Image Rendering Techniques in Postmortem Computed Tomography: Evaluation of Biological Health and Profile in Stranded Cetaceans

Summary

The Hong Kong cetacean stranding response program has incorporated postmortem computed tomography, which provides valuable information on the biological health and profile of the deceased animals. This study describes 8 image rendering techniques that are essential for the identification and visualization of postmortem findings in stranded cetaceans, which will help clinicians, veterinarians and stranding response personnel worldwide to fully utilize the radiological modality.

Abstract

With 6 years of experience in implementing virtopsy routinely into the Hong Kong cetacean stranding response program, standardized virtopsy procedures, postmortem computed tomography (PMCT) acquisition, postprocessing, and evaluation were successfully established. In this pioneer cetacean virtopsy stranding response program, PMCT was performed on 193 stranded cetaceans, providing postmortem findings to aid necropsy and shed light on the biological health and profile of the animals. This study aimed to assess 8 image rendering techniques in PMCT, including multiplanar reconstruction, curved planar reformation, maximum intensity projection, minimum intensity projection, direct volume rendering, segmentation, transfer function, and perspective volume rendering. Illustrated with practical examples, these techniques were able to identify most of the PM findings in stranded cetaceans and served as a tool to investigate their biological health and profile. This study could guide radiologists, clinicians and veterinarians through the often difficult and complicated realm of PMCT image rendering and reviewing.

Introduction

Virtopsy, also known as postmortem (PM) imaging, is the examination of a carcass with advanced cross-sectional imaging modalities, including postmortem computed tomography (PMCT), postmortem magnetic resonance imaging (PMMRI), and ultrasonography¹. In humans, PMCT is useful in investigating traumatic cases of skeletal alterations^2,3, foreign bodies, gaseous findings^4,5,6, and pathologies of the vascular system^7,8,9. Since 2014, virtopsy has been routinely implemented in the Hong Kong cetacean stranding response program¹. PMCT and PMMRI are able to depict patho-morphological findings on carcasses that are too decomposed to be evaluated by conventional necropsy. The non-invasive radiological assessment is objective and digitally storable, allowing second opinion or retrospective studies years later^1,10,11. Virtopsy has become a valuable alternative technique to provide new insights of PM findings in stranded marine animals^{12,13,14,15,16}. Combined with necropsy, which is the gold standard to explain the pathophysiological reconstruction and cause of death¹⁷, the biological health and profile of the animals can be addressed. Virtopsy has been gradually recognized and implemented into stranding response programs worldwide, including but not limited to Costa Rica, Japan, Mainland China, New Zealand, Taiwan, Thailand and USA¹.

Image rendering techniques in radiology use computer algorithms to transform numbers into information about the tissue. For example, radiological density is expressed in conventional X-rays and CT. The vast quantity of volumetric data is stored in the Digital Imaging and Communications in Medicine (DICOM) format. CT images can be used to produce isotropic voxel data using two-dimensional (2D) and three-dimensional (3D) image rendering in a postprocessing 3D workstation for high resolution visualization^18,19. Quantitative data and results are mapped to transform serially acquired axial images into 3D images with gray-scale or color parameters^19,20,21. Choosing an appropriate data visualization method from diverse rendering techniques is an essential technical determinant of the visualization quality, which significantly affects the analysis and interpretation of radiological findings²¹. This is particularly critical for stranding work that involves personnel without any radiology background, who need to understand the results in different circumstances¹⁷. The goal of implementing these image rendering techniques is to enhance the quality on the visualization of anatomical details, relationships and clinical findings, which boosts the diagnostic value of imaging and allows an effective rendition of the defined regions of interest^{17,19,22,23,24,25}.

