that cell as identiﬁed by the uPath HER2 Dual ISH image analysis for Breast algorithm. The cell will now be outlined in green,

and the corresponding count is highlighted in blue in the ROI Details Flipout Panel (Figure 13). Conversely, selecting one of the 20

cells from the Cell Count list found on the ROI Details Flipout panel will highlight the corresponding cell in green.

Figure 13

uPath HER2 Dual ISH image analysis for Breast Algorithm Guide 13

Quick ROI: Deletion

If a selected ROI is not optimal, it can be deleted. Select the Quick ROI by clicking anywhere on the ROI on the image and then

select the Delete icon within the Slide Panel (Figure 14) or within the slide image near the ROI (Figure 15). A conﬁrmation window

will appear. Select Conﬁrm to delete the selected ROI. Select Cancel to retain the ROI.

Quick ROI: Replacing Cells

If a selected cell(s) identiﬁed by the uPath HER2 Dual ISH image analysis for Breast algorithm does not meet the guidelines outlined

in the VENTANA HER2 Dual ISH DNA Probe Cocktail method sheet, select the cell by clicking on the center of the cell or selecting

the cell from Cell Count list. Delete the cell by selecting the Delete icon within the ROI Details ﬂipout panel above the Conﬁrm

button (Figure 16). Upon selection of the Delete icon, the cell HER2 and Chr17 count will be removed from the cell list and the

corresponding highlighted cell will be removed from the ROI (Row 8 in Figure 17). The Add icon will now be activated (Figure 16).

Select the Add button and draw around the new cell that should be scored.

Figure 15Figure 14

Figure 16 Figure 17

14 uPath HER2 Dual ISH image analysis for Breast Algorithm Guide

Internal Control Valid Conﬁrmation

The uPath HER2 Dual ISH image analysis for Breast algorithm requires the user to conﬁrm that valid internal controls are present

with the ROI (Figure 18). The deﬁnition of valid internal control cells can be found within the VENTANA HER2 Dual ISH DNA Probe

Cocktail Interpretation Guide (PN 1018386EN). If the user deems that the internal control within the ROI does not meet the criteria

deﬁned by the VENTANA HER2 Dual ISH DNA Probe Cocktail assay, the ROI should be deleted and a new Quick ROI selected. The

default selection for Internal control with the ROI will be Not Valid (Figure 19).

NOTE: In order to proceed to the Conﬁrmation step, 20 cells must be scored and the Internal Controls must be marked

as Valid within the ROI Details Panel.

ROI Conﬁrmation

If all the selected cells are acceptable and the user marks Internal Controls as Valid, the HER2/Chr17 Ratio can be conﬁrmed using the

Conﬁrm button (Figure 20).

Figure 18

Figure 19

Figure 20

uPath HER2 Dual ISH image analysis for Breast Algorithm Guide 15

If the HER2/Chr17 Ratio is greater than 2.2 or less than 1.8, the Slide Score Flipout will appear (Figure 21); an additional ROI is not

needed to sign out the case. If the score of the ROI is between 1.8 and 2.2, a second Quick ROI is required prior to signing out the case.

Once the second ROI is selected and conﬁrmed, the Slide Score Flipout will appear. The second Quick ROI cannot overlap with the

original ROI.

Within the Slide Score Flipout, the user has the ability to change Heterogeneity, HER2 Clusters and Chr17 Clusters. The default value

for all three is “No” and must be updated by the pathologist accordingly. In Figure 22, the HER2 Clusters have been changed to YES.

The Pathologist must select Reviewed to activate the Conﬁrm button. Upon conﬁrming, the Slide Score Flipout will be hidden.

The Slide Score can be toggled by pressing the hide/retrieval Button (Figures 23). The same button (Figure 23) can be then used to

show the ROI Details Panel.

Figure 21

Figure 22

Figure 23

16 uPath HER2 Dual ISH image analysis for Breast Algorithm Guide

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Roche uPath RUO User manual

Brand: Roche

Model: uPath RUO

Type: User manual

Using the uPath RUO enterprise software, the pathologist can:

View digital images at various magnifications.
Add annotations.
Make tissue section measurements.
Perform image analysis.
Generate reports.

Download PDF

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Roche uPath RUO User manual

Roche uPath RUO User manual

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