Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

Isolation or enrichment of small dispersed regions of interest from FFPE, or fresh frozen material, can be achieved with automated dissection more quickly and efficiently than with traditional methods. Extraction of genomic material from FFPE, or fresh frozen tissue, is a requirement for many clinical and translational research labs. Automated dissection fills a significant gap between macro dissection and laser capture micro dissection.

To begin, annotate the scanned slide images for the tumor regions of interest, using a vendor-provided viewing platform, or an open source viewer. Place the unstained sample tissue slide onto the stage in the first through fourth slide position when using a digital slide reference. Create a milling job using the automated tissue dissection software by opening Job list, then select Create new job, and enter case ID and name to enter the name of the job.

Go to Thickness, and select section thickness using the up or down arrow tab. Then, go to Tissue preparation, and select Paraffinized, or Deparaffinized. Select Reference image"From file, and Import image.

Then, select the file to import from the dropdown as the digital reference. Scan the stage by selecting the Scan stage"button in the bottom right corner to capture each sample tissue slide in the first through fourth position. For the on-stage reference, drag the box to create a rectangular area over the tissue, and select the circular bubble under the rectangular area to capture the stage reference image.

Copy this rectangle field onto the remaining sample tissue slides in the second through fourth slide positions by selecting the copy option in the upper right corner of the reference image. Then, align and resize as necessary. For the digital reference image, overlay the image on the rectangular area selected.

Then, resize and align the digital reference grossly, in course, zoom, to best match the size and the position over the sample tissue. Copy this rectangle field onto the remaining sample tissue slides in the second through fourth slide positions by selecting the copy option in the upper right corner of the reference image. Then, align and resize as necessary.

Select the Scan stage"button in the lower right corner to move into the fine adjustment step. Select the first stage position, and the Transform tool icon in the right hand tool bar, to make fine alignment and zoom adjustments of the reference to best match the sample slide overlay. Use the reference to sample sliding bar at the bottom of the screen, toggling between the reference and sample images.

And the zoom in and zoom out feature to adjust and achieve the alignment of each slide position. Once the optimal sample overlay is achieved, draw the milling path designations on the colored portion of the masked reference image by selecting the color picker tool icon in the right hand toolbar. Draw the milling paths onto the sample slides by selecting the Get annotations"button in the lower right corner of the screen.

Capture and collect the annotated region of interest with four or less milling tips when the milling path is calculated. The milling tip usage in the upper left corner is calculated based on the area covered, and the tip size selected. Tip size can be changed under the milling tip arrow, and tip usage will be recalculated.

When the milling path is selected in the first slide position, it will be copied onto the remaining slide positions. Select the Setup stage"button in the lower right corner of the screen for prompt loading of the milling tips from the collection tubes in their proper designation on the stage. Fill the reservoir with three milliliters of appropriate dissection buffer for the tissue type, and for downstream nucleic acid extraction.

Then, select the Dissect"button in the lower right corner of the screen. After the automated dissection, remove the collection tubes and the dissected sample slides from the stage. Place the collection tubes in the tube rack, and the slides in the slide rack.

The H&E stained formalin, fixed paraffin embedded, and fresh frozen mouse liver sections containing metastatic colorectal cancer in xenografts were scanned on a whole slide imager at 20x magnification, and used as the reference slides. The H&E stained reference slides were annotated and digitally masked for tumor regions for dissection and nucleic acid extraction. The post-dissected sample slides were H&E stained to confirm the dissection areas in 10 micrometer and 20 micrometer slides.

Captured metrics demonstrated the success of the automated dissection and nucleic acid extraction. When attempting this protocol, keep in mind that achieving a relatively accurate overlay of annotation onto the unstained slides is important for isolating areas of interest.