HIER, Further, Faster

Since 1893, formalin has been the standard fixative for tissue processing in histopathology (1). It’s certainly not the only fixative available but, so far, none have managed to supplant formalin in general use. Due to the chemical’s superior preservation of morphological detail, criteria for pathological diagnoses have been established through the observation of formalin-fixed, paraffin-embedded (FFPE) tissue sections stained with hematoxylin and eosin (H&E). Formalin is a cross-linking type of fixative, meaning that it forms methylene bridges between tissue proteins (2,3). This cross-linking reaction adversely alters the proteins’ structure, resulting in loss of antigenicity (4).

Although it was 1941 when Coons and colleagues published their groundbreaking work on immunofluorescence detection of antigens in frozen tissue (5), it took decades before the use of immunohistochemistry (IHC) in surgical pathology became routine. One reason for this lag was the absence of commercially available monoclonal antibodies. Over 30 years later, Kohler and Milstein published their seminal work (6) describing the generation of hybridomas to manufacture monoclonal antibodies “to order.” Along with the development of more sensitive detection systems, hybridoma technology generated renewed interest in diagnostic IHC. But one hurdle to widespread adoption still remained; antigen detection via visibly tagged antibodies only worked well on frozen tissue sections with inferior morphological detail.

In 1991, Shi et al. overcame that hurdle with the development (7) of heat-induced epitope retrieval (HIER). By heating slides in a buffered solution, HIER critically facilitated the use of FFPE tissues for IHC, retaining the utility of existing morphologic diagnostic criteria. The new technique dramatically enriched the value of archival FFPE tissue blocks with known clinical follow-up data – creating a valuable resource for translational clinical research, basic research, and diagnostic protocol development that cannot easily be reproduced. With HIER, antigenicity could now be restored in FFPE tissue while still retaining the key morphologic features that correspond to the H&E-stained sections that form the basis of diagnostic histopathology. The incubation time with the primary antibody in the IHC protocol was also shortened to less than one hour for most antigens – a major improvement over the overnight incubations required to drive suboptimal interactions without HIER.

The complexity of the IHC protocol necessitates a properly trained, highly skilled staff to achieve accurate, consistent diagnoses. As we have increasingly realized IHC’s enhanced diagnostic utility, histology laboratories have experienced a corresponding increase in demand. Automation, and the concomitant standardization and reduction of variability, allows laboratories to achieve the quality, reproducibility, and speed necessary to meet that increased IHC demand (8).

HIER we go

HIER, also known as antigen retrieval, is based upon biochemical studies suggesting that the cross-linking reaction between protein and formalin may be reversed by high-temperature heating or strong alkaline hydrolysis (2,3,9). The routine use of HIER prior to IHC has been shown to minimize inconsistency and standardize staining (10,11). The most critical aspects that influence the quality of HIER results are heating temperature, heating time, and the pH of the retrieval solution.

What exactly is HIER? It’s the process of heating the slide-mounted specimen material in an antigen retrieval solution, followed by a cooling-off period. There is no universal solution for restoring all antigens, but the most commonly used solutions are citrate-, tris-, or EDTA-based (12,13). The pH of the HIER solution influences the IHC staining intensity significantly and is critical to the protocol’s effectiveness for the specific antigen tested (14). Some antigens retrieve better in a lower pH solution, such as a citrate buffer, while others will retrieve better in a higher pH solution, such as a tris buffer. HIER at the appropriate pH allows the antigen to regain its natural shape, improving recognition and binding by its corresponding antibody – so the composition and the pH of retrieval buffers are critical for optimal retrieval, antibody binding, and subsequent staining.

HIER works by hydrolyzing the methylene bridge cross-links formed during the formalin fixation process (12,14,15,16,17). Breaking the formalin-induced methylene cross-links in the presence of the appropriate pH solution allows the fixed protein to undergo a series of conformational changes to restore, or partially restore, its native structure – allowing the antibody to better access and bind the antigen. The entire process is driven by heat (14); the methylene cross-links are hydrolyzed when their thermal energy threshold has been reached. The effectiveness of HIER is determined by heating time and temperature; the higher the temperature, the shorter the heating time needed to attain optimal results – but heating at a higher temperature for a shorter duration yields better staining results than heating at a lower temperature for a longer time (12).

There’s no need for specialized heat sources – anything from a microwave oven to a fully automated IHC stainer will work – but, of course, some perform better than others. Laboratory pressure cookers eliminate the irregular heating and temperature variation inherent to devices like steamers and microwave ovens (12,13,16), but the best solution to inconsistency problems is an automated IHC staining platform and online heat retrieval techniques. Automated platforms standardize protocol and reduce the inter-user variability seen with manual deparaffinization, HIER, and IHC staining processes. Online processes are more reproducible, less damaging to tissue sections, and save a great deal of hands-on technician time.

Beyond the 30-slide barrier

Over the past few decades, the adoption of HIER as a standard IHC enhancement technique has revolutionized the use of IHC as a diagnostic tool for FFPE tissue examination (18). However, to date, the mechanical requirements of HIER protocols have constrained the maximum performance features of automated IHC staining instruments, limiting their throughput capabilities.

Because HIER involves applying continuous high temperatures and specially formulated buffers to FFPE tissue on glass microscope slides, automated IHC instruments that support on-board HIER commonly use under-slide heaters to indirectly apply sustained heat (8). The inefficient thermal conductivity of microscope slide glass, combined with the rapid heating and sustained temperature requirements of HIER, necessitates the use of powerful heaters – and, when more than 30 slides are simultaneously undergoing HIER, those heaters can exceed the electrical power available from standard laboratory circuits (see Figure 1). The average lab wall outlet has a maximum power draw of 1,800 W – so, to remain beneath that threshold, automated IHC staining platforms are designed to use no more than 30 under-slide heaters in parallel.

Many IHC laboratories prefer the workflow advantages and consistency of automated staining, including on-board automation of the HIER process. The downside is the “30-slide barrier,” which impacts the lab’s overall daily slide throughput – leaving them with the choice to either purchase and accommodate additional machines or switch to a more labor-intensive and error-prone manual protocol. Ultimately, we need a better solution – one that allows automation without the capacity limitations of existing processes.

In a recent article, Schwedler and Basiji outline a way to deliver more power to the HIER process – the point at which energy demand typically exceeds availability (19). Their approach uses a rechargeable lithium-ion battery to augment the available wall power and boost parallel slide processing capability by 60 percent (see Figure 2). Following the HIER process, the excess energy available from the wall outlet (shaded green in Figure 1) is used to completely recharge the battery before the next run. In the event of a power outage, the battery can even serve as an electrical

This kind of hybrid power technology will allow clinical and research laboratories to maximize throughput and consistency without the need for additional staff. Overall, the technology not only saves time and cost, but also improves patient care through faster turnaround times and enhanced diagnostic accuracy.

With ever-increasing throughput and performance demands placed upon diagnostic laboratories to offset rising healthcare costs – not to mention expectations of accurate, consistent, high-quality staining results – laboratories must, in turn, demand maximum efficiency from IHC. To maximize return from a single automated platform, rather than needing to purchase and deploy several, we’ll need to turn to innovative technology. Integrating hybrid power systems into the next generation of automated IHC staining platforms will allow laboratories to meet and even exceed throughput and performance demands while maintaining the quality of their results.