TMA

HALO 3.0
TMA
Step-by-Step guide
May 2019
 TMA

Copyright © 2019 Indica Labs Inc.

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 TMA

This guide will show how to use the TMA module

After clicking the HALO icon to open you will be presented with the following. Please study the key features of the interface.

TMA tab: Tissue Micro Array caters for analysis of

Studies tab: This tab is for organization of images and
1 2 slides containing multiple tissue or cellular cores. Only
batch analysis mode is access from here.
available if purchased.
Annotations tab: Annotations allow you to identify Help Menu: Information about software version, help
3 4
specific regions to include or exclude from analysis. guides, and reference popups.
Classifiers tab: Classifiers instruct the algorithm to
Study Sort: Sort studies in ascending or descending
5 6 delineate tissue based on its type [tumor, stroma
order by creation date or alphabetical order.
etc.]. Only available if purchased.
Analysis tab: This tab is used to fine tune the analysis
7 8 Study Search: Free text search for studies.
settings and load algorithms.
Results tab: Results summary and object results are Refresh: Refresh HALO (if part of a group license or
9 10
located here. Halo Link installation).
11 Image Tab: Open image tab (file name if unchanged) 12 Slide Label: Contains information about the sample
Main image viewer: The main working area, displays Thumbnail: Used to rapidly navigate around the
13 14
images after opening. image, may be reduced in size or removed.
Snapshot: Take a snapshot of the current viewing Ruler: Measure the distance between two points on
15 16
window. the image screen.
17 Ellipse: Draw an elliptical annotation. 18 Rectangle: Draw a rectangular annotation.
Flood-Fill: Automated tool to identify tissue boundaries Magnetic Pen: Automatically outline tissues or regions
19 20
using RBG pixel intensity and R-value. which have a well-defined edge.
Exclusion Pen: construct annotation regions to be Pen Tool: Draw new polygon annotations or modify
21 22
excluded from analysis. existing ones by drawing lines.
Brush Tool: Draw new polygon annotations or modify
23 24 Pan tool: This allows you to pan around the image.
existing ones using brushstrokes.
Toggle Mark-up: Toggles between the analysis markup
25 26 Zoom buttons: Jump to a specified zoom.
overlay and classifier overlay. Can hide both overlays.
Image Actions pane: Images are organized and
27 28 Studies pane: Study folders are organized here
analyzed here.

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The TMA add-on module provides a productivity-enhancing workflow for tissue microarray analysis by enabling
automated, high-throughput, segmentation and batch analysis of whole slide TMA images. The add-on can be
used in conjunction with any of our cell-based analysis modules for brightfield or fluorescence, as well as our
tissue classifier add-on for automated tumor detection.

The workflow for this module is broken down into sections in this user guide:

Contents
Segment the TMA .................................................................................................................................................................. 3
Step 1. Choose the image to segment .................................................................................................... 3
Step 2. Select a block ................................................................................................................................ 3
Step 3. Block designer (if necessary) ....................................................................................................... 4
Step 3a. Design the block ..................................................................................................................... 4
Step 3b. Modify the block .................................................................................................................... 5
Step 3c. Label the TMA cores manually (optional) ............................................................................. 5
Step 3d. Save the block ........................................................................................................................ 6
Step 3e. Select a block .......................................................................................................................... 6
Step 4. Introduction to the alignment window....................................................................................... 7
Step 5. Align the TMA block ..................................................................................................................... 7
Step 6. Set the invalid threshold & save.................................................................................................. 8
Working in the TMA Tab ....................................................................................................................................................... 8
Step 7. Please study the TMA tab carefully..............................................Error! Bookmark not defined.
Step 8. Analyze the cores ......................................................................................................................... 9
Step 9. Select analysis settings ................................................................................................................ 9
Step 10. View the TMA heatmap ...........................................................................................................10
Step 11. Advanced export (optional) .....................................................................................................10
Step 11a. Select cores.........................................................................................................................11
Step 11b. Select desired outputs .......................................................................................................11
Step 11c. Select analysis jobs .............................................................................................................11
Step11d. Open the folder to view exported data .............................................................................11
Additional options ....................................................................................................................................................... 12
Reopening a TMA .......................................................................................Error! Bookmark not defined.
Set a scoring template.............................................................................................................................12
View individual core analysis results ......................................................................................................13
Simple export ...........................................................................................................................................13
Creating a TMA Block with the Import Wizard ............................................................................................................ 14
Step 1. Open the Import Wizard ............................................................................................................14
Step 2. Select the spreadsheet layout: Geometric or Tabular .............................................................14
Step 3. Copy the spreadsheet data onto the clipboard (outside of HALO) .........................................15
Step 4. Import the data into HALO ........................................................................................................15
Step 5. Configure Core Identifier #1 of 2 (optional) .............................................................................16
Step 6. Configure Core Identifier #2 of 2 (optional) .............................................................................16
Step 7. Finish the Wizard .......................................................................................................................17

