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Methods in Molecular Biology (Clifton,... 2019Immunofluorescence (IF) is an important immunochemical technique that allows for detection and localization of a wide variety of antigens in different types of tissues... (Review)
Review
Immunofluorescence (IF) is an important immunochemical technique that allows for detection and localization of a wide variety of antigens in different types of tissues of various cell preparations. IF allows for excellent sensitivity and amplification of signal in comparison to immunohistochemistry, employing various microscopy techniques. There are two methods available, depending on the scope of the experiment or the specific antibodies in use: direct (primary) or indirect (secondary). Here, we describe preparation of specimens preserved in different types of media and step-by-step methods for both direct and indirect immunofluorescence staining.
Topics: Antibodies; Antigens; Fluorescent Antibody Technique; Fluorescent Antibody Technique, Indirect; Humans; Immunohistochemistry; Staining and Labeling
PubMed: 30539454
DOI: 10.1007/978-1-4939-8935-5_26 -
Methods in Molecular Biology (Clifton,... 2019Immunohistochemistry (IHC) is a powerful technique that exploits the specific binding between an antibody and antigen to detect and localize specific antigens in cells... (Review)
Review
Immunohistochemistry (IHC) is a powerful technique that exploits the specific binding between an antibody and antigen to detect and localize specific antigens in cells and tissue, most commonly detected and examined with the light microscope. A standard tool in many fields in the research setting, IHC has become an essential ancillary technique in clinical diagnostics in anatomic pathology (Lin F, Chen Z. Arch Pathol Lab Med 138:1564-1577, 2014) with the advent of antigen retrieval methods allowing it to be performed conveniently on formalin fixed paraffin embedded (FFPE) tissue (Taylor CR, Shi S-R, Barr NJ. Techniques of immunohistochemistry: principles, pitfalls, and standardization. In: Dabbs DJ (ed) Diagnostic immunohistochemistry: theranostic and genomic applications, 3rd edn. Saunders, Philadelphia, 2010; Shi SR, Key ME, Kalra KL. J Histochem Cytochem 39:741-748, 1991) and automated methods for high volume processing with reproducibility (Prichard J, Hicks D, Hammond E. Automated immunohistochemistry overview. In: Fan L, Jeffrey P (eds) Handbook of practical immunohistochemistry: frequently asked questions, 2nd edn. Springer, New York, 2015). IHC is frequently utilized to assist in the classification of neoplasms, determination of a metastatic tumor's site of origin and detection of tiny foci of tumor cells inconspicuous on routine hematoxylin and eosin (H&E) staining. Furthermore, it is increasingly being used to provide predictive and prognostic information, such as in testing for HER2 amplification in breast cancer (Wolff AC, Hammond MEH, Hicks DG et al. Arch Pathol Lab Med 138:241-256, 2014) in addition to serving as surrogateĀ markers for molecular alterations in neoplasms, including IDH1 and ATRX mutations in brain tumors (Appin CL, Brat DJ. Mol Aspects Med. 45:87-96, 2015). In this chapter we describe the basic methods of immunohistochemical staining which has become an essential tool in the daily practice of anatomic pathology worldwide.
Topics: Biomarkers, Tumor; Female; Humans; Immunohistochemistry; Paraffin Embedding; Prognosis; Tissue Fixation
PubMed: 30539453
DOI: 10.1007/978-1-4939-8935-5_25 -
Cancer Communications (London, England) Apr 2020Conventional immunohistochemistry (IHC) is a widely used diagnostic technique in tissue pathology. However, this technique is associated with a number of limitations,... (Review)
Review
Conventional immunohistochemistry (IHC) is a widely used diagnostic technique in tissue pathology. However, this technique is associated with a number of limitations, including high inter-observer variability and the capacity to label only one marker per tissue section. This review details various highly multiplexed techniques that have emerged to circumvent these constraints, allowing simultaneous detection of multiple markers on a single tissue section and the comprehensive study of cell composition, cellular functional and cell-cell interactions. Among these techniques, multiplex Immunohistochemistry/Immunofluorescence (mIHC/IF) has emerged to be particularly promising. mIHC/IF provides high-throughput multiplex staining and standardized quantitative analysis for highly reproducible, efficient and cost-effective tissue studies. This technique has immediate potential for translational research and clinical practice, particularly in the era of cancer immunotherapy.
