Application Of Immunohistochemistry Research Articles

Looking through the same lens - Immunohistochemistry for salivary gland tumors: A narrative review on testing and management strategies

Salivary gland tumors are relatively uncommon neoplasms, presenting a diagnostic challenge for both clinicians and pathologists. When performing a biopsy, there is often an impasse between both specialists, with the clinician trying to remove as little tissue as possible and the pathologist in a perpetual quest for more. While immunohistochemistry (IHC), coupled with hematoxylin-eosin staining greatly helps in reaching a precise diagnosis and framing a management plan, there are no existing guidelines for the implementation of IHC panels in the diagnostic work-up of salivary gland tumors. The main objective of this review is to provide an up-to-date summary of the application of IHC in the diagnosis of salivary gland malignancies and the current treatment strategies. For this narrative review, we identified 50 meta-analyses and systematic reviews published between September 2013 and September 2023 using the PubMed and Google Scholar databases, with the keywords, “IHC in salivary gland tumors” and “immunohistochemistry in salivary gland tumors.” We excluded non-peer-reviewed articles, non-human studies, case reports, and series, and articles that were not in English or those for which the full text was not available; we included the remaining 18 articles in our qualitative data synthesis. To summarize the management strategy, we collated and included the key points from the National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology (Head and Neck Cancers, Version 1), 2023 and the American Society of Clinical Oncology guidelines. Our findings reflect that IHC markers, in addition to offering significant prognostic insight and guiding surgical strategy, also help plan hormonal and targeted therapies. A thorough understanding of the clinical implications of various IHC markers ensures informed multidisciplinary communication and helps the clinician and pathologist see things from a common perspective, that is, through the same lens.

Immunohistochemistry in pathology: A review

Immunohistochemistry (IHC) is an invaluable technique in pathology that allows for the localization and characterization of specific antigens within tissues using specific antibodies. Over the years, IHC has revolutionized the field of diagnostic pathology, providing critical information about the molecular and cellular composition of tissues. This comprehensive review article aims to provide an overview of the principles, methodologies, applications, and challenges of IHC in pathology. Immunohistochemistry (IHC) is a vital technique in pathology that allows for the detection and visualization of specific antigens within tissue samples. This article provides an overview of the principles, methodology, and applications of IHC in pathology. The principles of immunoreactivity, antibody selection, and detection methods are discussed, highlighting their importance in achieving accurate and reliable results. The methodology of IHC, including tissue preparation, antigen retrieval, and visualization techniques, is outlined to guide researchers and pathologists in implementing the technique effectively. The article also explores the role of antibodies in IHC, emphasizing the differences between monoclonal and polyclonal antibodies, as well as the significance of antibody validation and quality control. The applications of IHC in pathology are described, ranging from tumor classification and prognostic assessments to the detection of infectious agents and the characterization of autoimmune diseases. Furthermore, the article examines advancements and challenges in IHC, such as multiplex staining, digital pathology, quality control, and interpretation. Finally, emerging techniques, including the integration of next-generation sequencing and artificial intelligence, are discussed as future perspectives for IHC. Overall, this comprehensive overview demonstrates the indispensable role of IHC in pathology and its continuous evolution to meet the ever-expanding needs of diagnostic and research endeavors.

An overview of the detection of bovine respiratory disease complex pathogens using immunohistochemistry: emerging trends and opportunities.

The bovine respiratory disease complex (BRDC) is caused by a variety of pathogens, as well as contributing environmental and host-related risk factors. BRDC is the costliest disease for feedlot cattle globally. Immunohistochemistry (IHC) is a valuable tool for enhancing our understanding of BRDC given its specificity, sensitivity, cost-effectiveness, and capacity to provide information on antigen localization and immune response. Emerging trends in IHC include the use of multiplex IHC for the detection of coinfections, the use of digital imaging and automation, improved detection systems using enhanced fluorescent dyes, and the integration of IHC with spatial transcriptomics. Overall, identifying biomarkers for early detection, utilizing high-throughput IHC for large-scale studies, developing standardized protocols and reagents, and integrating IHC with other technologies are some of the opportunities to enhance the accuracy and applicability of IHC. We summarize here the various techniques and protocols used in IHC and highlight their current and potential role in BRDC research.