Although the primary axial CT/MRI images contain most information, they may limit accurate diagnosis or documentation of pathologies as structures cannot be viewed in various orthogonal planes. Image reformation at other anatomically aligned planes permits visualization of structural relationships from another perspective without having to reposition the body²⁶. As medical anatomy and forensic pathology data are predominantly 3D in nature, color-coded PMCT images and 3D reconstructed images are preferred to gray-scale images and 2D slice images in view of improved understandability and suitability for courtroom adjudications^27,28. With the advances in PMCT technology, a concern of visualization exploration (i.e., the creation and interpretation of 2D and 3D image) in cetacean PM investigation has been raised^12,29. Various volumetric rendering techniques in the radiology workstation allow radiologists, technicians, referring clinicians (e.g., veterinarians and marine mammal scientists), and even laymen (e.g., stranding response personnel, government officers and general public) to visualize and study the regions of interest. Yet, the choice of a suitable technique and confusion of terminology remain a major issue. It is necessary to understand the basic concept, strengths and limitations of the common techniques, since it would significantly influence the diagnostic value and interpretation of radiological findings. Misuse of techniques may generate misleading images (e.g., images that have distortions, rendering errors, reconstruction noises or artefacts) and lead to an incorrect diagnosis³⁰.

The present study aims to assess 8 essential image rendering techniques in PMCT that were used to identify most of the PM findings in stranded cetaceans in Hong Kong waters. Descriptions and practical examples of each technique are provided to guide radiologists, clinicians, and veterinarians worldwide through the often difficult and complicated realm of PMCT image rendering and review for the evaluation of biological health and profile.

Protocol

NOTE: In the framework of the Hong Kong cetacean virtopsy stranding response program, stranded cetaceans were routinely examined by PMCT. The authors were in charge of virtopsy scanning, data postprocessing (e.g., image reconstruction and rendering), data interpretation, and virtopsy reporting¹. This advanced technology emphasizes attentive findings and gives insights on the initial investigation of PM findings prior to conventional necropsy (https://www.facebook.com/aquanimallab).

1. Data preparation

1. Export the acquired CT datasets in DICOM 3.0 format. Copy the DICOM folder to computer (e.g., desktop).
2. Open a free or commercial DICOM viewer. The following steps are based on the TeraRecon Aquarius iNtuition Workstation (version 4.4.12).
3. Double-click the icon of Aquarius iNtuition Client Viewer (AQi) icon. Enter user name, password, and server name in the appropriate fields. Click the Login button.
   NOTE: Make sure that the server name field has the correct server IP address.
4. Click Import under the data management tool buttons and select the DICOM folder to import. Click the Update icon to renew the study list after the import status reaches 100%.
5. View the datasets by selecting 1 or multiple CT series from the Patient List by double-left-clicking the series.
6. After loading the designated series, click the Window Layout Button for the 2x2 display interface, showing a 2x2 default layout, a 3D volume rendered image (upper-right panel) and 3 MPR images in axial view (upper-left panel), coronal view (lower-left panel), sagittal view (lower-right panel), giving different orientations.
7. Evaluate the virtopsy datasets thoroughly using different image rendering techniques provided.

2. Multiplanar reconstruction (MPR)

1. Display the default MPR from axial view (upper-left panel), coronal view (lower-left panel), and sagittal view (lower-right panel) after loading the series. Change the rendering mode to MPR by either right-clicking the image and select MPR or click MPR in the Rendering Mode Mini-Toolbar.
2. Evaluate the virtopsy datasets from the first image to the last image using the axial view, followed by coronal and sagittal views, with the assistance of the following functions: Click Slice, left-click-hold mouse button and drag the mouse to view and adjust the CT image slice by slice.
3. Click Pan, left-click-hold mouse button and drag the mouse to adjust the location of the image inside the panel.
4. Click Zoom, left-click-hold mouse button and drag the mouse to magnify or minify the image.
5. Select the appropriate pre-set window/levels by clicking Abd 1 (window width: 350, window level: 75), Abd 2 (window width: 250, window level: 40), Head (window width: 100, window level: 45), Lung (window width: 1500, window level: -700), Bone (window width: 2200, window level: 200) in the Window/Level Mini-Toolbar, depending on the regions of interest.
6. Click Window/Level (W/L), left-click-hold mouse button and drag the mouse to manually adjust the window width and window level of the CT slice.
7. Click Rotate, left-click-hold mouse button and drag the mouse to rotate the MPR images.
8. Left-click-hold mouse button on the center of MPR Crosshairs to concurrently adjust the regions of interest and slices in 3 MPR images.
   NOTE: There are mouse modes for the 4 main functions of rotations, panning, zooming and window/level changes provided by AQi to facilitate the viewing process. For keyboard shortcuts, see Table 1.

