Does Cover Glass Matter in Digital Pathology?

Pathology is not infallible. Studies indicate that the absence of standardized tissue controls in immunohistochemistry (IHC) can lead to failure rates of up to 10 percent due to insufficient tissue availability. 

When comparing stained IHC slides across numerous labs, 10 to 30 percent of labs may exhibit borderline diagnostic performance or failure due to varying interpretations without established guidelines. 

With the onset of digital pathology and its already wide use across the globe, can errors occur that affect interpretation? This is a very sensitive application, and many known factors during preanalytical processing impact image quality: well-preserved and fixed tissue, tissue thickness, background staining, air bubbles or scratches, and more. Could imaging issues go beyond properly fixed tissues, staining, and clean slides? Cover glass is the first optical element that light encounters when capturing images. Can the choice in cover glass affect digital image quality? 

Glass could muddle digital images

Picture a fibroadenoma in breast tissue with multiple slides to view. Results are assessed using digital pathology, and the first slide image shows the tissue section as optically clear with brilliant, true-to-life colors that aid in an accurate interpretation. 

The second slide shows distortion and muddled colors but in an area of the tissue of great concern. An accurate interpretation cannot be made based on the poor image quality and can alter the diagnosis negatively. A known explanation for the differences in image quality cannot be determined.

The quality of cover glass may be of crucial consideration to maintain optical clarity, allowing light to pass through without distortion. Glass composition and thickness, refractive index range, and true-to-life color representation (Abbe value) are factors that may alter optical performance. While there are International Organization for Standardization (ISO) standards for each of these attributes, some cover glass options don’t adhere to the standards.

Although most are unaware of a difference in cover glass other than the size, there are two main types of cover glass: 

• Soda lime glass composition is a more economic choice but tends to have variations in glass uniformity due to how it is stretched in manufacturing, resulting in uneven dimensions. The differences in glass dimensions could result in distortion and a lower refractive index. 
• Borosilicate glass is comprised of high-quality raw ingredients, has exceptional clarity, and utilizes a down-drawn production process. This process creates a smooth, uniform surface for high-resolution imaging and a high refractive index.

Assessment of six different cover glass types has shown significant deviations from ISO standards in soda lime cover glass, according to a 2023 white paper from glass manufacture Schott called “Elevating microscopy workflows: How ISO 8255-1 compliant coverslips enhance efficiency and precision.” In many instances, deviations were found to be outside the accepted ISO tolerances, which can cause blurred or distorted imaging. Variation across one piece of cover glass can also result in warping or compromise the clarity in focal points in areas of greatest concern. 

On the other hand, analysis of borosilicate cover glass showed consistent results well within the accepted tolerance noted in ISO standards.

Cover glass influences digital pathology quality

So, which cover glass do you choose for digital pathology to achieve the best image quality? 

A higher refractive index can lead to less discoloration, and an Abbe value within certain parameters gives a true-to-life color representation. Consistent thickness and chemical purity both impact these attributes of glass. The information presented here suggests the best choice would be borosilicate cover glass.

As imaging capabilities become more advanced, in-depth studies will continue to show how the choice in cover glass can result in clear, reliable images that advance diagnostic pathology.