Flagship Biosciences Reports on Issues Faced by High Altitude Histology Laboratories Using Protocols Designed for Lower Elevations

Heat induced epitope retrieval (HIER) and enzyme induced epitope retrieval (EIER) are used to unmask proteins which have been cross linked by aldehyde fixatives on slide-mounted tissues that have been formalin fixed and paraffin embedded (FFPE). Cross linking of proteins with aldehyde fixatives preserves and protects them from being lost in paraffin processing, but it can also mask or prevent access to those “target” proteins keeping them from binding to their complimentary antibodies. HIER is used regularly with enzyme digestion as a way to improve the reactivity of various antigens within immunohistochemical and in situ hybridization staining reactions.

With the advent of new technologies, HIER has been incorporated into a number of autostaining machines. In Flagship’s CLIA certified histology laboratory, HIER is typically done through the online capabilities of Leica’s Bond Max autostainer. This machine improves the efficiency of the staining process and automates and regulates much of the tedium of slide preparation. Without online HIER, histologists are required to set up a heat source such as a pressure cooker, water bath, or steamer to unmask proteins before slides are stained. The offline HIER process can be problematic not only for histologists that have to complete the HIER manually, but also for researchers and funders dealing with delays and risks of inaccurate data from improperly prepared samples.

At lower altitudes, unmasking proteins by using HIER is accomplished by heating the tissue at temperatures near boiling (95-100dc is most often used) for a period of time in a buffer with a specific ph (6.5-9.0). In order to protect the tissue from damage during HIER it is important to avoid evaporation and boiling of the HIER solution. Because air pressure affects the temperature at which water boils, HIER done at high altitudes means that the usual 95-100dc temperatures used for HIER at lower altitudes can damage the tissue and should be avoided. The FDA creates specific protocols for antigen retrieval methods done in clinically approved test such as Dako’s HercepTest. These protocols define what temperatures must be reached during HIER and how long these temperatures must be maintained. The challenges to high altitude labs are in reaching and maintaining these temperatures while avoiding evaporation and boiling.

In order to better understand this issue, Flagship histologists have been involved in the monitoring and evaluation of the effects of temperature differences between high and low altitude labs for over a decade (P. Ruegg, 2001, Journal of Histopathology HER2/neu testing at altitudes above 5000 feet). Recent updated tests presented at USCAP 2013 have shown that HIER methods using 90dc, 95dc and 100dc on 3 separate antibodies showed significant performance loss above 90dc at high altitudes. The same methods were done at a partnered low elevation lab showing no performance loss. It is important to understand the environmental factors inherent to a laboratories elevation. In Colorado, water boils violently at 92dc, explaining the performance loss above 90dc.

Some high altitude labs have tried sealing water baths or insulating them to prevent tissue damage, but any success with this method depends on lower temperatures not in accordance with FDA protocol. At Flagships histology lab, temperatures are lowered during online HIER (typically from 95dc to 90dc) to prevent boiling of HIER solution and subsequent tissue damage. Because these methods are outside of the exact FDA protocols, tests are validated listing the alternative temperatures. All other protocol details are followed as closely as possible. By going the extra mile to address the issue of FDA HIER protocols being unmanageable at high altitudes, Flagship ensures that tissues are prepared properly and expeditiously.