Introduction to Digital Immunohistochemistry

Digital immunohistochemistry (IHC) is a significant advancement in the field of pathology. This article looks at how digital IHC differs from traditional IHC before discussing its benefits, challenges, and future directions.

What is digital IHC?

Being able to visualize intact tissue sections with techniques such as hematoxylin and eosin (H&E) staining, in situ hybridization (ISH), and IHC can reveal how cells organize and interact to drive disease progression. However, a limitation of traditional methods is that staining intensity is traditionally scored by eye, making the analysis highly subjective. As a result, traditional methods are advancing to digital solutions, namely digital pathology and digital IHC.

Digital pathology is the process by which a glass slide is digitized at high magnification using a specialized scanner, such as the Aperio GT 450 from Leica Biosystems*, to create a whole slide image (WSI), which can be viewed on a computer screen at resolutions equivalent to a traditional microscope. Digital IHC is a subset of digital pathology, focused on IHC stained slides, whereby an image of the entire IHC stained tissue section is captured, allowing for automated analysis with specialized software.

Advantages of digital pathology

“An important advantage of using a digital slide scanner is that you can achieve faster study turnaround times by having instant access to digital slides, rather than having to transport delicate glass slides between locations,” says Naveen Chandra, Vice President of Digital Pathology at Leica Biosystems. “What is even more exciting is automated image analysis. Not only does this help remove subjectivity or human error from data interpretation, but it also lets you move from a semi-quantitative result to something that is objective and truly quantifiable.”

Another benefit of digital IHC is that it allows for the remote viewing of images. This streamlines data sharing and can facilitate education by giving researchers and clinicians access to a digital repository of pathological cases, including rare samples. Furthermore, digital IHC simplifies the process of reanalyzing markers at a later date and is much better at managing multiplexed data than traditional methods.

“The human eye isn’t great at multiplexing as there’s too much information to interpret manually,” explains Chandra. “With digital IHC, researchers can more easily study multiple markers in parallel, as well as identify subtle differences in staining intensity for a deeper understanding of their samples.”

Digital pathology challenges

A main challenge for both traditional and digital IHC stems from the lack of a “standard” IHC protocol. “There are many factors that can impact the IHC assay, including the tissue thickness, fixation time, and antigen retrieval step,” reports Chandra. To help address these issues within the realm of digital IHC, Leica Biosystems has produced a guide to generating Digital Ready Slides, which covers steps such as microtomy, slide labeling, and staining. Tips include sectioning tissues to a thickness of 3–4 μm to enable image capture in a single focal plane without the need for z-stack scanning; implementing the use of barcoded slide labels to minimize the risk of data error; and using pre-formulated reagents and automated slide processors (e.g., the BOND RX) to reduce the variability commonly seen with manual staining protocols.

A challenge more specific to digital pathology, and subsequently digital IHC, centers on information technology (IT), owing to the vast quantities of data that are produced, as well as the diversity of file types. “For those choosing to go down the digital IHC path, I recommend focusing on two key items,” says Chandra. “First, ensure your solution has an open file format. The Aperio GT 450 slide scanner exports Digital Imaging and Communications in Medicine (DICOM) images, enabling operability between systems. Second, make sure to partner with an organization that can support you in your implementation journey. This means everything from preparing the initial infrastructure to helping you adapt as your digital IHC needs grow.”

Future perspectives

Digital IHC has been widely adopted in research applications across both academia and industry, particularly in biomarker discovery and pharmaceutical development. However, the transition to the clinic is slower, with just a handful of clinically cleared algorithms/applications globally.** Despite this discrepancy, Chandra notes that the increased use of AI-based image analysis tools for pathology opens the door for a massive ramp in the clinical utility of digital IHC, with prospective future applications including tumor grading and predicting patient responses to therapy through the use of companion diagnostics (CDx).

“Digital IHC also has the potential to accelerate the development of AI-enabled CDx, heralding a new era of discoveries and treatments based on biomarker expression profiles which are not discernible by the human eye,” he says. To this end, Leica Biosystems has made a significant strategic investment in Indica Labs, wherein they will develop a holistic digital pathology platform, as well as next-generation AI-enabled CDx assays.

Getting started with digital IHC

If you’re looking to go down the digital IHC route, it pays to consider your options. Key questions to ask include the following:

• Do you require brightfield or fluorescence-based scanning?
• What level of throughput would you like to achieve?
• Are you only considering digital IHC, or are there other slide types you would like to scan?
• Do you have the necessary IT infrastructure for a digital IHC platform?
• How much vendor support will you need?
• Do you want to integrate your digital slide scanner with automated upstream processing?

For answers to all these questions and more, researchers are advised to speak with an experienced provider of digital pathology solutions, such as Leica Biosystems. By guiding you toward a platform that not only meets your immediate needs, but also delivers on your future requirements, a digital pathology specialist can help you to maximize the amount of data obtained from a single, precious tissue section.

* For research use only. Not for use in diagnostic procedures.

** Market dependent