a step-by-step standard operating procedure manual for ...10.1007/s10334-015-0510... · a...

A step-by-step standard operating procedure manual for segmenting

magnetic resonance images of the lower leg

Supplementary Electronic Material

Karampatos, S., Papaioannou, A., Beattie, K.A., Maly, M., Chan, A., Adachi, J.D., Pritchard, J.M. A reliable segmentation methodology for assessing intramuscular adipose tissue and other soft-tissue compartments of the lower leg using 1 Tesla MRI images and sliceOmaticTM software. Prepared for MAGMA.

Corresponding author: Janet Pritchard, PhD. GERAS Centre, 88 Maplewood Ave. Hamilton ON, L8M1W9. Email: [email protected]

2 Segmentation Manual

Table of Contents 1.0 Introduction ............................................................................................................ 3

2.0 Identifying the Compartments of the Lower Leg ................................................ 3 2.1 Segmentation Definitions 5

3.0 Loading and Preparing Images ............................................................................ 5

4.0 Preparing For Segmentation ................................................................................ 7 4.1 Segmenting Subcutaneous Adipose Tissue Using Morpho and Edit 8 4.2 Segmenting the Tibia and Fibula Using Morpho and Edit 10 4.3 Segmenting the InterMAT Using Morpho and Edit 10 4.4 Segmenting the Muscle Using Morpho 11 4.5 Segmenting the IntraMAT Using Region Growing 11

5.0 Obtaining Measurements .................................................................................... 13

6.0 Key Board Shortcuts ........................................................................................... 13

7.0 Tips to Keep Your Segmentations Consistent ................................................. 13

8.0 Other Applications .............................................................................................. 14

1.0 Introduction Slice-O-MaticTM is a medical imaging software that is used to analyze computed

tomography (CT) and magnetic resonance (MR) images. There are many different uses of this software, but the purpose of this manual is to assist new users in segmenting the different compartments of the lower leg to obtain measurements of cross-sectional area. This manual was created using version 4.3 revsion 7c, but this protocol can also be used for newer versions of the software. For more information about sliceOmaticTM, please visit: www.tomovision.com/products/sliceomatic.htm Or contact TomoVision at (819) 843-1999 or e-mail: [email protected].

System requirements for downloading and using sliceOmaticTM for this manual: • A computer running Windows • License Dongle • sliceOmaticTM software available on CD or zip file from www.tomovision.com • MRI images in DICOM format

Note: sliceOmaticTM can be downloaded for free from the website. Users can practice segmenting images, but without a license dongle, segmentations can’t be computed or saved. sliceOmaticTM is not yet available for Mac computers. sliceOmaticTM Terminology: Segmenting- separating the compartments of the lower leg so that you can obtain a cross-sectional area of each compartment Tag- a mask that is applied to the image to seperate compartments of the lower leg.

2.0 Identifying the Compartments of the Lower Leg Identifying the different compartments of the lower leg is essential prior to

segmenting images because this will form the foundation of your segmentation definitions. For example, in the case of segmenting intermuscular adipose tissue (InterMAT), we used various muscle bellies as landmarks to locate and tag adipose tissue belonging to this compartment. In addition, although this manual does not guide you through segmenting each individual muscle, it may be relevant to your research to segment the individual muscles or segment the anterior versus posterior muscles. Therefore, knowing where each muscle is located is important in order to keep your segmentations consistent. Depending on the MRI sequence that you use to acquire your images, you may find that the muscle borders may be easier or harder to identify.


Figure 1. Typical axial/transverse MRI images of the lower leg acquired with a 2-dimensional fast spin echo (FSE) sequence. The total muscle cross-secitonal area = sum of the cross-sectional areas of each muscle. Some muscle bellies are grouped together, as borders can be difficult to identify. Abbreviations: TA: tibialis anterior (tag includes extensor hallucis longus and extensor digitorium longus and fibularis tertius), FLB: fibularis longus and brevis (tag includes longus and brevis), SOL: soleus, LG: lateral gastrocnemius, MG: medial gastrocnemius, FHL: flexor hallucis longus, TP: tibialis posterior (includes flexor digitorium longus), tib: tibia bone, fib: fibula bone Labeling completed with: Agur, A.M.R & Dalley, A.F. (2005). Grant’s Atlas of Anatomy. Philadelphia,PA: Lippincott Williams & Wilkins.