3. Curved planar reformation (CPR)

1. Decide the region of anatomical interest. Left-click-hold mouse button on the center of MPR crosshairs to the particular region of interest.
2. View the MPR from 3 different views. Ensure the MPR crosshairs is placed in a correct location. Adjust the MPR crosshairs if it is not.
3. Select 1 display panel from axial, coronal, and sagittal views as study panel, e.g., aiming to view the flipper from an axial view.
4. Depending on the study panel, adjust the extended line of MPR crosshairs (e.g., blue color) from coronal view perpendicularly to the region of interest by left-click-hold mouse button on the rotation point of extended line.
5. Adjust another extended line (e.g., red color) of MPR crosshairs from sagittal view parallel to the region of interest by left-click-hold mouse button on the rotation point of extended line.
6. Look at the axial view to check whether the region of interest is adjusted correctly. Adjust the extended lines if it is not. Evaluate the virtopsy datasets using the 4 main functions of rotation, panning, zooming and window/level changes.
   NOTE: There are 3 colored extended lines of MPR crosshairs (green, red, and blue), representing different alignments of the MPR plane (Figure 2).

4. Maximum intensity projection (MIP)

1. Change the rendering mode to MIP by either right-clicking the image and selecting MIP or by clicking MIP in the Rendering Mode Mini-Toolbar.
2. Adjust the slab thickness on the right upper corner (minimum: 1 mm, maximum: 500 mm) by clicking the green annotation and select a new thickness to visualize the regions of interest, e.g., the bronchial tree in the lung.
3. Evaluate the virtopsy datasets using the 4 main functions of rotation, panning, zooming, and window/level changes.

5. Minimum intensity projection (MinIP)

1. Change the rendering mode to MIP by either right-clicking the image and selecting MinIP or by clicking MinIP in the Rendering Mode Mini-Toolbar.
2. Adjust the slab thickness on the right upper corner (minimum: 1 mm, maximum: 500 mm) by clicking the green annotation and select a new thickness to visualize the regions of interest (e.g., the bronchial tree in the lung).
3. Evaluate the virtopsy datasets using the 4 main functions of rotation, panning, zooming, and window/level changes.

6. Direct volume rendering (DVR)

NOTE: As 1 of the default display 2x2 interfaces, DVR (upper-right panel) shows the 3D rendered images of the carcass. The default DVR template setting is AAA (abdominal aortic aneurysm; window width: 530, window level: 385), giving a gross skeletal structure of the carcass.

1. Automatically adjust the windowing setting by clicking Template under the Viewer and select the appropriate DVR template, e.g., Gray 10% (window width: 442, window level: 115), Fracture (window width: 2228, window level: 1414) if needed.
2. Click Window/Level (W/L), left-click-hold mouse button and drag the mouse to adjust the window width and window level of the CT slice manually, giving an outer layer (e.g., epidermal surface) to inner layer (e.g., internal structure).
3. Use the 4 main functions of rotation, panning, zooming, and window/level changes for further corrections.
   NOTE: All DVR templates provided by AQi are human clinical oriented, not designated for PM imaging of cetaceans.

7. Segmentation and Region-of-Interest (ROI) Editing

1. Segment the CT image slice using 3 different tools, Slab and Cube View tool, Free ROI tool, and Dynamic region growing tool.
2. For Slab and Cube View tool, click Slab under Tool, giving a parallel display line. Adjust the slab location by relocating the MPR crosshairs from the corresponding MPR views. Change the slab thickness (minimum: 1 mm, maximum: 500 mm) via the slab thickness bar, resulting a segmentation of 3D rendered images of the carcass.
3. For Free ROI tool, click FreeRO under Tool. Hold on the Shift key on the keyboard, and use either Draw Free Curve on MPR, Draw Circle on MPR, or Draw Sphere on MPR to exclude/include the region of interest from the MPR views and DVR.
4. For Dynamic region growing tool, click Region under Tool. Hold on the Shift key on keyboard, left-click-hold mouse button and scroll the middle button of the mouse (scroll-up: increase the selecting region, scroll-down: decrease the selecting region), giving a highlighted region. Click Exclude to delete the region. Click Include to keep the region.

