Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer’s Disease and Normal Elderly

1. Brain-Sizer Component

1.1 Total Intracranial Vault Extraction (TIV-E)

1. Open ITK-SNAP_sb, load T1 Click: File -> Open grayscale image -> Browse -> go to directory, Click -> Image -> Open -> Next -> Finish.
2. Click plus sign next to axial view to enlarge.
3. Turn off (or on) crosshairs with ‘x’ key.
4. Right click and drag mouse upwards to enlarge brain in window until it fits without small box appearing in bottom left corner.
5. Adjust intensity by clicking: Tools -> Image Contrast, then drag middle point up and slightly left until the image brightens to the appropriate level, Close.
6. Load TIV-E overlay by clicking: Segmentation -> Load from image -> Browse -> Select TIVauto -> Open -> Next -> Finish.
7. Begin editing TIVauto…
8. Click Paintbrush tool -> Select round -> Adjust size as necessary.
9. To add in / recapture brain that TIVauto has not accounted for, select ‘Active drawing label’ as ‘Label 1’, and ‘Draw over’ as ‘All Labels’.
10. To recapture colored TIV areas, or CAREFULLY recapture noncolored areas use paintbrush to repaint the TIV mask.
11. To undo a painting brush stroke, use <CTRL+Z> or click ‘Undo’ (on left).
12. Toggle TIVauto on/off by pressing ‘s’ to verify that brain tissue is appropriately captured.
13. To remove/delete TIVauto mask if it over-captures nonbrain tissue right click using “paintbrush tool”.
14. Use paintbrush and left click to repaint the TIVauto mask.
15. Check each and every slice carefully to make sure only brain tissue is Label 1 (green) and all nonbrain tissue is some label other than 1 (or not colored at all).
16. Recapture TIV as appropriate, and delete TIV as appropriate.
17. For superior slices make sure everything beneath the dura is kept to account for CSF.
18. If it is difficult to paint, use the closed polygon tool: Left Click to add points to the polygon and Right Click to close it such that everything contained within the polygon is what is being modified, then click “Accept” at the bottom, or if the tracing is incorrect, click “Delete”. Polygon changes can be undone by clicking undo or <CTRL+z>. See Figure 1.
19. When satisfied with TIV modifications click: Segmentation -> Save as image -> and modify file name ending from “TIVauto” to TIVedit” to indicate that it is ‘Done’, then click ‘Save’ (e.g. <name>_TIVedit).

1.2 Ventricular Reassignment

1. Load the T1_IHC.
2. Adjust the intensity.
3. Turn off the crosshairs (x).
4. Select only the axial image to view by clicking the plus symbol next to the axial window.
5. Zoom in (right click and drag).
6. Load the <name>_seg image over the T1 by selecting segmentation -> Load from image -> Browse -> <name>_seg -> Next -> Finish.
7. Adjust the drawing labels to the appropriate colors, through label editor.
8. Change the colors such that 5 is purple, 7 is magenta, and 3 & 4 are something easily distinguishable from the rest (e.g. Figure 2 shows 3=WM change to blue, and 4=GM change to yellow). Note: colors are arbitrary.
9. Reassign vCSF by using the floodfill tool. See Figure 2.
10. Go up slices through the brain to determine the most superior slice with ventricle and begin there.
11. Click the floodfill tool, Select ‘Active drawing label’ =7 and ‘Draw over’ =5.
12. Toggle back and forth between ‘Floodfilling’ and Drawing Limits by pressing the SPACEBAR. Limits are used to prevent the floodfill from filling certain areas of the ventricle that are considered periventricular black holes or part of white matter hyperintensities.
13. When floodfilling, a green arrow tip is visible, and when ready to draw a limit, a red arrow tip will be visible.
14. To fill, simply left click. Move down a slice, and repeat as necessary. Use limits as required to prevent floodfilling of nonventricle regions.
15. If floodfilling operations are incorrect, simply click ‘Undo”, or reverse the ‘Active drawing label’ and ‘Draw over’ colors.
16. Fill every voxel that connects to ventricle, knowing what not to fill is just as important as knowing what to fill.
17. Continue moving down until the 3^rd ventricle opens into the quadrigeminal cistern and draw a limit at the posterior edge of the quadrigeminal cistern until the posterior commissure separates the third ventricle from the quadrigeminal cistern.
18. A limit is necessary if the posterior commissure is not fully visible and does not create an enclosed space. Once the posterior commissure creates an enclosed space, discontinue relabelling the quadrigeminal cistern.
19. Limits may also be necessary if the anterior commissure does not enclose the 3^rd ventricle.
20. Stop filling the 3^rd ventricle once the cerebral peduncles are clearly visible on T1, and the central canal is round.
21. Limits may also be necessary with the anterior portion of the lateral ventricles around the brainstem, if they appear to connect to the sulcal CSF.
22. Use the T1 as a guide on what to fill and what not to fill for temporal lobe lateral ventricles (Toggle segmentation on and off with ‘s’ key).
23. When finished, save the segmentation as ‘<name>_seg_vcsf’ by clicking: Segmentation -> Save as image- > and then add _vcsf after <name>_seg -> Save.