2.1 Segmentation Definitions

1. Subcutaneous Adipose Tissue

Any tissue outside the fascia lata, including the vessels and skin

2. Tibia and Fibula

Cortical bone, including marrow cavity

Note: If you are interested in measuring cortical bone cross-sectional area, you may wish to create another category for bone marrow

3. Total Muscle Non-adipose tissue beneath the fascia lata, excluding the bone

4. Intramuscular Adipose Tissue

(IntraMAT)

Adipose tissue within the muscle bellies of the lower leg, including the deep vessels and nerves

5. Intermuscular Adipose Tissue

(InterMAT)

Adipose tissue beneath the fascia lata and between: 1. medial gastrocnemius and soleus 2. medial gastrocnemius and lateral gastrocnemius 3. lateral gastrocnemius and soleus 4. soleus and fibularis longus and brevis

3.0 Loading and Preparing Images The following steps will help you understand how to segment your MRI images.

Steps Explanation Image 1. Click File

2. File Open

3. Highlight all dicom images you wish to segment

4. Open

Hold down ‘shift’ key to highlight multiple files.


5. Click Tools

6. Image Info

7. Change the scale to

1.50, or until the image fills the screen

8. Display all images by clicking “ALL”

Using the scale function, you can increase the size of the MRI image for better segmentation performance as you are better able to see the compartment boundaries.

Note: Keep scaling consistent for all

participants

9. Click Tools

10. Gamma Correction

11. Slide bar to the point where the grey box turns one shade of grey

This calibrates the grey scale for all images. It is important because working on different monitors may make images appear lighter or darker.

4.0 Preparing For Segmentation Steps Explanation Image

1. Click Modes

2. Morpho

This will allow you to begin segmenting your images using mathematical morphology (Morpho).

3. Right click on ‘1’ to

change label to subQ (subcutaneous adipose tissue)

4. Press Enter

5. Repeat for all tags: 2) Tibia 3) Fibula 4) Muscle 5) Intermuscular Adipose Tissue (InterMAT) (inter) 6) Intramuscular Adipose Tissue (IntraMAT) (intra)

This will help you remember which tag colour corresponds to each compartment. You can save these names using the "Save as Script " option from the file menu. You can then drag & drop this script on the program to re-name the buttons, or use the "Startup Script File" option from the configuration menu to have the script loaded each time you start the program. When saving the script, create button names before you loading the images. The syntax in the script to rename a tag is: Tag: x label yyy Where: x is the tag number and yyy is the new label.

Note: if you wish to obtain cortical bone

cross-sectional area, you will need to create 2 more tags for tibia bone marrow and fibula

bone marrow


4.1 Segmenting Subcutaneous Adipose Tissue Using Morpho and Edit

Steps Explanation Image 1. Click the subQ

button

2. Click Thin

3. Click the Watershed #1 box

This allows you select your tag for subcutaneous adipose tissue. Thin means that the watershed lines will be thin, not thick, which makes it easier to distinguish between compartments while segmenting. Watershed #1 is the most detailed algorithm and is recommended for this protocol.

4. Select your brush size

5. Tag the subcutaneous adipose tissue by holding down the left mouse button

For the subcutaneous adipose tissue, choose the smallest or 2nd smallest paintbrush. If you make a mistake and go outside of the subcutaneous border, use the right mouse button to remove the tag

6. Click Modes

7. Edit and Tools

Use the ‘none’ tag to remove the red pixels outside of the subcutaneous border. Move your cursor around the subcutaneous region and edit so that the subcutaneous tag is not inside the muscle or outside the skin.

Note: it is ideal to compare your tagged

image with the original. Press F1 to show the original image, and press F2 to show the

segmented image.

8. Click Tools

9. Blow Up

You can use the ‘Blow Up’ function to help you edit the segmentations. This will help you identify the compartment borders, but may increase the length of time it takes to complete the entire segmentation.

10. Click Modes

11. Morpho

12. Propagate Box

13. Click ‘UP’ until the last slice number appears in the box

When satisfied with the segmentation for the subcutaneous fat on the first slice, propagate the work to the subsequent slices. Errors must be corrected after propagating

14. Click Modes

15. Edit Tools

The segmentation on each slice must be reviewed and edited where necessary.

16. Click Tools

17. Tag Lock

Once satisfied with the segmentation, lock the tag for the subQ compartment. If you lock a section, you will not be able to modify the segmentation unless the tag is unlocked. It is ideal to compare your tagged image with the original. Press F1 to show the original image, and press F2 to show the segmented image.


18. Click File

19. Save TAG Files

To ensure your work is being saved properly, save your work after segmenting each compartment.

4.2 Segmenting the Tibia and Fibula Using Morpho and Edit

Repeat Steps 1-19 in Section 4.1 to segment the tibia and fibula.

Note: it is ideal to compare your tagged image with the original. Press F1 to show the original image, and press F2 to show the segmented image.

