Pitfalls In Immunohistochemistry Research Articles

Algorithmic approach of immunohistochemistry in metastatic carcinoma of unknown primary site

Cancers of unknown primary site (CUPs) are histologically confirmed metastatic carcinoma for which primary site cannot be identified after standard diagnostic approach. It comprises 3-4% of all malignant neoplasms. The broad tumour type for CUPs is carcinoma. Metastatic tumours are more difficult to classify than primaries by Immunohistochemistry (IHC). Integration of morphology & IHC is the gold standard at diagnosing CUP. It is important to be aware of possible pitfalls of IHC and pay attention to correct interpretation. One should have a working algorithmic approach to identifying and classifying CUPs.

Interpretation pitfalls in immunohistochemistry of primary liver carcinoma: a retrospective analysis of liver biopsy specimens

Background: The diagnosis of primary liver carcinoma (PLC) primarily relies on routine histopathology with hematoxylin and eosin (H&E) and the supportive evidence of immunohistochemistry (IHC). Inappropriate use of immunostains without either careful histomorphological evaluation or clinical correlation may lead to misdiagnosis and inappropriate patient management. Objective: To retrospectively analyze the interpretation pitfalls in IHC as an adjunct in assessing PLCs in liver biopsy specimens at a tertiary hospital in Thailand. Materials and Methods: The archives of the Division of Pathology, Thammasat University Hospital, were searched between 2015 and 2020 using a search tool from the pathology software system and a combination of codewords, including PLC, hepatocellular carcinoma (HCC), and intrahepatic cholangiocarcinoma (iCCA). Histomorphology of PLCs in liver biopsy specimens and the corresponding immunophenotypes were retrospectively analyzed. Clinical data for each patient was retrieved from the electronic medical record. Results: One hundred fifty-three liver biopsy specimens were retrieved. There were 128 (83.7%) iCCA, 23 (15%) HCC, and 2 (1.3%) combined hepatocellular-cholangiocarcinoma (cHCC-CCA). Six cases (3.9%) with interpretation pitfalls in IHC were analyzed. These pitfalls included incorrect subtyping of PLCs in two cases (1.3%) and PLCs misdiagnosed as metastatic cancers in four cases (2.6%). Conclusion: Interpretation pitfalls in IHC of PLCs were noted in 3.9% of the present study cohorts. Pathologists should be familiar with the histomorphology of PLCs together with their rare variants. Appropriate use of IHC as adjuncts for evaluating PLCs and correlation with clinical details were essential for rendering the correct diagnoses of PLCs. Keywords: Immunohistochemistry; Primary liver carcinoma; Interpretation

Application and Pitfalls of Immunohistochemistry in Diagnosis of Challenging Genitourinary Cases.

Immunohistochemistry (IHC) has become increasingly important in the evaluation of pathologic conditions in the genitourinary (GU) organs. In addition to careful evaluation of hematoxylin-eosin sections and generation of a differential diagnosis, choosing the optimal panel of IHC markers becomes even more important when the biopsy material is very limited. The following summary of our experience supplemented with relevant literature review exemplifies how to use IHC to facilitate pathologic diagnosis in the GU system. To describe our experience with the most common immunohistochemical markers used in GU pathology. Institutional experience and literature search comprise our data sources. Application of IHC provides enormous benefits to the interpretation of GU pathologic conditions, including benign and malignant lesions. However, both insufficient and excessive types of use of IHC, as well as incorrect interpretations in common and rare GU conditions, could present pitfalls in diagnosis.

Avoiding pitfalls in diagnostic immunohistochemistry-important technical aspects that every pathologist should know.

This review focuses on technical aspects of diagnostic immunohistochemistry (IHC), with an emphasis on aspects of methodology and interpretation that may be problematic for practicing pathologists. Pitfalls in IHC are reviewed, and the importance of good controls and the the use of multi-tissue controls is discussed. Also covered is the optimal use of IHC in cytologic specimens and specimens where no paraffin block is available. Artifacts encountered in IHC are discussed and illustrated, and a number of useful techniques are described in detail, including preparation of multi-tissue control material, tissue and cell transfer techniques, and tissue protection techniques.

Utility and pitfalls of immunohistochemistry in the differential diagnosis between epithelioid mesothelioma and poorly differentiated lung squamous cell carcinoma

The aims of this study were to clarify the usefulness of immunohistochemistry in the differential diagnosis of epithelioid mesothelioma with a solid growth pattern [solid epithelioid mesothelioma (SEM)] and poorly differentiated squamous cell carcinoma (PDSCC), and to confirm the validity of a specific type of antibody panel. Additionally, we aimed to clarify the pitfalls of immunohistochemical analyses. Formalin-fixed paraffin-embedded specimens from 36 cases of SEM and 38 cases of PDSCC were immunohistochemically examined for calretinin, podoplanin (D2-40), Wilms' tumour gene product (WT1), cytokeratin (CK) 5/6, p40, p63, carcinoembryonic antigen (CEA), epithelial-related antigen (MOC31), claudin-4, thyroid transcription factor-1 (TTF-1), and napsin A. WT1 showed the highest diagnostic accuracy (85.1%) as a mesothelial marker, and CEA, p40 and claudin-4 showed higher diagnostic accuracies (95.9%, 94.6%, and 93.2%, respectively) as carcinoma markers. Calretinin (diagnostic accuracy: 75.7%), D2-40 (diagnostic accuracy: 67.6%), CK5/6 (diagnostic accuracy: 63.5%), TTF-1 (diagnostic accuracy: 55.4%) and napsin A (diagnostic accuracy: 52.7%) could not differentiate between SEM and PDSCC. Among these markers, the combination of calretinin and WT1 showed the highest diagnostic accuracy (86.5%) as a positive marker, and the combination of p40 and CEA showed the highest diagnostic accuracy (97.3%) as a negative marker. The combination of CEA and claudin-4 also showed relatively high diagnostic accuracy (94.6%) as a negative marker. We recommend the combination of WT1 and calretinin as a positive maker, and the combination of CEA and claudin-4 as a negative marker, for differential diagnoses of SEM and PDSCC.