1.3 Removal of Brain Stem, Cerebellum, and Subtentorial Structures

1. Select ‘Polygon tool’ from top left menu.
2. Toggle segmentation off.
3. Scroll to first slice on which cerebellum begins (if brainstem separates before cerebellum begins, see rule exceptions).
4. Select ‘Active drawing label’ = ‘Clear Label’ and ‘Draw over’ = ‘All Labels’.
5. These active drawing labels essentially DELETES data from the segmentation image, so exercise caution. Undo (CTRL+Z) still works, but only for a limited number of steps back.
6. Left click to draw a polygon over the dura surrounding the cerebellum, and along the base of the brainstem across the colliculi. Right click to close polygon.
7. Click ‘Accept’ to ‘Delete’ that area of the segmentation, which will now show the T1 beneath indicating it is no longer included in the segmentation.
8. Go to the next slice down and repeat. Always do the tracings on the T1, never on the seg.
9. Once the cerebral peduncles separate, begin also removing the brainstem and spinal cord.
10. At the anterior aspect, trace directly across the gap. Once there is a clear dural line at the anterior orbitofrontal end (generally beneath the level of the pituitary, start tracing an arch out along that dura line).
11. Once the occipital lobe separates from the temporal lobe, ensure that the tracing exits from the center, to remove any remaining ‘junk’ in this region. See Figure 3.
12. At some point, draw the polygons so that they only keep what is required, instead of removing what is unnecessary, using the ‘draw inverted’ option (while referring to the seg to assist in the tracing).
13. If only temporal lobes remain, simply draw a large poly around the cerebellum and remove that.
14. If it is certain that the polygon will only contain cerebellum on a subsequent slice below, use the “paste” button to paste on the previous tracing and use that to delete the cerebellum.
15. Once the cerebellum is all that remains in the image, paste the large tracing down each slice and “accept” to delete it until there is no more cerebellum in the image.
16. Now scroll up through the image slice by slice to verify that the ONLY portions of the segmentation that remain are supratentorial.
17. When finished, save the segmentation as ‘<name>_seg_vcsf_st’ by clicking: Segmentation -> Save as image- > and then add ‘_vcsf_st’ after ‘_seg’ -> Save.