4.3 Segmenting the InterMAT Using Morpho and Edit Repeat Steps 1-19 in Section 4.1 to segment the InterMAT

4.4 Segmenting the Muscle Using Morpho Repeat Steps 1-19 in Section 4.1 to segment the muscle

The Edit step can be skipped for segmenting the muscle, as the other tagged compartments will be “locked”. Do not tag lock the muscle.

4.5 Segmenting the IntraMAT Using Region Growing Steps Explanation Image

1. Click Modes

2. Region Growing

A histogram will appear on the right panel. Slide the y- axis slide bar on the right side of the histogram box to make the whole histogram visible. The maximum of the histogram should touch the top of the plotting field.


3. Click the intramuscular adipose tissue TAG

4. Click ON beside the lower limit

5. Slide Lower

Limit bar or use the mouse wheel to the point where the slope of the first histogram approaches 0 and begins to plateau

6. Click ON beside Upper Limit and slide bar to the maximum value

For each slice, move your cursor in a consistent motion from left to right and ensure that you cover the entire muscle area to tag the intramuscular adipose tissue. You cannot propagate this, so you will have to repeat region growing for each slice. The purpose of this step is to exclude pixels that are muscle. This means that the region growing will tag the pixels between 49-166 as adipose tissue within the muscle. Note: If the Upper Limit is turned "Off" it is automatically set to the maximum value.

Note: it is very important to be as consistent as possible between slices and participants when setting the lower limit. The more you

change where the lower limit is on the histogram, the greater possibility of variation

in your segmentation technique

7. Select the largest paint brush

8. Hold down the left mouse button and move your cursor over the muscle

Note: it is ideal to compare your tagged

image with the original. Press F1 to show the original image, and press F2 to show the

segmented image.

9. Click File

10. Save TAG Files

5.0 Obtaining Measurements Steps Explanation

1. Click Tools

2. Tag Surface/ Volume

3. Click DB Surfaces

4. Save File as a .txt file

5. Open .txt file in notepad or open directly into a database program (i.e., Microsoft Excel)

This will compute the cross-sectional area of each tagged compartment.

Note: If you would like to open the files with Excel, save in "DB" format. In sliceOmatic 5, the extension of the db file is .csv, a double click on these will open Excel automatically.

6.0 Key Board Shortcuts F1 Grey image (original, untagged) F2 Translucent tag F3 Solid tag Q Watershed computation 1 W Watershed computation 2 E Watershed computation 3 R Watershed computation 4 A Watershed computation is turned invisible S Watershed computation is turned visible R Delete

7.0 Tips to Keep Your Segmentations Consistent 1) Use the same workstation for segmenting your images

Even though there is the calibration tool for image grey-level, working on the same workstation can reduce the chance of variability in segmentation due to monitor contrast or grey-level settings.

2) Always follow this standard operating procedure, or a modified manual that is specific to your images

This will prevent deviations from the protocol and will ensure that you are segmenting each participant using the same methodology.


3) Always return to your segmentation definitions for guidance

This is essential for compartments such as intermuscular adipose tissue (InterMAT). This is a very difficult compartment to tag because the fascia border with the subcutaneous adipose tissue compartment is sometimes difficult to identify. We found that the best way to segment this compartment was to first locate the muscles that are included in the InterMAT definition and then tag the compartment.

4) Prior to sitting down and segmenting your images, review your images to come up with a reasonable definition based on your images

You may find that you want to modify the definitions provided in this manual because your images were acquired with a different sequence, which may make some compartments/borders more or less visible. Or, your participants have a different disease making compartments more or less visible.

5) Practice segmenting images before starting the analysis for a study and have an experienced reviewer ensure that the segmentations are suitable.

We found that having raters practice 100 segmentations was a sufficient training period to familiarize the rater with the software and protocol. A senior investigator should review the practice segmentations for quality assurance and provide feedback to the rater regarding performance.

8.0 Other Applications This manual was designed for users who would like to segment lower leg MR

images. However, many of the principles can be applied to other parts of anatomy. For example, if you have access to axial thigh images, the segmentation steps can be applied, but you must create your own definition of intermuscular adipose tissue (InterMAT). If you have access to MRI images acquired with a different sequence (i..e., IDEAL sequence) you may find that it is easier to differentiate compartments. For example, you may be able to separate the anterior muscles (tibialis anterior, extensor hallucis longus and extensor digitorium longus and fibularis tertius) versus combining these muscles into one tag, as described in this protocol. With higher resolution images, you may also find that you are able to identify the borders surrounding the deep vessels of the leg and therefore segment this compartment using a different tag versus including the deep vessels in the muscle tag or intramuscular adipose tissue (IntraMAT) tag.

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