During the past twenty-five years, immunohistochemistry (IHC) has progressed from a technique that was limited to specialized laboratories with strong research backgrounds to a technique that is commonly performed in most pathology laboratories. In fact, IHC has become an invaluable technique in the diagnostic pathology laboratory. The value of IHC is due to the power of this procedure to recognize and localize specific proteins (markers) within a tissue specimen. By determining the presence or absence of specific markers within a tumor the pathologist is provided with clues that may be helpful in accurately classifying or identifying the neoplasm.
The value or power of IHC frequently is compromised by tissue fixation and processing. While formalin based fixatives may provide excellent preservation of morphology, cytology and tissue architecture, formalin fixation greatly diminishes the sensitivity of the IHC technique. Formaldehyde covalently binds to tissue protein and also acts to crosslink adjacent proteins or peptides to form large aggregates of proteins. The cross-linking of proteins and peptides to the antigen is believe to block or “mask” the epitope and thus hinder the binding of the antibody.
Heat induced epitope retrieval (HIER) may be defined in the simplest terms as the use of heat coupled with specific buffered solutions to recover antigen reactivity in formalin fixed paraffin embedded tissue. The HIER procedure has had a huge impact on the way IHC is utilized in the diagnostic process. Many markers that are routinely analyzed by IHC in formalin fixed tissue could not be evaluated in such a manner 15 to 20 years ago.
Mechanisms of HIER
Although the mechanisms by which HIER act at are not clear, most agree that the procedure by some means reverses the formaldehyde mediated chemical modifications of the antigen. There are a number of theories as to how this may occur. Foremost among these theories is the belief that the thermal energy of the HIER process breaks the cross-links that binds surrounding proteins or peptides to the antigen. In this scenario HIER acts to “unmask” or open the epitope. In another theory, HIER is believed to act by removing bound calcium ions from the sites of cross-links. This theory is supported by the fact that several HIER buffers such as citrate and EDTA function as calcium chelators.
A variety of heating sources including the microwave, vegetable steamer, pressure cooker and water bath all have been used successfully for HIER. The temperature achieved by the sources is a critical factor in the HIER process. In general, the higher the temperature of the HIER solutions, the more effective the recovery of the epitope.
The steamer, water bath and microwave produce temperatures in the 94°C to 100°C range. Pressure cookers are capable of generating temperatures of 110-120°C. The differences in temperature achieved by these sources may be compensated for by adjusting the duration of the heating period. The lower the temperature, the longer the heat exposure required to produce an equivalent intensity of staining observed with a higher temperature. Thus, the appropriate adjustment of the heating time to compensate for maximum temperature differences allows for the use of any of the listed heat sources to produce comparable staining intensities.
While each of the heating sources is suitable for HIER, there are advantages and disadvantages associated with each source (Table). The microwave, which was the first widely used HIER heat source, is perhaps the least used of the heat sources today. This distribution of heat within a microwave frequently is uneven or inconsistent. Uneven heating results in a lack of reproducibility with respect to staining intensities. An additional drawback to the use of the microwave is the violent boiling that frequently occurs with this heat source. The agitating action from the boiling HIER solution can lead to tissue detachment from the slides.
In contrast to the microwave, the pressure cooker, steamer and water bath produce uniform and consistent heat distribution. While the higher temperatures produced by the pressure cooker are advantageous in that an effective recovery of epitope reactivity can be achieved in a short period of time, the higher temperature may damage or distort morphology. Connective tissue may appear shredded or burnt when using the pressure cooker.
Table 1: Heat source comparison
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Since the development of HIER, a wide range of buffered solutions have been employed. Currently, HIER solutions can be grouped in three categories based on pH and buffer compositions:
- Low pH (pH ~3-5) solutions frequently buffered by glycine-HCI.
- Low to neutral pH (pH ~ 6-7) solution buffered with citric acid.
- High pH (pH~ 8-10) buffered by Tris or EDTA.
Current evidence suggests that the pH of the HIER solutions is more important than the composition of the buffer. Optimal recovery for most epitopes occurs in alkaline buffers with a pH range of 8-10. EDTA buffers are particularly effective on over-fixed specimens and for the recovery of hard to detect antigens. The high pH and EDTA based buffers are not without drawbacks. Higher pH solutions are more likely to cause loss of sections from microscope slides. In addition EDTA solutions may result in distorted morphology as well as convoluted and bizarre shaped nuclei. At this time, there is no “universal” HIER buffer that is optimal for all antigens. Each laboratory should evaluate the effectiveness of different HIER solutions on the recovery of the various antigens commonly evaluated in the laboratory. A common approach is the use of a buffer such as citrate for most antigens. The high pH or EDTA based solution may be reserved for those antigens which may be difficult to retrieve with citrate.
The Role of the Histotechnologist
The HIER technique presents the technician or pathologist with a bewildering array of choices concerning heat sources and buffers. These choices or questions may be simplified by understanding the advantages and disadvantages of the equipment and basic reagents used in the process. These choices should be made in the context of the performance criteria or expectations set forth by the laboratory. The technician and pathologist should at a minimum understand the potential impact of different heat sources or retrieval buffers on reproducibility, sensitivity and specificity. In addition the potential detrimental effects of the heat sources and buffers should be considered.