Classification of Vulvar Squamous Cell Carcinoma and Precursor Lesions by p16 and p53 Immunohistochemistry: Considerations, Caveats, and an Algorithmic Approach

There is emerging evidence that vulvar squamous cell carcinoma (VSCC) can be prognostically subclassified into 3 groups based on human papillomavirus (HPV) and p53 status: HPV-associated (HPV+), HPV-independent/p53 wild-type (HPV−/p53wt), or HPV-independent/p53 abnormal (HPV−/p53abn). Our goal was to assess the feasibility of separating VSCC and its precursors into these 3 groups using p16 and p53 immunohistochemistry (IHC). A tissue microarray containing 225 VSCC, 43 usual vulvar intraepithelial neoplasia (uVIN/HSIL), 10 verruciform acanthotic vulvar intraepithelial neoplasia (vaVIN), and 34 differentiated VIN (dVIN), was stained for p16 and p53. Noncomplementary p16 and p53 patterns were resolved by repeating p53 IHC and HPV RNA in situ hybridization (ISH) on whole sections, and sequencing for TP53. Of 82 p16-positive VSCC, 73 (89%) had complementary p16 and p53 patterns and were classified into the HPV+ group, 4 (4.9%) had wild-type p53 staining, positive HPV ISH and were classified into the HPV+ group, whereas 5 (6.1%) had p53 abnormal IHC patterns (1 null, 4 overexpression), negativity for HPV ISH, and harbored TP53 mutations (1 splice site, 4 missense); they were classified as HPV−/p53abn. Of 143 p16-negative VSCC, 142 (99.3%) had complementary p53 and p16 patterns: 115 (80.4%) HPV−/p53abn and 27 (18.9%) HPV−/p53wt. One had a basal-sparing p53 pattern, positivity for HPV ISH and was negative for TP53 mutations—HPV+ category. The use of IHC also led to revised diagnoses—HSIL to dVIN (3/43), dVIN to vaVIN (8/34), and dVIN to HSIL (3/34). Overall, 215/225 VSCC (95.6%) could be easily classifiable into 3 groups with p16 and p53 IHC. We identified several caveats, with the major caveat being that “double-positive” p16/p53 should be classified as HPV−/p53abn. We propose an algorithm that will facilitate the application of p16 and p53 IHC to classify VSCC in pathology practice.

CD229 (Ly9) a Novel Biomarker for B-Cell Malignancies and Multiple Myeloma.

Simple SummaryCD229 is a cell-surface molecule predominantly expressed on lymphocytes. Its expression in B-cell malignancies is poorly known. We tested the presence of this immunoreceptor on a large number of malignancies and normal tissue using a new monoclonal antibody and tissue microarrays. Our data show that CD229 expression is restricted to hematopoietic cells. It was strongly expressed in myeloma and marginal-zone lymphomas. Because of the high expression on multiple myeloma cells, we also analyze the presence of soluble CD229 in the sera of these patients. We showed that serum levels of soluble CD229 in myeloma patients, at the time of diagnosis, could be useful as a prognostic biomarker. Altogether, our results indicate that CD229 represents not only a useful disease biomarker but also an attractive therapeutic target.CD229 (Ly9) homophilic receptor, which belongs to the SLAM family of cell-surface molecules, is predominantly expressed on B and T cells. It acts as a signaling molecule, regulating lymphocyte homoeostasis and activation. Studies of CD229 function indicate that this receptor functions as a regulator of the development of marginal-zone B cells and other innate-like T and B lymphocytes. The expression on leukemias and lymphomas remains poorly understood due to the lack of CD229 monoclonal antibodies (mAb) for immunohistochemistry application (IHC). In this study, we used a new mAb against the cytoplasmic region of CD229 to study the expression of CD229 on normal tissues and B-cell malignancies, including multiple myeloma (MM), using tissue microarrays. We showed CD229 to be restricted to hematopoietic cells. It was strongly expressed in all cases of MM and in most marginal-zone lymphomas (MZL). Moderate CD229 expression was also found in chronic lymphocyte leukemia (CLL), follicular (FL), classic mantle-cell (MCL) and diffuse large B-cell lymphoma. Given the high expression on myeloma cells, we also analyzed for the presence of soluble CD229 in the sera of these patients. Serum levels of soluble CD229 (sCD229) at the time of diagnosis in MM patients could be useful as a prognostic biomarker. In conclusion, our results indicate that CD229 represents not only a useful biomarker but also an attractive therapeutic target.