2. SABRE Component

2.1 ACPC Alignment

1. Open ITK-SNAP_sb.
2. Load ‘T1_IHCpre_iso’ as described in Brain-Sizer manual.
3. Adjust intensity as described in Brain-Sizer manual.
4. Select the ‘Navigation tool’ from the top left menu.
5. Then click on the ‘ACPC alignment tool’.
6. Load “T1_IHCpre_toACPC.mat” matrix file using load option in bottom left hand corner.
7. Zoom in to the image by right-clicking on the axial view and dragging the mouse upwards.
8. Change the position of the brain in the window (separate from zooming) by left-clicking on the image and moving the mouse around to better center the zoomed view. Also adjust the sagittal and coronal views. Make sure the sagittal view is close to mid-sagittal.
9. Click on ‘ACPC tool’ button.
10. Change the increment to 1.
11. Check Pitch, Roll and Yaw determined by T1_IHCpre_toACPC.mat matrix file, modify if necessary.
12. To find the ACPC plane, it is likely necessary to zoom in closely using the navigation tool. At any point, switch back and forth between the navigation tool and the ACPC tool (to adjust the view), and the ACPC tool will keep the position and return it to the previous position. When switching between these views, the image will change back and forth, but this is normal.
13. By using the pitch up/down and elevate up/down, adjust the axial view so that the AC is at its thickest (a nice u-shape of white matter fibers), and the PC straight across, which should end up forming a nice ‘key hole’ shape.
14. The AC-PC should also be visible with the crosshairs passing directly through both the AC and PC on the mid-sagittal view.
15. Do not adjust the pitch any further once this slice has been determined. However, the ‘elevate’ function can be used to move up and down through the image without losing the ACPC slice.
16. Now adjust the roll by balancing the eyeballs in the axial view. Readjust the view using the navigation tool to bring the eyeballs in to the field of view, then switch back to the ‘ACPC’ tool.
17. Use ‘Roll’ left or right to make sure that the eyeballs look evenly balanced (same size on both sides) while scrolling through the image one slice at a time using ‘Elevate’, making sure to adjust the roll as necessary. See Figure 4.
18. Once satisfied with the balance, Do not adjust ‘Roll’ any further.
19. Now move to a slice above the ventricles and corpus callosum in axial view (by using ‘Elevate’, or clicking the crosshairs at that level using ‘Navigation’) and place the crosshairs close to the center of the brain in axial view.
20. Adjust ‘Yaw’ by making sure that the vertical crosshair passes directly (or as close as possible) through the mid-sagittal plane in the axial view. Sometimes it may be difficult to get the plane to perfectly line up due to natural curvature of the brain at the poles – create the best fit possible.
21. Once satisfied with the position, Do not adjust ‘Yaw’ any further.
22. Now place crosshairs such that axial slice is just above the ventricles.
23. This should be approximately where it was from the previous step.
24. Now click: Save (make sure the filename is ‘T1_IHCpre_toACPC.mat’) -> OK.
25. NOTE: If “T1_IHCpre_toACPC.mat” matrix file does not require modification simply close without saving.
26. If changes were made to the matrix file, save over “T1_IHCpre_toACPC.mat” matrix file or save a new matrix file and delete the “T1_IHCpre_toACPC.mat” matrix file. The next command will not work correctly if there is more than 1 matrix file.

2.2 SABRE Landmark Identification

Part 1 – Grid File Coordinates

1. Load in ‘<name>__T1_IHC_inACPC’.
2. Adjust intensity.
3. Turn off crosshairs (x).
4. Zoom in to the image until it fills each window (right click and drag with crosshairs tool).
5. Adjust center of axial view if necessary, with navigation tool (may need to do several times during procedure).
6. Click on ‘2D-sabre land-marking’ tool.
7. In axial view, scroll up through the images/brain until you find the ACPC slice.
8. Click the ‘AC’ radio button on the left to select that landmark to define, then click on the AC in the axial view.
9. A small dot will appear on the spot you clicked, and the associated landmark coordinate will now appear next to the ‘AC’ button on the left.
10. If the placement is not desirable, click again and the point will update (this applies to any point during the creation of the grid file).
11. Click the ‘PC’ radio button on the left and then click on the PC on the axial image.
12. Click the ‘PE’ radio button to define the posterior edge of the brain on that slice, and then click on the most posterior part of the brain, either on the left or the right – this fills in values for ‘coronal slice’ which will be used momentarily. See Figure 5.
13. Click the ‘CA’ radio button to define the central canal. Scroll down 10 slices from the current axial view and click on the center of the central canal. This fills in the value for ‘sagittal slice’ which will be used now as a starting point for which to find the mid-sagittal plane.
14. Click on the ‘M’ radio button to define the mid-sagittal plane.
15. In sagittal view, scroll left and right a few slices to determine which slice has the least amount of brain and the maximal amount of falx cerebri. It should be within 2 or 3 slices of the value determined from the central canal point.
16. Click anywhere on the mid-sagittal slice and that slice number will be entered on the left next to ‘M’.
17. Click on the ‘LPRON’ radio button to define the left preoccipital notch. In coronal view, scroll to the slice indicated next to ‘coronal slice’.
18. Click on the most inferior part of the brain for the left hemisphere, which appears on the RIGHT side of the image (radiological convention).
19. Click on the ‘RPRON’ radio button to define the right hemisphere, and click on the most inferior part of the LEFT side of the image (radiological convention).
20. The values next to LPRON and RPRON should now be filled, and should be within a few points of each other.
21. The grid file is now ready to be saved. Click: Save -> _T1_IHC_inACPC_lobgrid.txt.

