We resume our journey and further discuss the various aspects of whole slide scanning such as virtual slide generation, image compression, pyramid representation along with storage and access. We hope you enjoy reading this part of our series and stay tuned for the upcoming parts on applications of digital pathology.
The process of digitization includes four sequential parts: image acquisition (scanning), storage, editing, and display of images (11). WSI uses slide scanners that consist of four main components: light source, slide stage, objective lenses, and a high-resolution camera for image capture (8,12-14).
Whole Slide Scanning
Whole slide scanners capture images of tissue sections tile by tile or in a line-scanning fashion. The multiple images (tiles or lines) are captured and digitally assembled to generate a digital image of the entire slide (15,16). WSI can be categorized as bright field, fluorescent, and multispectral and some scanners can accommodate more than one modality. Bright field scanning emulates standard bright field microscopy and is the most common and cost-effective approach. Fluorescent scanning is akin to fluorescent microscopy and is used to digitize fluorescently labelled slides (i.e., fluorescent immunohistochemistry [IHC], fluorescent in situ hybridization) (15,16). Multispectral imaging captures spectral information across the spectrum of light and can be applied to both the bright field and fluorescent settings (15-19). Line scanning exclusively uses focus maps; however, these can also be used with tile scanning. More recently, scanning processes have been developed that incorporate continuous automatic refocusing processes, further increasing the quality of scans (15-19). They have also incorporated tissue recognition features that allow for automatic detection of the histology specimen via a low-magnification overview scan (16). Different scanners vary in their scanning modality, slide-loading capacity and scan time with a capacity of holding 400 slides in high-throughput scanners (15,20). Scanning times per slide range mainly from 30 seconds to several minutes (21-23). If the camera sensor has a lower resolution than the objective’s numerical aperture allows for, information is lost. Therefore, quality of the capturing camera within a digital scanner should be taken into consideration (15-20). After digitisation, quality of scans need to be assessed as scanning artefacts can affect downstream results, and can be caused by improper cleaning of slides prior to scanning, poorly focused scans, or compensation lines from improper stitching of lines or tiles (24,25).
Whole slide scanning generates digital representations of glass slides that can be navigated in an interactive manner. The slide must be captured at sufficiently high resolution and with adequate color depth.
Many methods to reduce file size using image compression are available in WSI. Many vendors use picture formats like JPEG, JPEG 2000, or LZW compression to reduce file size, often resulting in a reduction of file size by a factor of 7 or more (26). However, information is lost in the conversion that cannot be recovered. Although morphologic assessments appear to be less affected, densitometric assessments are increasingly sensitive to this loss (27). Thus, users are discouraged from applying JPEG compression successively on the same image, as it further degrades image quality. Discarding blank regions of the slide reduces file sizes as well as scan times by identifying regions in the initial macro snapshot that do not need to be scanned (28).
Despite methods of reducing file size, a single whole slide image in practice often exceeds 1 GB in size which can be prohibitive to download and load into memory. This problem can be tackled by noting the intrinsic relationship between image scale and field of view. For large fields of view, resolution is limited by the computer monitor and therefore the image does not need to be loaded at the highest resolution. Conversely, when users examine tissue at high magnification, only a small field of view is visible on the monitor at any given time, and so the image does not need to be loaded in its entirety. Whole slide images are stored at multiple resolutions to accommodate a streamlined method for loading images. This multi-resolution representation is commonly referred to as an image pyramid. In this way, a viewer can retrieve a much smaller low-resolution component of the file when attempting to render large fields of view, therefore requiring less bandwidth to view the image. (23-28).
Storage and Access
The strategy for storing virtual slides is largely dependent on intended use. For applications with very few users and with no need for retention, local storage is often sufficient. However, if retention is important, a complete backup strategy including off-site storage, (RAID) storage, or optical/tape storage may be used. Hybrid solutions that involve local and cloud-based storage and access, or hub-and-spoke models for multisite organizations, can also be effective strategies (27-30).
Viewing and Managing Virtual Slides
WSI offers an opportunity to expand the tools available for users to include digital annotation, rapid navigation/magnification, and computer-assisted viewing and analysis (30). For example, when whole slide images are used for educational purposes, access to a dedicated image viewer enables us to annotate images for quick identification and navigation to regions of interest in the slide (30). Similarly, the use of WSI to support clinical diagnostics is often aided by the ability to view images in association with the patient’s clinical history, or alongside other slides or images that may have been acquired from the same patient (e.g., serial sections, IHC, gross photos, radiology) (31). For users who wish to apply image analysis algorithms to whole slide images, some of the viewers are packaged with algorithms that can detect cells, compute positive staining, perform regional segmentation, or perform nuclear segmentation in Hematoxylin and Eosin (H&E) images (32). Viewers often support the ability to annotate images, save regions of interest, take snapshots of selected regions, and export images to other formats. These can be integrated into department’s workflow in a seamless manner, providing on demand image analysis in conjunction with whole slide viewing (30,32,33).
Image Management Systems
Image management systems are software platforms that offer the ability to organize and access images using image metadata, patient information, or some other characteristic that can associate images into meaningful groups. For example, a common clinical workflow may organize slides in a hierarchy that provides users access to images in a manner not unlike laboratory information systems (LIS). Advanced features often include integrated image viewers and analysis routines, the ability to save and recall slide annotations, integration with information systems, storage of computed data (e.g., Human epidermal growth receptor 2 (HER2/neu score), authentication and user management, and modules that provide reports of results. As a result, image management systems are often a central component of a WSI system (28,30).
Preservation of Color Through the WSI Pipeline
Differences in color can have an influence on the diagnostic performance of pathologists. In a set of experiments examining the effect of a computer display’s age on color, Avanaki et al. (34) found that aging reduced the color saturation and luminosity of the display and produced a shift in the color point of white. Consequently, they found that the average time for pathologists to score digital slides increased from 41 to 50 seconds. Intersession percentage agreement of diagnostic scores for slides shown on a non-aged display was about 20% higher than that of aged slides. These findings indicate that preventing the degradation of color in the digitization and display process is important for optimal results. Additionally, color differences are commonly introduced by inter-display differences, which in turn can cause differences in color perception when the same slide, acquired by the same scanner and visualized in the same viewer application, is viewed on different displays. Absolute color calibration can be achieved with the use of the International Color Consortium (ICC) framework, an open, vendor-neutral, cross platform color management protocol. It begins by characterizing the whole slide scanner with a color calibration slide that contains a number of semi-transparent colored patches with known color attributes, such as that described by Yagi (35). After scanning the calibration slide, the relationship between the original color attributes of the reference patches and the values produced by the scanner is determined. This can then be characterized in the ICC source and destination profile and automatically attached to subsequently scanned slides, providing a complete reference to describe the color transformation introduced by the digitization process (36).
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Sambit K Mohanty, MD Nupur Sharma, MD