Diagnosis of giant cell-rich bone tumors on core needle biopsy: A practical approach

BackgroundThe differential diagnosis of giant cell-rich bone tumors comprises a broad spectrum of lesions with prominent reactive osteoclast-like and/or neoplastic giant cells, with substantial differences in biologic behavior and clinical management. Evaluation of giant cell-rich bone tumors on small biopsies can be challenging especially in specimens with limited representative material. An accurate diagnosis requires a high level of skill on the part of both radiologist and pathologist as correlation with clinical and radiologic characteristics is critical. The objective of the study was to assess the utility of touch preparations (TP), immunohistochemistry (IHC) for mutation-specific markers H3G34W and H3K36M, and fluorescence in situ hybridization (FISH) for USP6 rearrangements and MDM2 amplification in the diagnostic workup of core needle biopsy specimens. MethodsA total of 27 core needle biopsies with TPs from patients with primary giant cell-rich bone tumors (16 giant cell tumors of bone (GCTBs) (including 3 with lung metastasis), 3 chondroblastomas (CBs), 4 primary aneurysmal bone cysts (ABCs), 2 non-ossifying fibromas (NOFs), 1 low grade osteosarcoma (OS), and 1 conventional OS with tumor giant cells were analyzed with IHC for H3G34W and H3K36M and in select cases FISH for USP6 rearrangements and MDM2 amplification. ResultsIn all cases the core biopsies were sufficient for histologic examination and diagnostic workup. 16 of 16 GCTBs were positive for H3G34W and negative for H3K36M, and 3 of 3 CBs were positive for H3K36M and negative for H3G34W. All other cases were negative for H3G34W and H3K36M. 4 of 4 primary ABCs showed rearrangement of USP6 by FISH and the low grade OS showed amplification of MDM2 by FISH. ConclusionsOn-site adequacy assessment of TPs proved to be an accurate, simple, and fast method for obtaining sufficient material for complete diagnostic workup. The application of IHC for H3G34W and H3K36M and FISH for detection of rearrangements of USP6 and amplification of MDM2 can improve the diagnostic accuracy in core needle biopsy specimens.

Tilapia lake virus immunoglobulin G (TiLV IgG) antibody: Immunohistochemistry application reveals cellular tropism of TiLV infection

Tilapia lake virus (TiLV) is a notable contagious agent that causes massive economic losses in the tilapia industry globally. Evaluations of the histological changes associated with TiLV infection are not only crucial for diagnosis, but also to gain an understanding of the disease. We therefore synthesized a rabbit polyclonal immunoglobulin G antibody against TiLV and developed an immunohistochemical (IHC) procedure to detect TiLV localization in the tissues of infected fish for comparison with in situ hybridization (ISH) testing. A total of four different sample cohorts derived from TiLV-infected fish was used to validate the IHC procedure. The TiLV IHC application was successfully developed and facilitated nuclear and cytoplasmic immunolabelling in the intestines, gills, brain, liver, pancreas, spleen, and kidneys that corresponded with the ISH results. Apart from the ISH results, TiLV-IHC signals were clearly evident in the endothelial cells of various organs, the circulating leukocytes in the blood vessels, and the areas of tissue inflammation. Among the tested sample cohorts, the intestines, gills, and brain had IHC-positive signals, highlighting the possibility of these organs as common TiLV targets. Immunological staining pattern and distribution corresponded with the TiLV viral load but not the inoculation route. The TiLV IHC was also capable of detecting TiLV infection in the experimentally challenged ornamental cichlids, Mozambique tilapia, giant gourami, and naturally infected tilapia, indicating the dynamic range of IHC for TiLV detection. Overall, our study delivers the first IHC platform to detect TiLV infection and provides novel evidence of cellular tropism during TiLV infection. Our findings also reveal the TiLV distribution pattern of infected fish and propose the endotheliotropism and lymphotropism of this virus, which requires further elaboration. Importantly, this new IHC procedure could be applied to study the pathogenesis and interaction of TiLV in future research.