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Segment the TMA

Step 1. Choose the image to segment

〉 Navigate back to the Studies tab.

〉 Locate the image in the Image pane and left click to select. Right click on the
image name or thumbnail to reveal the image actions options list.
〉 Select Segment TMA... to open the Select Block window.

Analysis tip: Alternatively, click on the Image Actions drop down menu
to reveal these options. Make sure you have highlighted the correct image

Step 2. Select a block

〉 In the Select Block window, chose a pre-existing
block from the list and click the Select button to
close the Select Block window and open the TMA
Segmentation Alignment window.
〉 If the block is already available skip to step 4.

〉 Alternatively, click the New Block button at the

bottom left corner of the window to open the TMA
Block Designer window.

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Step 3. Block designer (if necessary)

Analysis
tip: Use the
Import Wizard
instead of
manual core
input to
improve
throughput. See
Page 15 for
details.

Step 3a. Design the block

〉 Starting in the
top left corner of
the grid hold the
left mouse button
and drag to
encompass a
number of cores
that represent
your TMA block
layout.
〉 Right click on
the region
highlighted in
blue to reveal a
drop-down menu
and select Add.
Empty squares in
the grid will now
be populated
with grey circles
indicating a core
is present.

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Step 3b. Modify the block

〉 Using the options Add,
Clear and Move recreate
the layout of your TMA
in the block designer
dialogue box. Ultimately,
the layout in the Block
Designer window should
match the layout of the
TMA on the slide.

Step 3c. Label the TMA cores manually (optional)

〉 Click on a core to
select it. When selected
it will be shown in blue.
It is possible to select
multiple cores if these
are to be labeled
identically.
〉 In the Core Identity
sub section input a label
for the core in the text
field adjacent to Id #1
and Id #2.
〉 Repeat this process for
all cores of the TMA.

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Step 3d. Save the block

〉 Select the Save
Block button to bring
up the Save Block
dialogue box.
〉 In the Save Block
dialogue box input a
name for the block
template.
〉 Click on the Save
button to close the
TMA block designer
and return to the
Select Block window.

Step 3e. Select a block

〉 In the Select Block window, chose the block
from the list.
〉 Click on the Select button to close the Select
Block window and open the TMA
Segmentation Alignment window.

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Step 4. Introduction to the alignment window

〉 The TMA Segmentation
Alignment window shows
the TMA image file
overplayed with the TMA
block template (red outline).
〉 The Mirror buttons will
mirror the TMA block
template (red outline)
across a single line of
symmetry.
〉 The Rotate button will
rotate the TMA block
template (red outline)
around the image focal
point in 90-degree
clockwise increments.
〉 The Spot Operation sub
section options are used to
adjust the size and position
of spots and invalidate cores
that are not suitable for
analysis.

Analysis tip: The cores in the template grid are elastically tied together. Place the cores on the corners before adjusting the
cores in the center of the TMA.

Step 5. Align the TMA block

〉 Left click and drag the
template cores to align the
template grid with the
underlying slide image.
〉 Set the size of the spot over
lay by clicking and dragging
the Size slider to ensure all
tissue is within the spot
overlay. Or type in a specific
value.
〉 Once loosely aligned, use
the Auto Center button to
center the cores.

Analysis tip: The cores are cropped into square boxes with the tissue core centered. If the template box overlaps neighboring
cores, the tissue from these neighboring cores will be included in the cropped image and in turn any image analysis results. Ensure the
square boxes do not overlap neighboring tissue cores.

