Improving the State of the Science of Immunohistochemistry

Immunohistochemistry in principle is no different from other techniques in molecular biology, an experimental approach for which the quality of the results rely entirely on the analyte, reagents, precise experimental technique, correct application of controls and, ultimately, accurate interpretation of the data based on all of the aforementioned factors. Despite this statement being self-evident, practitioners of immunohistochemistry nevertheless rarely afford immunohistochemistry the same attention to detail as might be the case for other techniques, and, unfortunately, immunohistochemistry often receives scant scrutiny by reviewers and editors. The consequence is that too many scientists publish poorly executed and uncontrolled immunohistochemistry, resulting in findings that are invalid or not reproducible; this carelessness subverts the respect that the technique deserves. The Histochemical Society has long championed the role of immunohistochemistry in biomedical research and considers the teaching and advancement of this technique central to its mission. Toward this end, The Histochemical Society, through formal and informal efforts, evaluated the current state-of-the-science of immunohistochemistry and will be publishing a series of articles aimed at researchers, reviewers and editors to delineate the critical elements required for the development and application of robust and valid immunohistochemical assays. The goals of these articles are to elevate the quality of immunohistochemistry in research. We seek to improve the reporting of assay methods in publications (beyond the inadequate “per standard protocol” or “as described elsewhere”), the validity of data derived from the technique, and the reproducibility of immunohistochemical assays. The goal is to maximize the unique molecular and spatial information that can be obtained from an in situ antibody binding assay.

Ultimately, this series of concise articles will provide a description of the application of scientific methodology to immunohistochemistry. Although the scientific method should never be elevated to the level of dogma, it nevertheless represents the fundamental paradigm guiding experimental biology. Here we are attempting to elevate the practice of immunohistochemistry for the benefit of all practitioners as well as the scientific community that relies on the validity of data derived from immunohistochemical methods.

Reading the current scientific literature leaves no doubt that immunohistochemistry is a powerful investigative tool but one that gets little respect. Papers that describe new biomarkers of disease routinely have as a final figure a Kaplan-Meier plot of survival based on immunohistochemical assessment of the biomarker, regardless of the method of discovery. Unfortunately all too commonly the methods sections of these articles lack sufficient detail for replication of the results, let alone replication of the test, even by those who are skilled in the field. Numerous journals have attempted to address this deficiency with well-meaning minimum standards of reporting but as a whole this has failed to improve the situation. As mentioned in the accompanying article (Hewitt et al. 2014), the lack of appropriate controls for immunohistochemical data in papers published in some high impact journals is dismaying. However, the fact that some high impact journals that publish immunocytochemical data have few or no requirements for reporting controls for immunohistochemical findings (or accept inappropriate controls) is truly a serious problem. This problem can be attenuated by proper adherence to the “Total Test” concept.

Very simply, a Total Test is the sum of its parts – the biospecimen, the reagents, the assay and the interpretation of the results. The concept of the Total Test is a paradigm used by the FDA and other regulatory agencies to evaluate a test (or assay) in clinical practice. It provides an experimental approach for dissecting test design, application and failure of an assay (Hewitt et al. 2012). Beyond its application for clinical assays, the Total Test paradigm also has utility for the application and interpretation of molecular biology based-assays. The success of the Total Test depends on treating each element of the assay with equal rigor. Only with an understanding of the specimen and quality control of its preparation, validated reagents (antibodies), a well-controlled assay, and a documented interpretation of the findings, can immunohistochemistry produce results that are reproducible and valid.

This series of articles will describe the challenges, common pitfalls and simple remedies encountered in each element of an immunohistochemical test. The articles will serve as guidelines for investigators, reviewers, and editors in the design, workflow, and valid interpretation of their immunohistochemical assays. Authors and reviewers often have conflicting views on what controls are appropriate to ensure the validity of an immunohistochemical assay. This is further complicated by the extreme diversity of the application of immunohistochemistry in biomedical research, from subcellular localization studies in living cells to clinical assays designed to predict response to a therapy. Our hope is that these articles will provide a standard of practice to encourage journals and their editors to adopt rigorous and standardized editorial policies for reporting data derived from immunohistochemical assays.