The Use of Immunohistochemistry for Biomarker Assessment—Can It Compete with Other Technologies?

Abstract

A morphology-based assay such as immunohistochemistry (IHC) should be a highly effective means to define the expression of a target molecule of interest, especially if the target is a protein. However, over the past decade, IHC as a platform for biomarkers has been challenged by more quantitative molecular assays with reference standards but that lack morphologic context. For IHC to be considered a ''top-tier'' biomarker assay, it must provide truly quantitative data on par with non-morphologic assays, which means it needs to be run with reference standards. However, creating such standards for IHC will require optimizing all aspects of tissue collection, fixation, section thickness, morphologic criteria for assessment, staining processes, digitization of images, and image analysis. This will also require anatomic pathology to evolve from a discipline that is descriptive to one that is quantitative. A major step in this transformation will be replacing traditional ocular microscopes with computer monitors and whole slide images, for without digitization, there can be no accurate quantitation; without quantitation, there can be no standardization; and without standardization, the value of morphology-based IHC assays will not be realized.

When Tissue Antigens and Antibodies Get Along

“…Two factors contribute to the use of formaldehyde as the fixative of choice for most histologic procedures: (1) for more than a century, pathologists have studied tissues fixed in formalin and have become accustomed to histologic examination through the artifacts it produces, and (2) archived paraffin blocks, an invaluable resource for disease studies, most commonly contain formalin-fixed tissues. 85 Formaldehyde preserves mainly peptides and the general structure of cellular organelles. It also interacts with nucleic acids but has little effect on carbohydrates.…”

Whole Slide Imaging for Analytical Anatomic Pathology and Telepathology: Practical Applications Today, Promises, and Perils

“…Developing techniques such as immunohistochemistry (IHC) and molecular diagnostics/theranostics have brought up the necessity and ability to measure discrete molecules or ''biomarkers'' within tissue; however, there are a number of analytical variables that can affect the results of these tests, including the accurate measurement of signals. 3,4 Given this inherent variability, there have been efforts to standardize these tests, and the US Food and Drug Administration, American Society of Clinical Oncology, and College of American Pathologists (CAP) have each provided special direction on some testing (eg, human epidermal growth factor 2 [HER2/neu]). 5,6 Whole slide imaging (WSI) with algorithmic analysis is one potential technology to improve the assessment of such markers (as shown in the Table).…”

Commentary

“…Therefore, a well-optimized staining protocol is essential not only to address the mere presence or absence of a biomarker but also to answer more detailed and sophisticated questions such as the intracellular compartmentalization, degree of expression, and sometimes colocalization with another biomarker (Dunstan et al 2011). Keystones for creating and sustaining well-optimized staining protocols that produce consistent staining results are the use of automated histostainers and the regular use of within-run controls, especially inclusion with each staining run of a positive control slide cut from the same tissue block to compare for consistency in labeling between staining runs (Dunstan et al 2011). A pathologist is best suited to assess that staining occurs in the expected cellular compartment of the target tissue and is absent in the nontarget tissues also present on the slide.…”

“…Even parameters that can be altered are often part of a ''black box'' solution, in which the end user cannot always predict how changing a certain setting will affect the algorithm performance or the final data extracted (Webster and Dunstan 2014). In contrast, other commercially available solutions are very complex and provide such a plethora of options for algorithm modifications that they require image analysis experts to be part of any analysis team in order to use the software to the extent of its abilities (Webster and Dunstan 2014;Dunstan et al 2011). In our experience, when used in a high-throughput setting, image analysis software requires the ability to manipulate multiple parameters to define the most suitable algorithm and necessitates the cooperation of a pathologist and experienced analytical scientist to achieve the best solution in the shortest time frame.…”