Multiplexed immunohistochemistry, imaging, and quantitation: A review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis

Abstract

Tissue sections offer the opportunity to understand a patient's condition, to make better prognostic evaluations and to select optimum treatments, as evidenced by the place pathology holds today in clinical practice. Yet, there is a wealth of information locked up in a tissue section that is only partially accessed, due mainly to the limitations of tools and methods. Often tissues are assessed primarily based on visual analysis of one or two proteins, or 2-3 DNA or RNA molecules. Even while analysis is still based on visual perception, image analysis is starting to address the variability of human perception. This is in contrast to measuring characteristics that are substantially out of reach of human perception, such as parameters revealed through co-expression, spatial relationships, heterogeneity, and low abundance molecules. What is not routinely accessed is the information revealed through simultaneous detection of multiple markers, the spatial relationships among cells and tissue in disease, and the heterogeneity now understood to be critical to developing effective therapeutic strategies. Our purpose here is to review and assess methods for multiplexed, quantitative, image analysis based approaches, using new multicolor immunohistochemistry methods, automated multispectral slide imaging, and advanced trainable pattern recognition software. A key aspect of our approach is presenting imagery in a workflow that engages the pathologist to utilize the strengths of human perception and judgment, while significantly expanding the range of metrics collectable from tissue sections and also provide a level of consistency and precision needed to support the complexities of personalized medicine.

Immunohistochemistry and mass spectrometry for highly multiplexed cellular molecular imaging

“…23 immune reaction. New multiplexing techniques that combine tyramide-signal amplification and multispectral imaging are being developed and permit complex analyses of the spatial interactions between tumors and many classes of immune cells, eg, see Stack et al 3 Prostate cancer diagnostics could also benefit from clinically directed multiplexing, as core biopsies could be routinely examined using antibodies defining basal and luminal cells and cells with upregulated racemase, 37,38 as well as antibody panels that might survey protein products of the Genomic Health prostate panel of 17 genes that has been developed to stratify patients into progression-risk categories. 39 It is difficult to get an accurate evaluation of the falsepositive and false-negative rates of prostate core biopsies, but it is clear that the use of combined 3-antibody IHC has become much more frequent and has probably decreased these rates.…”

“…Although two or more targets can be visualized simultaneously using different chromogens and amplification schemes, colorimetric detection of more than three antigens using multiple enzyme-linked secondary antibodies is challenging. 3 In practice, chromogenic multiplexing is usually limited to two targets because of difficulties encountered in sample preparation and imaging. Beyond this relatively low multiplexing ceiling, IHC has additional shortcomings.…”

Novel Platforms of Multiplexed Immunofluorescence for Study of Paraffin Tumor Tissues

“…It is an enzymelinked signal amplification method that conventional is using to detect and localize low copy number of proteins present in tissue by IHC, using most commonly alkaline phosphatase or horseradish peroxidase (HRP) to catalyse the deposition of labelled tyramide molecules at the site of probe or epitope detection. Tyramides can be conjugated to biotin or fluorescent labels and labeled with streptavidin enzyme HRP 6,18 . The HRP catalyzes the formation of tyramide into highly reactive tyramide radicals that covalently bind to electron-rich tyrosine moieties on FFPE tissue.…”

“…The HRP catalyzes the formation of tyramide into highly reactive tyramide radicals that covalently bind to electron-rich tyrosine moieties on FFPE tissue. Tissue surfaces with anchored biotinylated tyramide, must be further treated with fluorescent or enzyme tagged proteins that have a high affinity for biotin as streptavidin before to microscopic visualization 6,18 . The detection of the proteins is increased more than 10-times compared to standard biotin-based staining methods 19 .…”

“…Attempts are presently being made to develop even more comprehensive IHC-based technologies that allow simultaneous visualization of an even larger number of antigens from a single tissue section, as well as to streamline, automate, and reduce the time expended on tissue staining and processing. Multiplexed methods help achieve these technological goals to ultimately enhance disease diagnosis and better inform timely patient care 6 .…”