Part 2 – Object Map Creation

22. After grid file creation, the next stage is the creation of the first 4 tracings of the object map. All of these 4 tracings are performed in the sagittal plane. The slices for tracing are predetermined and based on the midline slice selected in the previous stages.
23. Click on the ‘RSC’ radio button to define the right superior central sulcus. Go to the slice indicated next to ‘Right Sagittal slice’. The left and right sagittal slices on which the tracings will be made: 7 slices peri-sagitally from the midline on each side.
24. Click a point directly above the center of the central sulcus, in the dura. The central sulcus on this slice generally appears as a small indentation, and is most commonly the first sulcus anterior to the marginal (ascending) branch of the cingulate sulcus. Scroll left or right to confirm the location of the landmark, but the tracing must always be made on the proper sagittal slice. Reclicking will relocate the landmark.
25. Click on the ‘ROP’ radio button to define the right occipito-parietal sulcus. This sulcus/tracing runs from the dura to the tentorium cerebelli.
26. A spline tool will now allow sulcus tracing. Left click to create new points along it, and right click to lock it and then click accept. Modifications or ‘undo’ functions can not be performed if there are errors made DURING the tracing. However, once the ‘right-clicking’ action is performed to complete the tracing, select ‘Delete’ to redo the tracing.
27. When the tracing is complete, select ‘Accept’ to lock it in.
28. Do the same for the left side at the appropriate slice, defining ‘LSC’ and ‘LOP’.
29. Click: Save (under object map) -> _T1_IHC_inACPC_lobtrace.obj.

Part 3 – Surface Rendered Tracings

31. Unload previous images (or close and open ITK-SNAP_sb again) and load in <name>_T1_IHC_erode_inACPC image.
32. Click on the 3D SABRE landmarking tool (the window should enlarge to only show 1 pane).
33. Click ‘LEFT’ under 3D Viewpoint to show the left rendered view (in radiological convention, whereby left and right are reversed, so it will appear as though it is the right hemisphere).
34. Load in object tracing from previous step by clicking: Load -> Select ‘<name>_T1_IHC_inACPC_lobtrace.obj’ (NOTE: a bug in the program automatically tries to anticipate loading the required file, but it incorrectly inputs ‘erode’ in the obj file name. Please select browse and then select the <name>_T1_IHC_inACPC_lobtrace.obj’ to load. Otherwise an error message will be displayed, ‘Error loading object map tracing: File cannot be opened for reading’).
35. To adjust the quality of the render, click: ‘Guess’, to have the program guess at the best parameters to use.
36. Click ‘LSF’ radio button to prepare to trace the Left Sylvian Fissure.
37. Now click ‘Landmark’ button at the bottom of the 3D render window to begin landmarking/tracing (you can toggle this on and off with the “x” key).
38. Additional points to the tracing can be added when the ‘Landmark’ button is shaded green.
39. When ‘Landmark’ is unselected, any mouse input will rotate the brain to examine it from a different angle. WARNING: Only trace the landmarks while in straight ‘LEFT’ or ‘RIGHT’ orientation by reclicking on the left or right 3D Viewpoint buttons.
40. Zoom in to or out of the image by right clicking and dragging when ‘Landmark’ is unselected.
41. Each click will add a point to the line.
42. Begin tracing the Sylvian fissure from the superior to posterior end, at the point at which it bifurcates into small ascending and descending rami.
43. Continue tracing the Sylvian down the superior aspect of the temporal lobe until it trails off the end.
44. If an error is made, simply click the ‘Undo’ button to move backwards step by step (or press CTRL+Z).
45. Once satisfied with the tracing, click on ‘Accept’ to lock in the tracing. See Figure 5.
46. IMPORTANT: If redo is required for one of the tracings, first select the radio button (on the left) of the incorrect tracing. Then click ‘SABRE3D’ on the menu bar at the top and select ‘Delete CURRENT Accepted Tracing’. If at some point all of your tracings require removal, click on ‘Delete ALL accepted tracings’ from this drop down menu.
47. Now click the ‘LC’ radio button to trace the Left Central Sulcus.
48. Start from the inferior end at the point of Sylvian fissure directly below the termination of the sulcus.
49. The line will only allow superior and posterior movement- meaning the program prevents placing points that are anterior to any previous point.
50. Finish tracing the sulcus at the superior end until it is difficult to follow the curvature of the brain.
51. Once complete, click ‘Accept’ to lock it in.
52. Now click on the ‘RIGHT' button under ‘3D Viewpoint’ and repeat the steps for the right Sylvian Fissure and Central Sulcus.
53. Remember to click on the ‘RSF’ radio button to trace the right Sylvian Fissure, and click on the ‘RC’ radio button to trace the right central sulcus, clicking ‘Accept’ after each tracing is complete.
54. Once all tracings are completed, click: Save -> Browse -> select ‘<name>_T1_IHC_erode_inACPC_lobtrace.obj’.
55. Close ITK-SNAP_sb.