8. Transfer Functions (TF)

1. Click 3D Setting under Viewer, select Copy to create a new 3D reconstructed model.
2. In the new 3D reconstructed model, click FreeRO or Region under Tool. Hold on the Shift key on the keyboard, use 3D VR to include the region of interest and then click Select.
3. Configure the 3D settings, including W/L Slider, W/L Text-input Boxes, VR Pull-down Menu, Opacity Slider (minimum: 0, maximum: 1), Opacity Text-input Box, and HU Range Color Slider under 3D Setting.
4. Right-click 1 of the sliders in the color slider bar to change the color of the DVR. Select Change Color and define a custom color from the color palette if needed.

9. Perspective Volume Rendering (PVR)

1. To launch the Flythrough Module, right-click on the selected series and select Flythrough from the right-click menu.
2. Choose the Primary 3D of Reading Style Preference Wizard for primary view selection. Click the 2x2 screen layout and OK, resulting in an automatically RVR, e.g., colon. Make sure the region of interest is selected.
3. Build a flight path by placing the start and end of control points by drawing a path. Correct the path by clicking the Edit Connection/Edit Path radio button in the tool panel if there is a broken path or missing structure, editing the control points for smoother sections of the curve or correcting problems. Create new control points by clicking on the flight path. Once the flight path is correct, click OK.
4. View the Flythrough window displayed, showing a Main flythrough window, MPR views and Flat view.
5. Use Cine Tools by clicking the Tool Panel located on the right side of the screen to evaluate the luminal structure. Adjust the speed and direction of the flythrough using Fly backward, Pause, Fly Forward, Slow down flythrough, and Speed up flythrough under the Cine tools.

10. Data evaluation

1. Conduct virtopsy evaluation systematically from head to tail. It is generally within 30 minutes, acting as a reference to guide veterinarians for subsequent necropsy.
2. After necropsy, compare virtopsy findings and necropsy findings. Based on the site report, virtopsy, necropsy, and sample analysis (e.g., histopathology and microbiology), conclude the PM investigation on the biological health and profile of the stranded cetacean.

Representative Results

From January 2014 to May 2020, a total of 193 cetaceans that stranded in Hong Kong waters were examined by PMCT, including 42 Indo-Pacific humpback dolphins (Sousa chinensis), 130 Indo-Pacific finless porpoises (Neophocaena phocaenoides) and 21 other species. A whole-body scan was performed on 136 carcasses while 57 were partial scans on skulls and flippers. Anatomical features and pathologies commonly observed were illustrated with the 8 image rendering techniques for the evaluation of the stranded cetaceans’ biological health and profile.

Figure 1: MPR function displaying a deceased Indo-Pacific humpback dolphin in (A) axial, (B) reconstructed 3D, (C) reconstructed coronal, and (D) reconstructed sagittal views. Area measurements of the atlanto-occipital space are demonstrated in the axial plane. Linear measurements of the ventral tubercle to outer margins of the occipital condyle (coronal), basion-dorsal arch and opisthion-ventral arch (sagittal) for the diagnosis of atlanto-occipital dissociation are demonstrated. Please click here to view a larger version of this figure.

Figure 2: CPR function displaying curved structures in the flipper of a deceased Indo-Pacific finless porpoise in planar view. Please click here to view a larger version of this figure.

Figure 3: MIP function highlighting hyperattenuated pulmonary nodules (intense white dots) in both lungs of a deceased Indo-Pacific finless porpoise. Please click here to view a larger version of this figure.

Figure 4: MinIP function highlighting hyperattenuated gas-filled structures, i.e., tracheobronchial trees in both lungs of a deceased Indo-Pacific finless porpoise. Please click here to view a larger version of this figure.