English

BACKGROUND Cytological examination of intra-abdominal lesions is crucial in diagnosis, staging, and prognosis of suspected malignancies. Fine needle aspiration cytology (FNAC) is a routine diagnostic modality for diagnosis. FNAC is a reliable, rapid, cost effective procedure and plays an important role particularly in low resource centres. FNAC along with cell block has shown remarkable result in diagnosis of malignant lesions and its sub-classification using various antibody markers. METHODS A total of 102 cases of intra-abdominal lesions were studied over a period of one year in this descriptive study. Ultrasound sonography (USG) guided FNACs were done in 89 cases and ascetic / peritoneal fluid analysis in 13 cases. Cell blocks were made in 95 cases and immunohistochemistry (IHCs) were done in 69 cases of suspected / malignant intra-abdominal lesions to confirm the diagnosis. RESULTS Out of 102 cases, cytological smears in 89, fluid smears in 13 and cell blocks in 95 cases were available respectively. Most common intra-abdominal organ involved was liver (21.56 %) followed by retroperitoneum (17.65 %) and lymph nodes (14.70 %). The intra-abdominal lesions were classified into non - neoplastic (16.67 %) and neoplastic lesions (83.33 %). Neoplastic lesions (83.33 %) were further sub classified into benign (15.68 %) and malignant (67.65 %). IHC was done in all malignant lesions for confirmation of primary and / or metastasis. Complete concordance between cell blocks and smears was observed in 95 / 102 cases (93.14 %), while discordances were noted in 05 out of 102 cases (06.86 %). CONCLUSIONS Cell blocks play a vital role in cytopathology and as an adjunct to the routine cytology smears and body fluids. Cell buttons are cost effective and simple to make without involving much higher technicality. Cell block sections offer advantages with respect to cellular architecture, archival storage and application of IHCs which help in pin-pointing the diagnosis. Hence, cell blocks must be considered to increase the diagnostic yield and efficacy. KEYWORDS Cytopathology, Malignant Lesions, Immuno - Histochemistry, Diagnostic Utility

Immunohistochemistry as an important tool for exploring the insights of various aspects of gastro-intestinal tract

The concepts in immunology and techniques in histology have come together in a novel way to create a pioneering discipline known as ImmunoHistoChemistry (IHC), to discover new ways in detecting cell and tissue antigens related to amino acids, proteins and infectious agents by using labeled antibodies. These amalgamation techniques are applied in the disciplines of endocrinology, entero-biology, neurobiology, pathology, tumor biology and pharmaceutical research as a diagnostic tool. The simultaneous advancements in the field of imaging techniques further assisted and widened the application of IHC in molecular studies, thereby facilitating the development of novel therapeutic strategies. This paper attempts to discuss the different aspects of gastro-intestinal tract in relation to its cellular diversity, cellular differentiation, physiology and pathology, through the application of IHC methods.

Clinical Applications of Immunohistochemistry in Germ Cell Tumors in Men.