3. Lesion-Seg Component

3.1 For Scans with PD/T2 (no FLAIR)

1. Open ITK-SNAP_sb, load <name>T1_IHC, <name>_PD_inT1­_IHC, <name>_T2_inT1_IHC, Click: File -> Open grayscale image -> Browse -> go to directory, Click -> Image -> Open -> Next -> Finish.
2. Click plus sign next to axial view to enlarge.
3. Turn off the crosshairs (x).
4. Zoom in (right click and drag).
5. Adjust intensity by clicking: Tools -> Image Contrast, then drag middle point up and slightly left until the image brightens to the appropriate level, Close.
6. Load lesion-seg on PD_inT1_IHC by clicking: Segmentation -> Load from image -> Browse -> Select <name>_LEauto -> Open -> Next -> Finish.
7. Adjust intensity of all 3 images as described in Brain-Sizer manual.
8. Click the paintbrush tool, Select ‘Active drawing label’ =2 and ‘Draw over’ =Visible labels.
9. Use T1, PD and T2 to inform decision about what to capture as lesion.
10. Use paintbrush tool to paint label 2 over label 1 to signify lesion (positives) (Toggle segmentation on and off with ‘s’ key).
11. Use paintbrush tool to paint label 1 over label 2 to signify false positives. See Figure 6.
12. When satisfied with lesion-seg modifications click: Segmentation -> Save as image -> and modify file name by replacing “auto” with “edit” to the end of the file to indicate that it is ‘Done’, then click ‘Save’ (i.e. <name>_LEedit)

NOTE: Label 2 (default color is RED) is used to signify lesion.

3.2 For Scans with FLAIR Imaging

1. Open ITK-SNAP_sb, load <name>_FL_inT1_IHC Click: File -> Open grayscale image -> Browse -> go to directory, Click -> Image -> Open -> Next -> Finish.
2. Click plus sign next to axial view to enlarge.
3. Turn off the crosshairs (x).
4. Zoom in (right click and drag).
5. Adjust intensity by clicking: Tools -> Image Contrast, then drag middle point up and slightly left until the image brightens to the appropriate level, Close.
6. Load lesion-seg on FL_inT1_IHC by clicking: Segmentation -> Load from image -> Browse -> Select <name>_FLEXauto -> Open -> Next -> Finish.
7. Adjust intensity as described in Brain-Sizer manual.
8. Click the paintbrush tool, Select ‘Active drawing label’ =2 and ‘Draw over’ =Visible labels.
9. Use FL (use T1, PD, T2 if necessary) to inform decision about what to capture as lesion.
10. Use paintbrush tool to paint label 2 over label 1 to signify lesion (positives) (Toggle segmentation on and off with ‘s’ key).
11. Use paintbrush tool to paint label 1 over label 2 to signify false positives. See Figure 7.
12. When satisfied with lesion-seg modifications click: Segmentation -> Save as image -> and modify file name by changing “auto” to “edit” to indicate that it is ‘Done’, then click ‘Save’ (i.e. <name>_FLEXedit).

NOTE: Label 2 (default color is RED) is used to signify lesion.