Manual Scanning Capabilities of the Aperio GT 450 Scanner

The manual scan interface for a slide is accessed after the Aperio GT 450 completes the standard automated scan workflow. Regardless of whether an image is successfully generated, users may launch the manual scan interface from the Slide View on the Aperio GT 450 touch screen (Figure 1). 

With the Manual Scan feature, the user can: 

• Adjust the bounding box that defines the scan area
• Add or remove focus points
• Verify that the calibration point is positioned correctly, and re-position it if needed
• Zoom into the macro image of the slide to assess potential problem areas 

When the user is finished setting these parameters, tapping “Submit” in the bottom right corner of the screen will add the slide back into the scanning queue. The user may also move this scan up in the queue by assigning priority to the rack. The manual scan interface will remain accessible for any slide while the rack remains on the scanner, allowing for quick adjustments and iterative rescans as needed.

Of the 15 identified “challenging” slides, 6 were successfully scanned with existing automated scanning workflow on the Aperio GT 450, yielding in-focus, high quality images at 40X magnification. 7 were able to be scanned with the automated workflow, but either the automated tissue finder was unable to detect all samples on the slide or had populations in differing focal planes that was beyond the capacity of Real Time Focus to resolve. 2 slides were too faint or sparse to be detected by the automated tissue finder, and no scan was generated by the automated workflow. All nine of the incomplete scans were successfully reimaged with a singular scan using the Manual Scan interface – resulting in a 100% scan rate for the samples selected.

a) Manual Scanning for Faint and Sparsely Cellular Samples

The Aperio GT 450 automated workflow utilizes a highly tuned tissue finding algorithm that identifies tissue and excludes dust, debris, and other slide preparation artifacts. However, there are cases in which the sample being scanned may be too small or sparse to be distinguished from artifacts. This can be further compounded by treatment condition and staining protocol, as shown in Figure 2. This preparation of cells was negative for the IHC marker being utilized and had only very faint uptake of the counterstain. Despite the cell pellet being hardly visible at low magnification (Figure 2A), with the manual scan feature, a 40X scan was generated with sufficient quality and resolution to distinguish cell types via nuclear morphology (Figure 2B).

b) Manual Scanning for Samples with Prominent Artifacts

Conversely, there are cases in which an artifact is too prominent or too close to the tissue to be excluded by the automated tissue finder. Such artifacts, by increasing the total scan area, contribute to increased scan time and image file size. Figure 3 demonstrates such a scenario. Excess stain and mounting media left over from slide prep resulted in a larger scan area for this lung tissue stained with a multiplex IHC panel (Fig 3A). Utilizing manual scan, a second scan (Figure 3B) was acquired, reducing the total file size by 37%.

c) Manual Scanning for Samples with Multiple Focal Planes

The Aperio GT 450 automated workflow also utilizes Leica’s patented Real Time Focus algorithm, which utilizes a feedback loop to fine adjust the objective position and create consistent focus along the length of the scan area. In our validation studies this results in a greater than 99% first time scan success rate. However, our beta test participants presented us with 7 examples of slides where the specimens were in substantially different focal planes, either due to cell type, slide preparation, or stain uptake. In Figure 4, three different cell pellets were spotted onto the slide, representing 3 different experimental treatments. The cell pellet on the rightmost side had minimal cells adhering to the slide and required annotation with permanent marker on the slide to be imaged with their current scanning workflow (Figure 4A). Scanning with the Aperio GT 450 automated workflow resulted in an appropriate scan area; however, the middle and right cell pellets were not adequately focused due to the discrepancy in focal planes from the pellet at the top of the scan area (Fig 4B). With the manual scan interface allowing placement of focal points on all 3 specimens, a whole slide image was successfully generated (Figure 4C).

In some cases, re-scanning of the entire slide may not be necessary, which can again save on scan time and image storage. In Figure 5, a small population on the original scan was out of focus relative to the rest of the slide (Figure 5A). Utilizing the manual scan feature, the user was able to capture the specific region of interest (Figure 5B).

4. Participant Feedback

Feedback on the manual scan feature was overwhelmingly positive, with users expressing their enthusiasm for the ease of use and efficacy in scanning challenging slides (Figure 6). Beyond the manual scan feature, users were impressed with several aspects of the Aperio GT 450. These included its continuous slide loading capability, the ease of loading and unloading racks, the rapid scan speed, and the 40X magnification. 

5. Conclusion – Potential of Aperio GT 450 to Enhance Productivity

In summary, the Beta test provided invaluable voice of customer feedback and confirmed the potential of the Aperio GT 450 to enhance users' productivity. The manual scan feature allows users to take advantage of the innovative automation, rapid scanning, and high-quality optics that has made the Aperio GT 450 a staple of high throughput imaging facilities, with the added flexibility to fine tune if needed. With an intuitive touch screen interface, users can quickly and easily tailor their scan setup to accommodate highly challenging samples. The Aperio GT 450 automated, high-capacity digital pathology scanner featuring manual scan is for research use only.