This is Part I of a two-part series on the evolution of multiplex methods.
Over the last 5 years or so, there have been some major technological advances in the practice of histotechnology, especially compared to when I started my first histology career 33 years ago. At that time, the most common methods were routine hematoxylin and eosin (H&E) staining and special stains. The term “special stains” refer to application of histochemical dyes to stain structural elements such as collagen and muscle or microorganisms such as bacteria and fungus or other endogenous features like mucin and minerals. Around the same time, the use of immunohistochemistry (IHC), which is an antibody to epitope labeling technique and not a traditional “stain”, was growing in its development and application. It was becoming more widely used in (supportive) diagnostic and research practices; however, they were limited to single color brightfield assays using chromogens such as DAB and AEC. Some of my very first IHC assays were to detect vimentin, cytokeratin of various molecular weights and proliferating cell nuclear antigen (PCNA). These early assays were primarily used to support a diagnosis and to build our understanding of cancer differentiation and metastases. Microscopic images of these histology methods were captured using a standard 35mm camera that was mounted to a microscope. Photomicrographs were developed in a darkroom, and these used slide projector presentations and included a reference image in medical records. There was very limited use of image analysis at that time since the software packages of today didn’t exist. Most of the image analysis was done by pathology using a microscope reticle and on the photographs, using grids. These were overlaid on the pictures and cells and other structures were counted manually in addition to being qualitatively described. During the last three decades we have seen an explosion in the variety of antibodies that are available for use in IHC along with tools, such as automated platforms and detection reagents. In fact, research and diagnostic labs have benefited from the increased development and application of automated platforms that, for the most part, replace manual assays that were previously run on the bench. In parallel to the developments in automation, there has been significant improvement in the detection reagents as the technology advanced into labeled polymer detection systems. These detection chemistries are much more sensitive than those that were previously available such as the once state-of-the-art avidin\biotin complexes. As the photography industry moved away from film and embraced digital imaging, a similar movement was occurring in histology. Today we use whole slide digital scanning systems that capture brightfield as well as fluorescently labeled tissue sections. We are witnessing another evolution in histology as we embrace the value of having multiple protein targeting antibodies in the same tissue section. A few of the major drivers for multiplexing include utility in drug discovery and development, the use of image analysis software, the limited availability of tissue biopsies and the application of immunophenotyping to clinical trials. Within many diagnostic histopathology laboratories, individual IHC markers are performed on serial sections that are collected from a paraffin tissue block. The single IHC stains are evaluated by a pathologist and typically used for differential diagnosis, especially in cases with tumors of unknown origin. Since this is a qualitative or semiquantitative assessment performed by a pathologist, there is limited value to have these multiple markers on the same tissue section. However, in image analysis, we want to evaluate the same cells with the different markers in a way that is like the approach used in flow cytometry. Since the average size of a cell is between 15 and 20 µm, and each tissue section is approximately 5 µm thick, cell populations are lost as sections are cut deeper into the tissue block so the ability to track specific cells is lost. Since we are interested in quantifying the number of cells of a given phenotype, it is critical that we measure the same cell populations. This is of even greater importance in clinical trial biopsies because we are trying to measure changes in these cell populations in response to drug treatment. When considering multiplexing IHC assays there are several options. In brightfield microscopy there are chromogenic dyes that are used in combination with horseradish peroxidase or alkaline phosphatase enzymatic detection systems. These methods require anti-species secondaries that are conjugated to polymers linked with the enzymes that react with reactive dyes collectively known as chromogens. While these assays are easy to visualize using traditional brightfield microscopy and brightfield whole slide scanning, there are a few disadvantages. For example, when performing a dual or triple IHC method it is important to avoid overlapping dyes as these can create a masking effect that makes it difficult to discern colocalization. This issue is especially noted when using DAB chromogen in combination with other chromogens. The most successful multiplex IHC are those assays that use antibodies that are directed against different compartments of the cell such as the nucleus, cytoplasm or membrane. An additional point to consider is the limited dynamic range that can be measured by optical density using image analysis. To move beyond the limitations of brightfield IHC assays, immunofluorescence is being employed to a greater extent within research environments. In part II we will explore the advantages and limits of Immunofluorescence. Written by: David Krull, HT(ASCP), QIHC (ASCP)
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