135IN - The Importance of the Preanalytical Phase for Immunohistochemistry

ABSTRACT The importance of the preanalytical phase for immunohistochemistry Preanalytical treatment of tissues was studiedto clarify pitfalls in immunohistochemistry (IHC) and, in general, in molecular analysis in fixed and paraffin-embedded tissues, which are the only tissues available for any patient for diagnostics and for clinical research. Specific attention was given to this issue because of the important role recently played by the biomarkers predictive of treatment efficacy, and not only for diagnosis definition and prognosis evaluation. The preanalytical phase is quite complex and is not only related to pathologists but also to surgeons. The warm ischemia time, the use of specific techniques or simply the traction and distortion of tissues can influence gene expression modification, protein coagulation or their diffusion into extracellular spaces with consequences for IHC results. Warm ischemia and tissue transport conditions can differently influence genes with increased or decreased expression, with loss of reactivity in IHC. On the other hand, many genes can be totally indifferent to these conditions. IHC reliability can also be affected by common pathological processes such as hemorrhagic diffusion in tissues, necrosis, inflammation or apoptosis. Antigenetic epitopes of proteins are modified by fixation in formalin with the formation of methylenic bridges among different aminoacid residues, but also tertiary and quaternary structures are affected. Protein alterations are quantitatively related to time of fixation and enzymatic or heat related demodification procedures for antigen retrieval. This variability should be carefully taken into account. An insufficient time of fixation can also affect IHC results, because tissue protein alteration is related to over-fixation, but at the same time the inner part of the tissue is altered by hypoxia with initial autolytic phenomena due to released lysosomal enzymes. These hypoxic areas can also be affected by inclusion procedures and alcohol treatment coagulates the proteins. Inclusion process temperatures can also affect the same more sensitive antigens. All these possible alterations, together with methods and interpretation pitfalls, make internal and external quality controls and audit procedures mandatory for IHC laboratories. Disclosure: The author has declared no conflicts of interest.

Indications and pitfalls of immunohistochemistry in head and neck cancer

Immunohistochemistry (IHC) has been employed in the differential diagnosis of tumors. ObjectiveTo assess the use of IHC in cases of head and neck tumor. MethodThis is a retrospective study of the cases included in the Cancer Registry of the institution. ResultsIHC was used in 76 (11%) of 704 pathology tests. Most cases were carcinomas (85.80%), and 83.66% of them were squamous cell carcinomas. All tests were done with diagnostic purposes. The most frequently used antibodies were 34BE12 (37.18%), AE1/AE3 (35.9%), 35BH11 (28.21%), CD45 (25.64%), CD20 (24.36%), CD30 (24.36%), CK7 (23.08%) and CD3 (23.08%). ConclusionsIHC was used in 10.67% of the head and neck tumor cases submitted to pathology testing, mostly for carcinoma (5.26%). In the determination of squamous cell carcinoma, IHC accounted for 18.42% of all tumors.

Is my antibody‐staining specific? How to deal with pitfalls of immunohistochemistry

Immunohistochemistry is a sensitive and versatile method widely used to investigate the cyto- and chemoarchitecture of the brain. It is based on the high affinity and selectivity of antibodies for a single epitope. However, it is now recognized that the specificity of antibodies needs to be tested in control experiments to avoid false-positive results due to non-specific binding to tissue components or recognition of epitopes shared by several molecules. This 'Technical Spotlight' discusses other pitfalls, which are often overlooked, although they can strongly influence the outcome of immunohistochemical experiments. It also recapitulates the minimal set of information that should be provided in scientific publications to allow proper evaluation and replication of immunohistochemical experiments. In particular, tissue fixation and processing can have a strong impact on antigenicity by producing conformational changes to the epitopes, limiting their accessibility (epitope masking) or generating high non-specific background. These effects are illustrated for an immunoperoxidase staining experiment with three antibodies differing in susceptibility to fixation, using tissue from mice processed under identical conditions, except for slight variations in tissue fixation. In these examples, specific immunostaining can be abolished depending on fixation strength, or detected only after prolonged postfixation. As a consequence, antibody characterization in immunohistochemistry should include their susceptibility towards fixation and determination of the optimal conditions for their use.

Biomarkers in diagnostic obstetric and gynecologic pathology: a review.

Until recently, the histologic diagnosis of obstetrical and gynecologic neoplasia was based principally on morphologic criteria. However, interobserver reproducibility for entities such as squamous intraepithelial, endometrial, and trophoblastic disease varies widely between observers. This inherent variability in interpretation between individuals has led to wide ranges in diagnostic precision between practices, and in many cases, between recognized experts. The advent of immunohistochemistry, and the more recent accelerated discovery of new genes and their functions has resulted in the discovery of cellular proteins or nucleic acids that are differentially expressed in tumors. When applied in conjunction with existing histologic criteria, these "biomarkers" have the potential to enhance diagnostic consistency and reproducibility. The gains expected are to practicing diagnostic pathologists (who will enjoy greater diagnostic consistency) and to academics (for whom biomarkers may uncover new pathways unappreciated by histologic diagnosis alone). However, fundamental to the success in both arenas will be critical analysis of the potential pitfalls in immunohistochemistry, strict validation of new markers as they arrive in the field, and a realistic view of their value in the laboratory management of obstetrical and gynecologic diseases.