Topics: Fluorescent Antibody Technique; Humans; Immunohistochemistry; Immunotherapy; Neoplasms
PubMed: 32301585
DOI: 10.1002/cac2.12023 -
The Oncologist May 2018Chemotherapy has been the historical mainstay of treatment for patients with breast cancer, with immunohistochemical markers and tumor characteristics driving treatment... (Review)
Review
PURPOSE
Chemotherapy has been the historical mainstay of treatment for patients with breast cancer, with immunohistochemical markers and tumor characteristics driving treatment decisions. The discovery of different intrinsic subtypes of breast cancer has advanced the understanding of breast cancer, with gene-based assays shedding further light on tumor behavior and response to treatment.
DESIGN
This review focuses on the landscape of the luminal A subtype, its definition based on immunohistochemistry (IHC) and gene assays, the prognostic and predictive value of these assays, guideline recommendations, and treatment implications.
RESULTS
Clinical studies of the prognostic value of gene-based and IHC-based assays in patients with luminal A-subtype breast cancers suggest a better prognosis for these patients compared with those with breast cancers of other subtypes.
CONCLUSION
In today's era of precision medicine, the best treatment regimen for patients with luminal A-subtype tumors is still undetermined, but available data raise the question whether chemotherapy can be omitted and endocrine therapy alone is sufficient for this patient population.
IMPLICATIONS FOR PRACTICE
Immunohistochemical markers have traditionally guided treatment decisions in breast cancer. However, advances in gene-expression profiling and availability of gene-based assays have launched these newer tests into everyday clinical practice. Luminal A-subtype tumors are a unique subset that may have favorable tumor biology. Properly defining this tumor subtype is important and may identify a subset of patients for whom endocrine therapy alone is sufficient.
Topics: Breast Neoplasms; Female; Humans; Immunohistochemistry
PubMed: 29472313
DOI: 10.1634/theoncologist.2017-0535 -
Viruses Nov 2019Feline infectious peritonitis (FIP) is a fatal disease that poses several challenges for veterinarians: clinical signs and laboratory changes are non-specific, and there... (Review)
Review
Feline infectious peritonitis (FIP) is a fatal disease that poses several challenges for veterinarians: clinical signs and laboratory changes are non-specific, and there are two pathotypes of the etiologic agent feline coronavirus (FCoV), sometimes referred to as feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV) that vary fundamentally in their virulence, but are indistinguishable by a number of diagnostic methods. This review focuses on all important steps every veterinary practitioner has to deal with and new diagnostic tests that can be considered when encountering a cat with suspected FIP with the aim to establish a definitive diagnosis. It gives an overview on all available direct and indirect diagnostic tests and their sensitivity and specificity reported in the literature in different sample material. By providing summarized data for sensitivity and specificity of each diagnostic test and each sample material, which can easily be accessed in tables, this review can help to facilitate the interpretation of different diagnostic tests and raise awareness of their advantages and limitations. Additionally, diagnostic trees depict recommended diagnostic steps that should be performed in cats suspected of having FIP based on their clinical signs or clinicopathologic abnormalities. These steps can easily be followed in clinical practice.
