The overall goal of this positron emission tomography computer tomography or PET/CT image analysis procedure, is to study the disease process and potential treatment methods of Mycobacterium tuberculosis infections in non-human primates. This method can help answer key questions in the study of tuberculosis, including infection location over time, the reactivation from latency and the outcome of specific treatment regiments. The main advantage of this technique is that Mycobacterium tuberculosis infection and treatment can be tracked in real time without the need for periodic animal necropsies.
Begin by exporting the co-registered PET/CT images to the appropriate software and opening the images from the software database in the axel orientation. Click on the CT scan and change the window level, window width to CT pulmonary. The lung tissue will become dark and the anatomical features will appear lighter with the airways in black and the vasculature in white.
Focusing on one lobe at a time, scroll through the fused PET/CT image to identify the lesions. Small lesions can be distinguished from the vessels by hovering the cursor over the structure in question and scrolling up and down a slice or two. If the structure remains under the cursor, it is a lesion.
To measure a lesion of interest, remove the PET signal, so that only the CT is visible and select the length tool. Scroll until the slice that contains the largest portion of lesion is visible and draw a long line across the longest length of the lesion. The data included in this region of interest will represent the length in millimeters of the diameter of the lesion.
In the window level, window width and color lookup table menu, select set window level, window width manually in the window level, window width drop down menu and enter zero for from, and 20 for to, in the dialog box. Select the oval tool from the mouse button function tool drop down menu and scroll over the lesion to assess the hardest portion of the tissue. Then draw an oval around the lesion to obtain descriptive statistics for all of the specific threshold levels within the region in voxels.
To measure the total lung FDG avidity, click anywhere on the CT scan. Under the region of interest drop down menu, select grown region 2D 3D segmentation and set the lower threshold to 1024 and the upper threshold to 200. When the thresholds have been set, click anywhere inside the lung to highlight the entire slice in green.
In the segmentation parameters dialog box, click compute to expand the grown region from one slide to the entire lung volume. Next, click on the small icon to the left of the name of the CT scan and drag the icon to the PET scan. Then, select copy regions of interest to overlay the lungs onto the PET scan.
To fill in the high density areas of the lung that appear as gaps on the PET scan. Under the region of interest drop down menu, select brush regions of interest and closing. In the pop up window, slide the arrow to three so that the top of the box reads, structuring element radius 3 and check apply to all regions of interest with the same name.
Close any remaining gaps manually as necessary. When the entire lung is visible on the PET scan, delete all of the pixels outside of the lung, open the region of interest menu and select, set pixel values to, click the outside region of interest checkbox and set all of the pixels outside of the region of interest to zero. To isolate the hot"pathology, select set pixel values to again and click the inside regions of interest checkbox, clicking the end box, so that all of the values between zero and 2.3, are set to zero.
To make sure that only the disease pathology is measured, under the region of interest menu, select delete all regions of interest in this series and grow region 2D 3D segmentation. Change the lower threshold to 2.3 and the upper threshold to 100 and scroll through the entire PET window, clicking on any disease pathology and compute. After repeating for every area of heart disease, open the regions of interest menu and select save regions of interest, to save the whole lung region of interest.
Then export the raw data values into a spreadsheet. To determine the FDG uptake in the hot"lymph nodes, highlight the PET/CT fusion image, open the color lookup table drop down box in the toolbar and select the UCLA lookup table setting. To draw a manual region of interest around the lymph node of interest, click the drop down menu on the right side of the mouse button function in the main toolbar and select closed polygon.
Click on the edge of the lymph node to establish the first point of the region of interest, continuing to click and trace the tissue segment until the entire lymph node is almost enclosed. Then double click to close the region of interest and record the specific threshold values in a separate spreadsheet. To normalize the disease pathology FDG data values, click on the co-registered PET and CT image and scroll through the image, until the slice containing the meeting point of the main bronchial tube is reached.
Use the oval tool to draw regions of interest of approximately the same size on the back muscle and the muscle posterior and lateral to the spinal column, to obtain background specific threshold values. Track the icon to the left of the co-registered PET/CT scan to the PET window and select, copy regions of interest. The regions of interest should appear in the PET scan window.
Under the main menu, select the mode check box and make sure that max intensity projection is selected in the dropdown menu. Then confirm that the thick slab sliding scale is set to 10 and record the mean specific threshold values of the two muscle regions of interest in a new spreadsheet. Individual granulomas can be visualized for number, size and FDG uptake qualitatively, to understand the general scope of the infection process.
Counting granulomas from the images over time, allows a quantitative measure of the disease spread. For example, of the 10 animals in this representative experiment, three developed active disease and six developed latent infection. From six weeks after infection and thereafter, animals that would later develop active disease, demonstrated statistically higher numbers of granulomas than the animals that would develop latent infection.
In all of the active infection animals, an increase in FDG uptake was measured in every granuloma from three to six weeks post infection. While the animals that went on to latency, exhibited a variation in FDG uptake. With some lesions increasing, decreasing or demonstrating the same uptake over the same period.
While the FDG uptake in the mediastinal lymph nodes is similar between active and latent animals at three weeks. The lymph nodes from active animals exhibit a significantly higher uptake at six, eight and 12 weeks. Indeed, animals with a higher total lung FDG avidity are overall more likely to reactivate with over 90%of the animals demonstrating a more than 1 x 10 to the three lung FDG avidity or with at least one scan visible extrapulmonary site of infection reactivating after tumor necrosis vector neutralization in this experiment.
Once mastered, this technique can be completed in one to three hours per animal, depending on disease severity, if it is properly performed. While attempting this procedure, it is important to pay attention to the details and always save your regions of interest. Following this procedure, other assessments such as, determining the immunologic and bacteria logic parameters can be performed to answer additional questions about the relationship of the size and SUV of granulomas to infection outcome.
After its development, this technique paved the way for researchers in the field of tuberculosis to evaluate specific treatments and vaccines in a variety of non-human primate species. After watching this video, you should have a good understanding of how to qualitatively and quantitatively evaluate PET/CT scans from M tuberculosis infected non-human primates. Don't forget, that working with Mycobacterium tuberculosis can be extremely hazardous and precautions such as, wearing the appropriate bio-safety level 3 PPE and following approved protocols should always be taken while performing imaging on infected non-human primates.