Germ cell tumors (GCT) in men comprise of tumor subtypes with distinct histomorphologies, genetic and genomic alterations, and clinical behavior. Immunohistochemical (IHC) markers, including many newly described nuclear transcription factors, are often applied in challenging cases to arrive at a correct diagnosis and classification, and to establish germ cell origin for metastatic tumors. However, there is no established role for IHC markers in prognosis and therapy response prediction in GCTs. This chapter briefly reviews the clinical utility of IHC in diagnosis and classification of GCTs, including technical aspects of performing IHC and clinical applications of commonly used IHC markers in the workup of common and clinically relevant diagnostic scenarios.

The diagnosis of primary thyroid lymphoma by fine-needle aspiration, cell block, and immunohistochemistry technique.

Primary thyroid lymphoma (PTL) is a rare malignant disease. Its prognosis depends on early diagnosis. The role of fine-needle aspiration (FNA), including smear cytology, cell block (CB) techniques, and immunohistochemistry (IHC) sections in the diagnosis of PTL is still unclear. Here we reported 19 cases of PTL and literature review to evaluate the diagnostic accuracy for lymphoma by cytology. Our study retrospectively reviewed 19 patients diagnosed with PTL at the affiliated hospital of Southwest Medical University in China from June 2011 to May 2019. According to the Bethesda system for reporting thyroid cytopathology, the CB sections were evaluated for the presence of single tumor cells. IHC was performed on CB. The diagnostic accuracy for PTL of FNA, CB with smears, and the joint application of the three methods (FNA + CB + IHC) of our study with 19 cases was 68.4% (13/19), 83.3% (15/18), and 100% (17/17), respectively. The present study demonstrates that FNA has low sensitivity in diagnosing PTL, but the joint application of FNA, CB, and IHC might provide high diagnostic accuracy for lymphoma and should be applied in all cases where the clinical suspicion is high regardless of the FNA findings.

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.

Combined Application of Immunohistochemistry and Warthin-Starry Silver Stain on the Pathologic Diagnosis of Cat Scratch Disease.

Cat scratch disease (CSD) is an infectious disorder caused primarily by the bacterium Bartonella henselae (B. henselae). Immunohistochemistry (IHC) and Warthin-Starry silver stain (WS) are considered to be indispensable to diagnose CSD in combination with morphologic characteristics. In this study, we retrieved and reviewed 46 cases of paraffin-embedded lymphadenitis with histologic and/or clinical suspicion of CSD between 2014 and 2018, and detected B. henselae by IHC and WS, respectively, and evaluated the application significance of IHC and WS for the detection of B. henselae and validated their values in the pathologic diagnosis of CSD. B. henselae was detected by IHC and WS; validation of 2 methods for detecting B. henselae was evaluated by sensitivity, specificity, false-positive rate, false-negative rate, precision, negative predictive value, and agreement rate. Microscopically, suppurative granulomas and/or multiple stellate microabscesses were observed in the accessory cortex of lymph nodes, especially near the subcapsule. Our results showed that 80.4% (37/46) of cases were positive for B. henselae by IHC, manifesting mainly punctuate, granular, or linear to outline the shape of bacteria. However, the positive rate of B. henselae by the WS method was 52.2% (24/46). There was a significant difference between IHC and WS (P=0.023). Moreover, a positive percentage of B. henselae was 97.8% (45/46), which was detected by the combined application of IHC and WS. The combination of IHC and WS exhibited high sensitivity (97.8%) and good agreement rate (86.5%). The combined application of the IHC and WS method may have important clinical advantages, which is with the highest sensitivity and agreement rate for pathologic diagnosis of CSD.

Assessment of quality and consistency of monoclonal antibodies for CB1 and CB2 in head and neck squamous cell carcinoma.