Topics: Animals; Antibodies, Viral; Antigen-Antibody Complex; Antigens, Viral; Biomarkers; Cats; Coronavirus, Feline; Diagnostic Tests, Routine; Feline Infectious Peritonitis; Immunoassay; Immunohistochemistry; Macrophages; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 31731711
DOI: 10.3390/v11111068 -
Advances in Anatomic Pathology May 2020Immunohistochemistry represents an indispensable complement to an epidemiology and morphology-driven approach to tumor diagnosis and site of origin assignment. This... (Review)
Review
Immunohistochemistry represents an indispensable complement to an epidemiology and morphology-driven approach to tumor diagnosis and site of origin assignment. This review reflects the state of my current practice, based on 15-years' experience in Pathology and a deep-dive into the literature, always striving to be better equipped to answer the age old questions, "What is it, and where is it from?" The tables and figures in this manuscript are the ones I "pull up on the computer" when I am teaching at the microscope and turn to myself when I am (frequently) stuck. This field is so exciting because I firmly believe that, through the application of next-generation immunohistochemistry, we can provide better answers than ever before. Specific topics covered in this review include (1) broad tumor classification and associated screening markers; (2) the role of cancer epidemiology in determining pretest probability; (3) broad-spectrum epithelial markers; (4) noncanonical expression of broad tumor class screening markers; (5) a morphologic pattern-based approach to poorly to undifferentiated malignant neoplasms; (6) a morphologic and immunohistochemical approach to define 4 main carcinoma types; (7) CK7/CK20 coordinate expression; (8) added value of semiquantitative immunohistochemical stain assessment; algorithmic immunohistochemical approaches to (9) "garden variety" adenocarcinomas presenting in the liver, (10) large polygonal cell adenocarcinomas, (11) the distinction of primary surface ovarian epithelial tumors with mucinous features from metastasis, (12) tumors presenting at alternative anatomic sites, (13) squamous cell carcinoma versus urothelial carcinoma, and neuroendocrine neoplasms, including (14) the distinction of pheochromocytoma/paraganglioma from well-differentiated neuroendocrine tumor, site of origin assignment in (15) well-differentiated neuroendocrine tumor and (16) poorly differentiated neuroendocrine carcinoma, and (17) the distinction of well-differentiated neuroendocrine tumor G3 from poorly differentiated neuroendocrine carcinoma; it concludes with (18) a discussion of diagnostic considerations in the broad-spectrum keratin/CD45/S-100-"triple-negative" neoplasm.
Topics: Algorithms; Biomarkers, Tumor; Humans; Immunohistochemistry; Neoplasms
PubMed: 32205473
DOI: 10.1097/PAP.0000000000000256 -
Cancer Cytopathology May 2019With an escalating number of predictive biomarkers emerging in non-small cell lung carcinoma (NSCLC), immunohistochemistry (IHC) is being used as a rapid and... (Review)
Review
With an escalating number of predictive biomarkers emerging in non-small cell lung carcinoma (NSCLC), immunohistochemistry (IHC) is being used as a rapid and cost-effective tool for the screening and detection of many of these markers. In particular, robust IHC assays performed on formalin-fixed, paraffin-embedded (FFPE) tumor tissue are widely used as surrogate markers for ALK and ROS1 rearrangements and for detecting programmed death ligand 1 (PD-L1) expression in patients with advanced NSCLC; in addition, they have become essential for treatment decisions. Cytology samples represent the only source of tumor in a significant proportion of patients with inoperable NSCLC, and there is increasing demand for predictive biomarker testing on them. However, the wide variation in the types of cytology samples and their preparatory methods, the use of alcohol-based fixatives that interfere with immunochemistry results, the difficulty in procurement of cytology-specific controls, and the uncertainty regarding test validity have resulted in underutilization of cytology material for predictive immunocytochemistry (ICC), and most cytopathologists limit such testing to FFPE cell blocks (CBs). The purpose of this review is to: 1) analyze various preanalytical, analytical, and postanalytical factors influencing ICC results; 2) discuss measures for validation of ICC protocols; and 3) summarize published data on predictive ICC for ALK, ROS1, EGFR gene alterations and PD-L1 expression on lung cancer cytology. Based on our experience and from a review of the literature, we conclude that cytology specimens are in principal suitable for predictive ICC, but proper optimization and rigorous quality control for high-quality staining are essential, particularly for non-CB preparations.
