Technical pitfalls potentially affecting diagnoses in immunohistochemistry

Abstract

Result of the immunohistochemical reactions routinely used in diagnostic surgical pathology should be properly interpreted, since false results, related to technical and interpretative pitfalls may lead to incorrect diagnosis. The main sources of such pitfalls are reviewed, analytically described and related to different steps (fixation, tissue processing and embedding, decalcification, antigen retrieval) which may affect the accuracy of immunohistochemistry. In addition, the presence of endogenous enzyme activity, improper binding of avidin to endogenous biotin, incorrect use of antibodies, chromogen and detection systems, as well as incorrect interpretation may produce unreliable data. The high frequency and extension of such pitfalls make mandatory the use of internal and external controls and adoption of cross-validation programmes. The present study, supported by an extensive review of the related literature, is intended as a guideline leading to proper interpretation of immunohistochemical data, an essential component of the diagnostic process. Experience on the antigen retrieval procedures for different antigens is also presented.

Improved decision making for prioritizing tumor targeting antibodies in human xenografts: Utility of fluorescence imaging to verify tumor target expression, antibody binding and optimization of dosage and application schedule

“…The tissue fixation and staining problems that occur with in situ hybridization and IHC of human tissue samples were addressed in previous work. 9,10,12,[48][49][50] We confirmed the IHC results in a preclinical NSCLC tumor xenograft model, illustrating high HER2 staining variability for different anti-HER2 detection antibodies, with staining intensities ranging from 0C to 3C, and also the substantial effect of the selected antigen retrieval buffer on receptor staining results. In addition to these fixation and antigen retrieval problems, the fact that TAAs can be modulated during the growth of tumor cells must be taken into consideration.…”

“…The quality, reproducibility and validity of immunostains depend substantially on 3 major factors: 1) tissue fixation and processing; 2) unmasking of epitopes, which is also termed antigen retrieval (AR); and 3) the affinity and specificity of the primary detection antibody to the relevant epitope. [9][10][11][12][13][14] In particular, the loss of immunoreactivity of TAAs after fixation is a critical factor in FFPE tissue section. The chemical fixation process can mask and modify antibody binding sites, resulting in staining variations that may lead to an inaccurate determination of the antigen expression level.…”

A 92-gene cancer classifier predicts the site of origin for neuroendocrine tumors

“…Protein IHC has additional variation in antibody retrieval methods and antibody clone differences, as well as wide variation in techniques and controls used between labs; all these factors can all affect the rate of false positive and false negative IHC results. [68][69][70][71][72][73][74] In addition, selection of specific IHC markers and the interpretation of the protein expression patterns can be subjective and is dependent on the individual pathologist.…”

Association of the stramenopilan protists, the aplanochytrids, with zooplankton of the equatorial Indian Ocean

“…Care was taken to inhibit endogenous ALP activity by pre-treatment with DTT, a strong reducing agent. In addition, the specimens had been fixed in formalin, which destroys endogenous alkaline phosphatase activity (Bussolati & Leonardo 2008). Despite these precautions, the streptavidinalkaline phosphatase conjugate appeared to yield false positives wherein the colour reaction appeared in a diffused manner in many parts of the body of the zooplankton (Fig.…”