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Step 6. Set the invalid threshold & save

〉 Adjust the Invalid
Threshold bar slider
to adjust the OD
density level for a
core to be considered
for analysis. Cores
determined to be
invalid are
demarcated by a red
X through the spot
overlay.
〉 Cores can also be
individually marked
as valid/invalid after
segmentation.
〉 Click on the Save
button to close the
TMA segmentation
Alignment window
and complete the
TMA segmentation.
Working in the TMA Tab
Step 7. Please study the TMA tab carefully.

〉 The TMA is now displayed in the TMA tab.

Double clicking on each core will cause it to be
loaded as an image in the main image viewer.
〉-When a core is open in the main image viewer
use the directional keys to navigate through the
TMA to study each core in turn. Use the space
bar to progress through the cores. Note: If you
adjust the magnification of the image the
directional keys will only function within the
image and not transition through the TMA.
〉 The Block Actions drop down list will allow you
to create New…, Load… (if pre-existing), Edit… or
Delete…. After analysis the Block Actions drop
down list will allow the Export of results as
Summary data, Object data or Advanced… which
links the TMA results to the Export Manger tool.
〉 The Slide Actions drop down list will allow you
to Segment TMA… to adjust your segmentation,
Set Stain…, Set Scoring Template, Export results,
create a Heat Map… after analysis to visualize
the characteristics of each core for each core,
and delete results or create spatial plots with
Spot Results….
〉 The Analyze drop down list will allow you to
analyze Selected Spot(s), All Spots (current
slide), or All Spots (all slides).

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Step 8. Analyze the cores

〉 Select the Analyze drop down menu and click
on Selected Spot(s), All Spots (current slide), or
All Spots (all slides).. Selected spots are shown in
blue on the TMA image.
〉 Saved settings are required to run a batch of
TMA spots.

Step 9. Select analysis settings

〉 The Submit Analysis Jobs window will open.
Ensure Analysis Settings is selected and the
chosen algorithm is highlighted.
〉 In the Region of Analysis sub section select
either Entire Image or Annotation Layers to
analyze.
〉 Check the box labelled Skip Invalid TMA
Spot(s) to remove invalidated cores from the
analysis.
〉 Click the Analyze button.

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Step 10. View the TMA heatmap

〉 Click on the Slide Actions drop down-menu and select Heat Map….

〉 In the TMA Heat Map window, select the analysis of interest from the first drop-down list and the second drop-down list to
view the cores with a heat map overlay to identify which cores have the highest and lowest values for the selected result.

Step 11. Advanced export (optional)

〉 Select the Slide Actions drop down menu.

〉 Select Export.

〉 Select Advanced to link to the Export Manager.

〉 If Advanced is selected, please proceed to the next

step.

Analysis tip: All of the block data can be

exported in one step if this step is followed from the
Block Actions drop down menu.

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Step 11a. Select cores

〉 Select Yes to export data for all the spots on the
slide.
〉 Select No to export data for only the selected
spots (selected spots are shown in blue).

Step 11b. Select desired outputs

〉 In the Images sub-section check
boxes to export image Labels, Analysis
Markup image, Classifier Markup
image. Check boxes to export
Thumbnail image, Mid-sized images or
Actual sized images. Enter values to set
the pixel dimension of the Mid-Sized
image if this option is selected. Enter
values to set transparency of classifier
mark up image.
〉 In the Filter Results by Index sub-
section use the drop down menu to
select 1st, LAST or ALL results for
export
〉 In the Results Data sub-section check
boxes to export Summary Data (.csv
files), Object Data (.csv files) or
generate Analysis Report (.docx files).
〉 In the Other sub-section check boxes
to export Annotations (if present),
Analysis Settings in the form of an
Analysis protocol and Classifiers in the
form of a non-editable classifier file
〉 In the Folders sub-section input the
file path in which HALO will create an
archive.
〉 Click the Generate button.

Step 11c. Select analysis jobs

〉 Select the Export All or Export Selected ONLY options to determine
which data is exported
〉 HALO will generate the archive. The Jobs Processed % bar will indicate
how many TMA cores have had data exported

Step11d. Open the folder to view exported data

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Additional options
Reopening a TMA

〉 Navigate to the TMA tab.

〉 From the Block Actions drop-down list,

select Load….
〉 In the Select Block window, chose the block
from the list.
〉 Click on the Select button to close the Select
Block window and open the selected block.

Set a scoring template

〉 From the Slide Actions

drop-down list, select Set
Scoring Template….
〉 In the Select Score
Template window, select
an existing template or
create a new template.
Each template field can be
configured as text, list,
decimal, or integer.
〉 Click the Select button.

