Diagnostic Immunohistochemistry: What Can Go Wrong and How to Prevent It

Abstract

Context.—There are a number of critical factors that can lead to incorrect results if the diagnostic pathologist performing immunohistochemistry is unaware of, or not vigilant about, their influence. Objective.—To highlight 3 arenas in which errors may be introduced. Data Sources.—For choosing the correct primary antibody, selection of the most appropriate antibodies for a given clinical application can be aided by obtaining information from the vendor; however, this can yield incomplete information. There are a number of online databases that have comparisons of antibodies from different vendors, particularly with respect to their use and properties. Reading the published literature can assist in this process, particularly with respect to determining antibody sensitivity and specificity, but it is a daunting task to keep up with all of the immunohistochemistry-related papers published. Finally, Web sites of a number of quality assurance organizations are accessible and can provide a wealth of information comparing the “real world” performance characteristics of different antibodies to the same target protein. False-positive signals can result from a number of factors, including the use of inappropriately high antibody concentration, and “pseudospecific” signal that is in the wrong compartment of the cell. False-negative signal can result from factors such as use of a nonoptimized epitope retrieval method. It is critical that epitope retrieval methods be optimized for each antibody employed in the laboratory. Conclusions.—By paying attention to these potential problems, the “black box” of diagnostic immunohistochemistry can be made more transparent.

HER2 immunohistochemistry in endometrial and ovarian clear cell carcinoma: discordance between antibodies and with in‐situ hybridisation

“…Although this occurred with all three antibodies, there were also considerable differences between these antibodies. Possible explanations for different antibody performances are individual antibody sensitivity and specificity, but also technical differences in staining methods (such as dilution, epitope retrieval method and incubation times) . Additionally, pre‐analytical factors such as tissue size, fixative type, fixation time and temperature during fixation and processing could influence antibody sensitivity .…”

“…Possible explanations for different antibody performances are individual antibody sensitivity and specificity, but also technical differences in staining methods (such as dilution, epitope retrieval method and incubation times). 26,[48][49][50] Additionally, pre-analytical factors such as tissue size, fixative type, fixation time and temperature during fixation and processing could influence antibody sensitivity. 59 We included samples from only two laboratories, in which tissue processing has been consistent for years.…”

“…Additionally, IHC overexpression by different HER2 antibodies can vary. 26,[48][49][50] Although concordance between HER2 antibodies in breast-and gastroesophageal cancer is high, 22,47,51 this has not been established for endometrial and ovarian cancer. Studies on endometrial and ovarian CCC to date have applied different criteria, using either IHC or ISH and often not both.…”

Sensitivity of HOXB13 as a Diagnostic Immunohistochemical Marker of Prostatic Origin in Prostate Cancer Metastases: Comparison to PSA, Prostein, Androgen Receptor, ERG, NKX3.1, PSAP, and PSMA

“…Also, this study lacks data on the Gleason scores of biopsies of primary tumors, which precludes further correlation analyses of biomarker expression. Finally, we did not aim to verify the specificity of our candidate biomarkers, which would have necessitated an additional large analysis of non-prostatic neoplasms [38]. …”

Immunohistochemical validation studies in effusion cytology: A cautionary tale

“…Antibody selection is another relevant factor in IHC assay performance. It is important that the pathologist signing out the case be aware of the sensitivity and specificity of different monoclonal antibodies because there can be a variety of clones targeting different epitopes of the same protein . For example, for TTF1, the SPT24 clone is more sensitive but less specific for the detection of all lung adenocarcinomas in comparison with the 8g7g3/1 clone .…”