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Review
. 2014 Jun;8(4):783-98.
doi: 10.1016/j.molonc.2014.03.008. Epub 2014 Mar 21.

Garbage in, garbage out: a critical evaluation of strategies used for validation of immunohistochemical biomarkers

Gillian O'Hurley  1 , Evelina Sjöstedt  2 , Arman Rahman  3 , Bo Li  4 , Caroline Kampf  2 , Fredrik Pontén  5 , William M Gallagher  6 , Cecilia Lindskog  2
Affiliations
Review

Garbage in, garbage out: a critical evaluation of strategies used for validation of immunohistochemical biomarkers

Gillian O'Hurley et al. Mol Oncol. 2014 Jun.

Abstract

The use of immunohistochemistry (IHC) in clinical cohorts is of paramount importance in determining the utility of a biomarker in clinical practice. A major bottleneck in translating a biomarker from bench-to-bedside is the lack of well characterized, specific antibodies suitable for IHC. Despite the widespread use of IHC as a biomarker validation tool, no universally accepted standardization guidelines have been developed to determine the applicability of particular antibodies for IHC prior to its use. In this review, we discuss the technical challenges faced by the use of immunohistochemical biomarkers and rigorously explore classical and emerging antibody validation technologies. Based on our review of these technologies, we provide strict criteria for the pragmatic validation of antibodies for use in immunohistochemical assays.

Keywords: Antibody reliability; Antibody validation; Biomarker discovery; Immunohistochemistry; Workflow.

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Figures

Figure 1
Figure 1
A schematic representation of various factors which may influence the standardization and reproducibility of the IHC process.
Figure 2
Figure 2
A lung cancer TMA stained with antibodies towards PTPRC (DakoCytomation) and CD99 (DakoCytomation), utilizing both brightfield IHC (A and B) and darkfield IF (C) on consecutive sections. (A) and (B), IHC staining of PTPRC and CD99, respectively. PTPRC shows distinct cytoplasmic positivity in lymphoid cells, while CD99 is strongly expressed in both tumour cells and surrounding tumour stroma. The IHC stained images show clear tissue morphology and manual interpretation of staining intensity can be easily determined. (C), IF staining of PTPRC (red) and CD99 (green). The IF stained images show autofluorescence and not as clear morphology; however, the different dyes and antibodies can be easily distinguishable from each other.
Figure 3
Figure 3
A schematic representation of recommended techniques to use for antibody validation in high‐throughput systematic investigations, such as the Human Protein Atlas project.
Figure 4
Figure 4
A schematic representation of recommended techniques to use for antibody validation in mainstream biomarker development projects, oriented towards clinical application.
See this image and copyright information in PMC

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