Figure 5: DVR function displaying different components of a deceased Indo-Pacific finless porpoise. (A) Vasculatures overlaid with the skeletal system are highlighted by AAA. (B) The respiratory system is highlighted by Lung. (C) The skeletal system including the vertebral physeal plates is highlighted by Bone plus Plate. (D) Hyperattenuated ear bones and fish hooks are highlighted by Hardware. Please click here to view a larger version of this figure.

Figure 6: ROI editing function displaying a deceased Indo-Pacific finless porpoise (A) with the CT couch and (B) with the CT couch removed. Please click here to view a larger version of this figure.

Figure 7: TF function displaying different components of a deceased Indo-Pacific finless porpoise. Sand in an air sac is highlighted in cyan. Stomach content is highlighted in green. A parasitic granulomatous mastitis lesion is highlighted in red. Please click here to view a larger version of this figure.

Figure 8: PVR function demonstrating a virtual bronchoscopy of a deceased Indo-Pacific humpback dolphin with the Flythrough function. Please click here to view a larger version of this figure.

Table 1: Keyboard shortcuts of the software for different image postprocessing functions. Please click here to download this table.

Discussion

For the clear visualization of virtopsy datasets, 8 image rendering techniques, consisting of both 2D and 3D rendering, were routinely applied to each stranded carcass for the PM investigation of their biological health and profile. These rendering techniques included MPR, CPR, MIP, MinIP, DVR, segmentation, TF, and PVR. Diverse rendering techniques are complementarily used together with windowing adjustment. The concepts of each image reformation technique and advantages are also described.

Multiplanar reconstruction (MPR)
MPR is the process of creating non-axial 2D images, including the coronal, sagittal, and any anatomically aligned oblique plane image^24,30, which is not acquired directly during the acquisition in an axial plane. This dominant 2D rendering technique is especially helpful in assessing any intact anatomical structure or pathology in the required plane with high quality images^31,32. With the help of MRP, cetacean PM investigations of the entire body, orthopedic, and neurological/spine were routinely performed in 3 directions simultaneously, which significantly improved the accuracy of findings (Figure 1). Through comprehensive observation from the 3 planes, the error rate of misidentifying minute pathologies is reduced. In addition, MPR also supports linear and area measurement at the axial, coronal, and sagittal plane. However, it is operator-dependent, and requires sufficient anatomical knowledge to identify both normal structures and pathological conditions, which avoid misinterpretation of the rendered images.

Curved planar reformation (CPR)
CPR is also called curved MPR. Despite being treated as MRP in some peer-viewed literatures, CPR is a distinct 2D rendering technique. Using isotropic imaging that aligns the long axis of the image plane with a selected anatomic structure, 2D images are reformatted with no loss of image quality^18,24. This allows the operator to manually define a centerline path for a curved reconstruction within the volumetric dataset. This is particularly crucial when the subject cannot be placed in a true or relatively true anatomical position in reference to the PMCT detectors (i.e., true reconstructed coronal/sagittal/axial image), especially for frozen or mummified carcasses. The alignment of complicated, tortuous or calcified structures is needed to obtain a more symmetrical image for diagnosis. Due to its flexible flattening and distortion characteristics, misinterpretation can be easily induced. The operator must clearly remember the position and shape of the anatomical structures of interest. Flippers are 1 of the most difficult body parts to obtain a true anatomical position as they are curved towards the body flanks, unless resected before the PMCT scan. With the utilization of CPR, most of the anatomical features in the flippers were demonstrated at 1 plane and for skeletal age estimation (Figure 2).

Maximum intensity projection (MIP)
MIP projects only the highest attenuation value in each pixel of the volumetric datasets within the viewer’ sight³² and selects the voxel with the maximum intensity as the value of the corresponding display pixel¹⁸. Originally, this technique is recognized to evaluate the osteological material, metallic implants, and contrast-filled structures for CT angiography in clinical radiology antemortem^17,33. Due to decomposition of internal structures and organs, and the absence of blood perfusion in stranded carcasses, the adoption of MIP in evaluating the contrast-filled structures for CT angiography become very difficult in virtopsy. However, MIP still takes a dominant character in examining osteological materials, foreign bodies (e.g., food bolus, fish remains, stone, metallic entanglement) and calcifications within soft tissues, as well as highly attenuated, narrow, and blood- or water-filled structures such as the major arteries and veins. Through the adjustment of slab thickness (i.e., image thickness for data reconstruction) subjective to the size of the evaluated target, the visualization of lesions could be emphasized. For instance, using different sliding thin-slab³⁴, the identification of small pulmonary nodules in collapsed lungs of a stranded carcass was intensively improved, as MIP emphasized these minutes of hyperattenuated speckles, which evidenced the presence of lung consolidation and parasitic pneumonia (Figure 3).

