Technologies in WSI have been improving for the last decade . A variety of scanners are now available with faster scanning speed and higher image quality . The usages of WSI are varied such as for developing decision support system, image analysis, education, conference and remote diagnosis. However, there are still many issues we must solve before implementation in the clinical environment such as the system stability, consistency of image quality, etc. The issues are becoming even more serious as WSI is becoming popular and used as a part of clinical practice. One of the most important issues in WSI is the color. Standardization and validation of the color of digital slides on the display is an important aspect of digital pathology implementation. While the most common reason for color variation is the variance in the protocols and practices in the histology lab, the color displayed can also be affected by variation in capture parameters (for example, illumination and filters), image processing and display factors in the digital systems themselves. All processes are very important and influence each other. Five major reasons of color variation are thickness of specimen, staining, scanner, viewer and display. To realize that the color we are looking at is not optimized or standardized is the first step towards standardization. Because no one who is involved in the process, i.e. between making a slide and displaying it, looks at the slide color at each step. Most of them are only responsible for 1-2 processes. For example, a histology technician looks at the physical stain dyes and a stained slides only; the person scanning a slide checks the stained slide and scanned image, and a reviewer looks at the images on her/his display in remote server. The advantage of WSI is to minimize the physical distance between the slide and the reviewer and also among reviewers. It is difficult to know whether the appropriate color of the WSI is displayed at the reviewers’ end, or even at a local display station. It is a huge challenge to standardize the color in the entire process (staining to displaying the scanned slides), for anyone at anywhere.
Five major causes of color issues are following.
Thickness of specimen
Generally the thickness of the specimen in the USA is 4-7 um. However this number is not measured thickness, it is targeted and expected thickness. The thickness of across the tissue section is often not uniform especially when the tissue size is relatively large such as surgical resection samples. An automated staining machine is used for H&E stain at major histology lab in most countries. Figure 1 shows the difference of stain dyes absorbance by tissue thickness. The tissues were sectioned by an automated sectioning machine to have good consistency in thickness and quality of tissue. Figure 2 shows digitized images of slides in Figure 1. Thicker tissue slides show darker and unclear details of tissue. Thinner tissue slides show clearer details with lighter color. Thus, the thickness of specimen influences the color appearance of stained slide and scanned image.
The appearance of H&E stained slide varies between laboratories or institutions. So that there is more confusion from viewing digitized slides (WSI) compared to observing the slides under a microscope because the actual stained slides can not be seen. Figure 3 shows the color variations of H&E stained slide. It has to be standardized or adjusted to the preferred color of each pathologist. Another critical effect of staining is the image analysis results. To have consistent image analysis results, the appearance of staining should be standardized.
Scanner and scanning process
Scanner and Scanning process also produce differences of color appearance. A scanner is a combination of many components such as optics, image acquisition device and image acquisition algorithm; this is the most complicated part in the implementation of color standardization. Figure 4 shows sample images highlighting the color differences between scanners. Both images in Figure 4 were displayed on the same display.
Some of viewers can show multiple WSI produced by other scanners and it is very useful functions. However, the image quality and color appearance often differ with different viewers. Figure 5 shows an example of the difference by the viewer. Original image and display are same.
Display is another cause of color variations. Recently there is a variety of display types having varying size and resolution, and each has many settings to change. There are also many choices of display cards. The matching between display card, display type and computer specification is important to see the original imaging data best. However, to attain the best performance of each device is often difficult. Most of the time, we use it inefficiently without noticing.
Figure 6 shows the example of the differences between three displays. Original image and computer connected to the 3 displays are the same.
Users including pathologists and engineers at the imaging facility [like us] have limited control of the thickness of the specimen, staining process, the color of an image which a scanner produced and the viewer. However, if it is possible to know how and how much is the acquired inaccuracy then we could optimize the displayed images. The staining condition can be controlled and standardized using spectral information. This is discussed in our other papers . Therefore, the aims of this study were to understand the variation of the color in WSI environment and to establish a simple protocol to improve the color towards Color standardization.