〉 Template values can now

be updated on a core by
core basis in the TMA tab.
These values will be
exported along with any
analysis data.

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View individual core analysis results

〉 Once the analyzed core is open in the viewing
window, navigate to the Results tab.

Simple export 〉 Select the Block Actions or Slide Actions drop

down menu.
〉 Select Export.

〉 Select Summary Data or Object Data.

〉 In the next step, select which analysis results to

export and where to save the exported csv file.

Analysis tip: All of the block data can

be exported in one step if this step is
followed from the Block Actions drop down
menu.

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Creating a TMA Block with the Import Wizard

Step 1. Open the Import Wizard

〉 Click the Import
Wizard… button on the
TMA Block Designer
popup window to open
the TMA Import Wizard.
〉 This series of steps
replaces Step 3 in this
user guide.

〉 The wizard will guide

you through the following
steps for the layout style
selected.

Note: The Import Wizard uses data imported from a spreadsheet format outside of HALO.

Step 2. Select the spreadsheet layout: Geometric or Tabular

〉 Specify the layout style of the spreadsheet

〉 Geometric: cores are represented by their

position in the spreadsheet

〉 Tabular: cores are represented by one row of

information in the spreadsheet

Geometric Example Tabular Example

CTRL1 BC4257-1 BC4257-2 BC4257-3 BC4257-4 BC4257-5 position sex age organ grade stage type
A1 F 64 Lung 1I Malignant
BC4250-1 BC4250-2 BC4250-3 BC4250-4 BC4250-5 A2 F 64 Lung 1I Malignant
BC4300-1 BC4300-2 BC4300-3 BC4300-4 BC4300-5 A3 F 56 Lung 1I Malignant
A4 F 56 Lung 1I Malignant
CTRL2 BC4225-1 BC4225-2 BC4225-3 BC4225-4 BC4225-5
A5 M 70 Lung 1I Malignant
BC4237-1 BC4237-2 BC4237-3 BC4237-4 BC4237-5 A6 M 70 Lung 1I Malignant
BC4352-1 BC4352-2 BC4352-3 BC4352-4 BC4352-5 A7 M 65 Lung 1I Malignant
A8 M 65 Lung 1--2 I Malignant
BC4111-1 BC4111-2 BC4111-3 BC4111-4 BC4111-5 A9 M 72 Lung 2I Malignant
BC4125-1 BC4125-2 BC4125-3 BC4125-4 BC4125-5 … … … … … … …

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If Geometric Layout, specify the number of cells per core.

Step 3. Copy the spreadsheet data onto the clipboard (outside of HALO)

〉 Select all the cells that contain data and 〉 Alternatively, use the CTRL + C 〉 The data is now copied to the
right click to bring up a drop-down menu function to copy the selected cells. clipboard and may be imported into
to copy the data. the HALO.

Step 4. Import the data into HALO

〉 Click the Next > button to import the data
into HALO.

〉 In the TMA Block Designer window, grey

circles indicating the core positions identified
by the wizard will appear after this step.

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If Tabular Layout, specify the layout and position.

〉 Specify the number of header rows so that the
Import Wizard will not attempt to count these
header rows as cores.
〉 Indicate how the core position is identified in the
spreadsheet: position identified by one column or
two columns.
〉 Select the column(s) containing the core
positions from the dropdown menu(s).

Step 5. Configure Core Identifier #1 of 2 (optional)

〉 Configure the first of two core identifiers in this
step.

〉 These identifiers will be tied to each individual

core in HALO.

〉 Create the identifier by adding a single piece of

information or create a string of information by
combining data from multiple columns or static
text.
〉 This step may be skipped.

Step 6. Configure Core Identifier #2 of 2 (optional)

〉 Configure the second of two core identifiers in
this step.

〉 These identifiers will be tied to each individual

core in HALO.

〉 Create the identifier by adding a single piece of

information or create a string of information by
combining data from multiple columns or static text.

〉 This step may be skipped.

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Step 7. Finish the Wizard

〉 Click Finish to exit the Wizard.

〉 The TMA block designer grid can be modified

manually at this point.
〉 The core identifiers can be viewed by clicking on
individual cores and modified manually.
〉 Return to Step 4 to save the block template.

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