Minimum intensity projection (MinIP)
In contrast to MIP, MinIP projects only the lowest attenuation value encountered along a ray pass through a volume toward the viewer’ sight within a volume^18,24. Although MinIP is not commonly used in clinical radiology²⁴, this technique still served as an excellent visualization tool on hypoattenuated structures and gas-filled structures, such as the respiratory and gastrointestinal tract. The examination of the morphology and pulmonary parenchymal abnormalities, started from the blowhole down to the tracheobronchial tree, in the stranded cetaceans were significantly enhanced (Figure 4). Similar to MIP, additional control should be taken on the slab thickness, subject to the examined pathologies, to generate a more distinguishable image³⁵, as the slab thickness is critical to determine the distinction of presented structures on the studied structures.

Direct volume rendering (DVR)
DVR is an algorithm that converts an entire 3D image set into 2D images directly without discarding any information¹⁸. The final displayed 2D image is created based on its Hounsfield units by assigning each voxel in the image a specific color and opacity value along with other voxels in the same projection ray. As the opposition of creating an intermediate representation (e.g., an extracted surface model by soft-tissue removal tool), the internal and external conditions of a stranded carcass at all depths with the 3D method can be examined at once, without obscuring each other. This 3D rendering technique was a quick, versatile and interactive tool for a whole-body carcass evaluation from any angle. Bony lesions, complex fractures, body fragmentation, and foreign bodies caused by human interaction (e.g., traumatic injuries caused by vessel collision and fisheries) were possible to be identified (Figure 5). The challenge of DVR is that the operator needs to adjust the rendering parameters, i.e., the opacity and brightness, to display the vasculature more accurately^21,36.

Segmentation and Region-of-Interest (ROI) Editing
Irrelevant structures, objects (e.g., body bag and CT couch), and artefacts (e.g., metallic zippers) displayed on the DVR model may degrade the image quality and obscure radiological diagnosis. To illustrate certain areas of anatomy or pathology in a better manner, segmentation is used to include or exclude selected volumetric data on either 2D or 3D images^18,24. Although automated segmentation programs are available, manual segmentation which requires high tissue recognition and delineation by the operator was performed in most circumstances to assist the identification of radiological findings on the DVR of stranded carcasses. ROI editing was the most common segmentation tool used in the present study, which allowed the operator to include or exclude a region of interest manually by drawing a rectangular, elliptical or other shape to define the precise spatial boundary of the target (Figure 6). Similar to DVR templates provided in the 3D workstation, automated segmentation is based on the rules of connectedness and thresholding, and subjected to clinical radiology, which was mostly unsuitable for this study, except for the automatic body bone removal function.

Transfer Functions (TF)
TF is an algorithm to control the threshold of opacity, brightness, and color of the selected volume^18,24. This tool allows the operator to selectively reveal the relevant structures on the DVR model, by selecting the threshold value, range, and shape, to serve different purposes at the defined region. For instance, choosing a lower opacity threshold removes the external low-opacity soft tissues (skin and fat) and obscures the abdominal content, while a high opacity threshold keeps high opaque objects (e.g., bone, calcium, and excreted contrast materials); changing the color, brightness, and contrast scale highlights the region of interest, and makes the appearance of the DVR model to look different. These controls give a better elucidation and quicker differentiation of structures based on their attenuation. However, these are vulnerable to interobserver variability and dependent on operator mastery in optimization of rendering parameters²¹. With the contribution of segmentation and TF, the relationship of displayed tissues, organs, and foreign bodies in scanned carcasses were well-classified (Figure 7). Fast and clear preliminary findings on stranded cetaceans were demonstrated on the edited DVR model, which gave veterinarians and stranding response personnel an overview on the internal and external condition, as well as the initial PM investigation findings, and facilitated subsequent conventional necropsy.