Topics: Biomarkers, Tumor; Cytodiagnosis; Humans; Immunohistochemistry; Lung Neoplasms; Predictive Value of Tests
PubMed: 31050216
DOI: 10.1002/cncy.22137 -
Anatomical Record (Hoboken, N.J. : 2007) Mar 2013
Topics: Fluorescent Antibody Technique; Image Processing, Computer-Assisted; Immunohistochemistry; Microscopy; Microscopy, Fluorescence; Software
PubMed: 23382140
DOI: 10.1002/ar.22641 -
International Journal of Molecular... Feb 2023Traditional immunohistochemistry (IHC) has already become an essential method of diagnosis and therapy in cancer management. However, this antibody-based technique is... (Review)
Review
Traditional immunohistochemistry (IHC) has already become an essential method of diagnosis and therapy in cancer management. However, this antibody-based technique is limited to detecting a single marker per tissue section. Since immunotherapy has revolutionized the antineoplastic therapy, developing new immunohistochemistry strategies to detect multiple markers simultaneously to better understand tumor environment and predict or assess response to immunotherapy is necessary and urgent. Multiplex immunohistochemistry (mIHC)/multiplex immunofluorescence (mIF), such as multiplex chromogenic IHC and multiplex fluorescent immunohistochemistry (mfIHC), is a new and emerging technology to label multiple biomarkers in a single pathological section. The mfIHC shows a higher performance in cancer immunotherapy. This review summarizes the technologies, which are applied for mfIHC, and discusses how they are employed for immunotherapy research.
Topics: Humans; Fluorescent Antibody Technique; Immunohistochemistry; Neoplasms; Biomarkers; Immunotherapy; Biomarkers, Tumor
PubMed: 36834500
DOI: 10.3390/ijms24043086 -
Journal For Immunotherapy of Cancer Jul 2021Emerging data suggest predictive biomarkers based on the spatial arrangement of cells or coexpression patterns in tissue sections will play an important role in...
BACKGROUND
Emerging data suggest predictive biomarkers based on the spatial arrangement of cells or coexpression patterns in tissue sections will play an important role in precision immuno-oncology. Multiplexed immunofluorescence (mIF) is ideally suited to such assessments. Standardization and validation of an end-to-end workflow that supports multisite trials and clinical laboratory processes are vital. Six institutions collaborated to: (1) optimize an automated six-plex assay focused on the PD-1/PD-L1 axis, (2) assess intersite and intrasite reproducibility of staining using a locked down image analysis algorithm to measure tumor cell and immune cell (IC) subset densities, %PD-L1 expression on tumor cells (TCs) and ICs, and PD-1/PD-L1 proximity assessments.
METHODS
A six-plex mIF panel (PD-L1, PD-1, CD8, CD68, FOXP3, and CK) was rigorously optimized as determined by quantitative equivalence to immunohistochemistry (IHC) chromogenic assays. Serial sections from tonsil and breast carcinoma and non-small cell lung cancer (NSCLC) tissue microarrays (TMAs), TSA-Opal fluorescent detection reagents, and antibodies were distributed to the six sites equipped with a Leica Bond Rx autostainer and a Vectra Polaris multispectral imaging platform. Tissue sections were stained and imaged at each site and delivered to a single site for analysis. Intersite and intrasite reproducibility were assessed by linear fits to plots of cell densities, including %PDL1 expression by TCs and ICs in the breast and NSCLC TMAs.
RESULTS
Comparison of the percent positive cells for each marker between mIF and IHC revealed that enhanced amplification in the mIF assay was required to detect low-level expression of PD-1, PD-L1, FoxP3 and CD68. Following optimization, an average equivalence of 90% was achieved between mIF and IHC across all six assay markers. Intersite and intrasite cell density assessments showed an average concordance of R=0.75 (slope=0.92) and R=0.88 (slope=0.93) for breast carcinoma, respectively, and an average concordance of R=0.72 (slope=0.86) and R=0.81 (slope=0.68) for NSCLC. Intersite concordance for %PD-L1+ICs had an average R value of 0.88 and slope of 0.92. Assessments of PD-1/PD-L1 proximity also showed strong concordance (R=0.82; slope=0.75).
CONCLUSIONS
Assay optimization yielded highly sensitive, reproducible mIF characterization of the PD-1/PD-L1 axis across multiple sites. High concordance was observed across sites for measures of density of specific IC subsets, measures of coexpression and proximity with single-cell resolution.
Topics: Biomarkers, Tumor; Female; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Laboratories, Clinical; Male; Tissue Array Analysis
PubMed: 34266881
DOI: 10.1136/jitc-2020-002197