The Gold Standard for Immunohistochemistry

Introduction of Frozen Tissue Section

Frozen tissue sections are the standard for immunofluorescence and immunohistochemical analysis. There has been increasing attention regarding the application of immunohistochemical analysis relating to formalin fixed paraffin embedded (FFPE) tissues as it represents the samples that are available for it. FFPE tissue blocks are an invaluable resource used in translational research of cancer and other disorders. Although immunohistological methods with antigen retrieval can be applied to FFPE for a wide range of diagnostic work and research, many still think that the gold standard to authenticate immunohistochemical results are fresh cell and tissue samples. Many assume this especially when new markers and reagents are being evaluated. This is due to the assumption that formalin fixation is always bad.


The increasing use of FFPE and antigen retrieval have led to isolated reports of anomalies of immunohistochemical results between FFPE and frozen tissue sections. For example, a study by Yamashita and Okada that examined the immunostaining results of acetone and aldehyde frozen tissue sections showed that there is stronger intensity of staining of antibodies in aldehyde fixed frozen tissue sections compared to the acetone fixed tissue sections.

Based on the study by Shi et al, 2008; the traditional concept of gold standard achieved by acetone is not fully correct. Following acetone or ethanol-based fixation of tissue samples, many proteins were seen to be lost compared to the FFPE preparations. It has been acknowledged that low molecular weight proteins and lipoproteins are extracted easily by coagulant fixatives. One study showed that approximately 13% of total protein was lost after acetone fixation. It is proposed that fixatives used for coagulation dehydrates and disrupts the protein conformation which leads to denaturation and loss of function.

Compared to coagulating fixatives, formaldehyde can be used as a cross-linking fixative that fixes proteins via formation of elaborate intramolecular and intermolecular covalent cross-links. Many studies have proven that the majority of proteins can be preserved quite well through formalin fixation. This can also be seen by the abundance of immunohistochemical research that utilizes antigen retrieval treatment and recent proteomic studies that analyzed the extraction of proteins from FFPE tissue sections. Studies have also supported that extensive intramolecular and intermolecular cross-links of tissue proteins fixed through formalin is indeed a reliable way to stabilize proteins in tissue to protect against denaturation. The boiling antigen retrieval method also serves to recover and restore formalin fixed protein structures that are satisfactory and sufficient for immunohistochemical staining.

The antigen retrieval technique for immunohistochemical staining has been globally used on FFPE tissue sections. From Shi et al, 2008; they observed that FFPE tissue sections have a strong immunohistochemical signal with extra advantages of superior morphologic features along with a clean background. In a separate study, the researchers concluded that scraped cell smears from a freshly cut surface of unfixed tumor samples showed that FFPE tissue sections with the use of antigen retrieval has the best immunohistochemical results.

Currently, more literature has been reporting that immunohistochemical studies are based FFPE tissue section usage, assuming that authentication through frozen section analysis is not necessary. Ultimately, the FFPE tissue section along with an optimal AR treatment have been accepted as the standard for immunohistochemical research. Theoretically, it has been argued for a long time that frozen tissue sections ought to produce better preservation of natural proteins. However, in practice, stored frozen tissue sections probably will not maintain satisfactory localization or preservation of proteins used in the immunohistochemical staining for research of extracted proteins. There are several variables that may cause possible losses in frozen sections such as degradation, autolysis, and diffusion that happens before effective freezing; insufficient or non-uniformity of low temperature storage conditions; and the heating of tissues by compression when the frozen tissue sections are being prepared.

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However, there is a crucial issue that ought to be addressed for proteins that show negative immunohistochemical staining results when utilizing acetone or ethanol fixation of specimens but positive staining results when neutral buffered formalin fixed samples with antigen retrieval treatment are used. These variable immunohistochemical results may need to be validated by other objective methods.

Through the study by Shi et al, 2008; it was also observed that all neutral buffered formalin fixed frozen sections and FFPE tissue sections with antigen retrieval were seen to be dramatically reduced nonspecific background staining. Although the exact mechanism is yet to be clear, it is speculated that antigen retrieval treatment may have changed the overall electrostatic charge of tissue samples which leads to less nonspecific binding. The cleaner background that is achieved through formaldehyde fixation can be used in to detect disseminated tumor cells in the bone marrow. Neutral buffered formalin fixed frozen tissue sections can also be utilized to determine the degree of sensitivity or proteins to formalin fixation. In conclusion, the examination of new antigens found that it would be best to use a combination of acetone fixed frozen sections and neutral buffered formalin. FFPE tissue sections can also be considered to be the standard for the majority of antigens for immunohistochemical staining.