Perspective Volume Rendering (PVR)
PVR, also called endoluminal imaging or immersive rendering, is mainly applied to air-containing structures such as trachea, colon, esophagus, and arteries. It allows the operator to visualize the internal conditions of the lumen by virtual navigation³⁵. The operator designates the start point, end point, and a centerline path to fly through. By displaying an animation of flying through the structure, the relationships between anatomic structures and endoluminal abnormalities such as polyps or cancerous growths on the walls can be identified as in a non-invasive virtual endoscopy¹⁹. The corresponding MPR images displayed alongside allow concurrent reviewing of particular lesions^37,38. By extending PVR beyond the lumen, adjacent extraluminal structures can also be visualized²⁴. In the present study, PVR was only applicable on fresh carcasses with uncollapsed structures, which permitted the reconstruction of the endoluminal view (Figure 8).

In the present overview of rendering techniques, only 8 techniques commonly used in the routine virtopsy of stranded cetaceans were described, while others were disputed due to their limited usefulness. The techniques mentioned could also give insight and be applied to other animals in general. In clinical radiology, there are many other rendering techniques and DVR templates, built on threshold-based algorithms with preset values for opacity, brightness, lighting, heat scale, window level and window width, provided in most 3D workstations. Those are designed to emphasize the illustration of different tissue types and body parts for special examinations, for instance, vascular contrast, airways, stomach or thrombus^18,24,31. However, in the case of stranded carcasses, there is gas accumulation caused by decomposition with no organ perfusion. Most DVR presets of clinical CT examination, especially CT angiography, require contrast injection and thus could not be applied in the present study. The self-designed DVR templates combined with single or multiple DVR models for cetacean PM investigation could be established after standardization of the threshold-based algorithms in terms of species and their level of decomposition. Nevertheless, based on our experience, the 8 rendering techniques listed were able to identify most of the PM findings in stranded cetaceans, and were sufficient to investigate their biological health and profile.

Preparation and scanning of carcasses is critical for subsequent postprocessing and visualization of virtopsy data. Operation of a CT machine, an ionizing radiological unit, must be performed by a certificated radiological technician or clinician in compliance with the law. Although the scanned subjects were carcasses, the radiation dose should be kept to as low as reasonably achievable. The control of scanning parameters, especially slice thickness, would highly influence the accuracy of the reconstructed coronal and sagittal planes. Moreover, reduction in CT slice thickness permits more precise diagnosis. For instance, acquiring PMCT images at 3 mm thickness may neglect a 1×1×1 mm parasitic granuloma, commonly observed in the mammary glands of stranded cetaceans. To avoid missing any finding and improve the resolution of 2D and 3D rendering, a standardized scanning protocol was used. The slice thickness was controlled at 1 mm, and down to 0.625 mm whenever possible, which is the minimum slice thickness available for the CT machine used.

A proper postprocessing visualization and manipulation of virtopsy datasets requires clear understanding of the principles and pitfalls of the common rendering techniques used for cetacean PM investigation, e.g., the identification of strength and weakness between the techniques²¹. The choice of rendering techniques depends on the anatomical structures and the underlying pathologies to be illustrated, there is no single technique that can comprehensively recognize all the PM findings. Knowing the pros and cons and choosing the appropriate rendering techniques can boost image quality and interpretability of virtopsy datasets, which aid to obtain a correct diagnosis. Carefully reviewing virtopsy datasets and correlating them with other techniques can avoid potential rendering and segmentation error¹⁸. Still, the final judgement and diagnosis should be made by veterinary radiologists or radiological clinicians who are certificated and experienced to report virtopsy findings.

Materials

Name	Company	Catalog Number	Comments
Aquarius iNtuition workstation	TeraRecon Inc	NA
Siemens 64-row multi-slice spiral CT scanner Somatom go.Up	Siemens Healthineers	NA