Marijuana has numerous roles as an agonist in the endocannabinoid signaling system (ESS). This study evaluated monoclonal antibodies across experimental techniques to establish a framework for studying ESS receptors, CB1 and CB2. Tissue from five patients with head and neck cancer were used to generate cell lines and formalin-fixed paraffin-embedded (FFPE) sections, which were analyzed by western blot (WB), immunohistochemistry (IHC), and immunofluorescence (IF). Subgroup analysis was performed on FFPE sections from 8 marijuana users and 10 controls by IHC. Results were compared across methods for consistency. In all patients, WB and IF were CB1 positive, whereas IHC was negative. Select samples were CB2 positive by WB, but failed IF and IHC applications. In subgroup analysis, 1 of 8 users and 3 of 10 nonusers were CB1 positive. Interpretation of CB1/CB2 antibody data should be performed cautiously and confirmation of findings across multiple experimental methods is recommended.

Immunohistochemistry-Enabled Precision Medicine.

Immunohistochemistry (IHC) can be applied to diagnostic aspects of pathologic examination to provide aid in assignment of lineage and histologic type of cancer. Increasingly, however, IHC is widely used to provide prognostic and predictive (theranostic) information about the neoplastic disease. A refinement of theranostic application of IHC can be seen in the use of "genomic probing" where antibody staining results are directly correlated with an underlying genetic alteration in the tumor (somatic mutations) and/or the patient (germline constitution). All these aspects of IHC find their best use in guiding the oncologists in the optimal use of therapy for the patients.

Best Practices Recommendations for Diagnostic Immunohistochemistry in Lung Cancer

Since the 2015 WHO classification was introduced into clinical practice, immunohistochemistry (IHC) has figured prominently in lung cancer diagnosis. In addition to distinction of small cell versus non–small cell carcinoma, patients’ treatment of choice is directly linked to histologic subtypes of non–small cell carcinoma, which pertains to IHC results, particularly for poorly differentiated tumors. The use of IHC has improved diagnostic accuracy in the classification of lung carcinoma, but the interpretation of IHC results remains challenging in some instances. Also, pathologists must be aware of many interpretation pitfalls, and the use of IHC should be efficient to spare the tissue for molecular testing. The International Association for the Study of Lung Cancer Pathology Committee received questions on practical application and interpretation of IHC in lung cancer diagnosis. After discussions in several International Association for the Study of Lung Cancer Pathology Committee meetings, the issues and caveats were summarized in terms of 11 key questions covering common and important diagnostic situations in a daily clinical practice with some relevant challenging queries. The questions cover topics such as the best IHC markers for distinguishing NSCLC subtypes, differences in thyroid transcription factor 1 clones, and the utility of IHC in diagnosing uncommon subtypes of lung cancer and distinguishing primary from metastatic tumors. This article provides answers and explanations for the key questions about the use of IHC in diagnosis of lung carcinoma, representing viewpoints of experts in thoracic pathology that should assist the community in the appropriate use of IHC in diagnostic pathology.

Characterization and Validation of Antibodies for Immunohistochemical Staining of the Chemokine CXCL12.

Chemokines and their receptors have been implicated in cancer biology. The CXCL12/CXCR4 axis is essential for the homing and retention of hematopoietic stem cells in bone marrow niches, and has a significant role in neonatal development. It is also implicated in multiple facets of cancer biology including metastasis, angiogenesis/neo-vasculogenesis, and immune cell trafficking at the tumor microenvironment (TME). Immunohistochemistry (IHC) is an ideal method for investigating involvement of CXCL12 in the TME. Three antibodies were evaluated here for their suitability to stain CXCL12. Both D8G6H and K15C gave apparent specific staining in both lymphoid and tumor tissue, but with converse staining patterns. D8G6H stained cells in the parafollicular zone whereas K15C showed staining of lymphoid cells in the interfollicular zone of tonsil tissue. Using a cell line with high CXCL12 expression, TOV21G, as a positive control, it was found that D8G6H gave strong staining of TOV21G cells whereas no staining was observed with K15C indicating that D8G6H specifically stains CXCL12. Significant staining of CXCL12 in the ovarian TME using tissue microarray was observed using D8G6H. These data demonstrate the importance of antibody characterization for IHC applications, and provide further evidence for the involvement of CXCL12 in ovarian cancer biology.

Applications of Immunohistochemistry to Endocrine Pathology.

The role of immunohistochemistry (IHC) in endocrine pathology is similar to that in other organ systems in that it can aid in the subclassification of tumors within an organ, confirm site of primary in metastatic disease, provide prognostic information, identify underlying genetic alterations, and predict response to treatment. Although most endocrine tumors do not require IHC to render a diagnosis, there are certain scenarios in which IHC can be extremely helpful. For example, in thyroid, IHC can be used to support tumor dedifferentiation, in the adrenal it can aid in the diagnosis of low-grade adrenocortical carcinomas, and in paragangliomas it can help identify tumors arising as part of an inherited tumor syndrome. This review will focus on the applications of IHC in tumors of the thyroid, parathyroids, adrenals, and paraganglia in adults.

Comparison of Immunohistochemistry and Direct Sanger Sequencing for Detection of the BRAF(V600E) Mutation in Thyroid Neoplasm.

BackgroundThe BRAFV600E mutation is the most common genetic alteration identified in papillary thyroid carcinoma (PTC). Because of its costs effectiveness and sensitivity, direct Sanger sequencing has several limitations. The aim of this study was to evaluate the efficiency of immunohistochemistry (IHC) as an alternative method to detect the BRAFV600E mutation in preoperative and postoperative tissue samples.MethodsWe evaluated 71 patients who underwent thyroid surgery with the result of direct sequencing of the BRAFV600E mutation. IHC staining of the BRAFV600E mutation was performed in 49 preoperative and 23 postoperative thyroid specimens.ResultsSixty-two patients (87.3%) had PTC, and of these, BRAFV600E was confirmed by direct sequencing in 57 patients (91.9%). In 23 postoperative tissue samples, the BRAFV600E mutation was detected in 16 samples (70%) by direct sequencing and 18 samples (78%) by IHC. In 24 fine needle aspiration (FNA) samples, BRAFV600E was detected in 18 samples (75%) by direct sequencing and 16 samples (67%) by IHC. In 25 core needle biopsy (CNB) samples, the BRAFV600E mutation was detected in 15 samples (60%) by direct sequencing and 16 samples (64%) by IHC. The sensitivity and specificity of IHC for detecting the BRAFV600E mutation were 77.8% and 66.7% in FNA samples and 99.3% and 80.0% in CNB samples.ConclusionIHC could be an alternative method to direct Sanger sequencing for BRAFV600E mutation detection both in postoperative and preoperative samples. However, application of IHC to detect the BRAFV600E mutation in FNA samples is of limited value compared with direct sequencing.

Pipetting-driven microfluidic immunohistochemistry to facilitate enhanced immunoreaction and effective use of antibodies.

Immunohistochemistry (IHC), which has been used to detect antigens in cells of a tissue section using an immunoreaction between an antibody and an antigen, is a practical tool for identifying the type and stage of diseases in cancer diagnosis and scientific research. However, conventional IHC requires long, laborious process times and high costs. Although microfluidic IHC platforms have been developed to overcome these limitations, the application of microfluidic IHC in real-world environments is still limited due to the additional equipment needed to operate the microfluidic systems. In addition, continuous flow in a microfluidic channel leads to a waste of unbound antibodies. In this study, we demonstrate a novel and easy-to-use microfluidic IHC platform operated only using a manual pipette that is commonly available in research laboratories or hospitals. No other device such as a pump or a controller is required to operate our system. Bidirectional flows of the antibody solution in a microfluidic device are induced by repetitive manual pipetting which facilitates the enhanced antigen-antibody reaction and enables the effective use of a limited amount of antibody. When breast cancer cell and tissue sections are reacted with antibodies using our platform, pipetting for less than 2 min is sufficient to obtain immunostaining results without damaging the sample. The staining intensity by our method is similar to that of the sample stained for 1 h by a conventional batch process. We believe that this pipetting-based approach to the operation of a microfluidic system allows end users to use microfluidic IHC more conveniently and easily in real-world environments.