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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 -
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 -
Anais Brasileiros de Dermatologia 2019Bullous pemphigoid is the most frequent autoimmune bullous disease and mainly affects elderly individuals. Increase in incidence rates in the past decades has been... (Review)
Review
Bullous pemphigoid is the most frequent autoimmune bullous disease and mainly affects elderly individuals. Increase in incidence rates in the past decades has been attributed to population aging, drug-induced cases and improvement in the diagnosis of the nonbullous presentations of the disease. A dysregulated T cell immune response and synthesis of IgG and IgE autoantibodies against hemidesmosomal proteins (BP180 and BP230) lead to neutrophil chemotaxis and degradation of the basement membrane zone. Bullous pemphigoid classically manifests with tense blisters over urticarial plaques on the trunk and extremities accompanied by intense pruritus. Mucosal involvement is rarely reported. Diagnosis relies on (1) the histopathological evaluation demonstrating eosinophilic spongiosis or a subepidermal detachment with eosinophils; (2) the detection of IgG and/or C3 deposition at the basement membrane zone using direct or indirect immunofluorescence assays; and (3) quantification of circulating autoantibodies against BP180 and/or BP230 using ELISA. Bullous pemphigoid is often associated with multiple comorbidities in elderly individuals, especially neurological disorders and increased thrombotic risk, reaching a 1-year mortality rate of 23%. Treatment has to be tailored according to the patient's clinical conditions and disease severity. High potency topical steroids and systemic steroids are the current mainstay of therapy. Recent randomized controlled studies have demonstrated the benefit and safety of adjuvant treatment with doxycycline, dapsone and immunosuppressants aiming a reduction in the cumulative steroid dose and mortality.
Topics: Aged; Autoimmunity; Diagnosis, Differential; Fluorescent Antibody Technique; Humans; Pemphigoid, Bullous; Steroids
PubMed: 31090818
DOI: 10.1590/abd1806-4841.20199007 -
Annals of the Rheumatic Diseases Jul 2019The indirect immunofluorescence assay (IIFA) on HEp-2 cells is widely used for detection of antinuclear antibodies (ANA). The dichotomous outcome, negative or positive,...
The indirect immunofluorescence assay (IIFA) on HEp-2 cells is widely used for detection of antinuclear antibodies (ANA). The dichotomous outcome, negative or positive, is integrated in diagnostic and classification criteria for several systemic autoimmune diseases. However, the HEp-2 IIFA test has much more to offer: besides the titre or fluorescence intensity, it also provides fluorescence pattern(s). The latter include the nucleus and the cytoplasm of interphase cells as well as patterns associated with mitotic cells. The International Consensus on ANA Patterns (ICAP) initiative has previously reached consensus on the nomenclature and definitions of HEp-2 IIFA patterns. In the current paper, the ICAP consensus is presented on the clinical relevance of the 29 distinct HEp-2 IIFA patterns. This clinical relevance is primarily defined within the context of the suspected disease and includes recommendations for follow-up testing. The discussion includes how this information may benefit the clinicians in daily practice and how the knowledge can be used to further improve diagnostic and classification criteria.
Topics: Antibodies, Antinuclear; Autoimmune Diseases; Biomarkers; Fluorescent Antibody Technique, Indirect; Humans; International Cooperation
PubMed: 30862649
DOI: 10.1136/annrheumdis-2018-214436 -
Clinical Reviews in Allergy & Immunology Oct 2022Circulating autoantibodies are a key diagnostic tool in autoimmune hepatitis (AIH), being positive in 95% of the cases if tested according to dedicated guidelines issued... (Review)
Review
Circulating autoantibodies are a key diagnostic tool in autoimmune hepatitis (AIH), being positive in 95% of the cases if tested according to dedicated guidelines issued by the International Autoimmune Hepatitis Group. They also allow the distinction between type 1 AIH, characterized by positive anti-nuclear and/or anti-smooth muscle antibody, and type 2 AIH, characterized by positive anti-liver kidney microsomal type 1 and/or anti-liver cytosol type 1 antibody. Anti-soluble liver antigen is the only AIH-specific autoantibody, and is found in 20-30% of both type 1 and type 2 AIH. Anti-neutrophil cytoplasmic antibody is frequently positive in type 1 AIH, being associated also with inflammatory bowel disease and with primary/autoimmune sclerosing cholangitis. The reference method for autoantibody testing remains indirect immunofluorescence on triple tissue (rodent liver, kidney and stomach), allowing both the detection of the majority of liver-relevant reactivities, including those autoantibodies whose molecular target antigens are unknown. Of note, the current knowledge of the clinical significance of autoantibodies relies on studies based on this technique. However, immunofluorescence requires trained laboratory personnel, is observer-dependent, and lacks standardization, leading to ongoing attempts at replacing this method with automated assays, the sensitivity, and specificity of which, however, require further studies before they can be used as a reliable alternative to immunofluorescence; currently, they may be used as complementary to immunofluorescence.
Topics: Antibodies, Antineutrophil Cytoplasmic; Autoantibodies; Fluorescent Antibody Technique, Indirect; Hepatitis, Autoimmune; Humans; Immunologic Tests
PubMed: 34491531
DOI: 10.1007/s12016-021-08888-9 -
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 -
The Journal of Investigative Dermatology Jan 2013
Topics: Antigen-Antibody Complex; Autoantibodies; Fluorescent Antibody Technique; Humans; Skin Diseases
PubMed: 23299451
DOI: 10.1038/jid.2012.455 -
Molecular Oncology Oct 2020Multiplex immunofluorescence is a powerful tool for the simultaneous detection of tissue-based biomarkers, revolutionising traditional immunohistochemistry. The Opal...
Multiplex immunofluorescence is a powerful tool for the simultaneous detection of tissue-based biomarkers, revolutionising traditional immunohistochemistry. The Opal methodology allows up to eight biomarkers to be measured concomitantly without cross-reactivity, permitting identification of different cell populations within the tumour microenvironment. In this study, we aimed to validate a multiplex immunofluorescence workflow in two complementary multiplex panels and evaluate the tumour immune microenvironment in colorectal cancer (CRC) formalin-fixed paraffin-embedded tissue. We stained CRC and tonsil samples using Opal multiplex immunofluorescence on a Leica BOND RX immunostainer. We then acquired images on an Akoya Vectra Polaris and performed multispectral unmixing using inform. Antibody panels were validated on tissue microarray sections containing cores from six normal tissue types, using qupath for image analysis. Comparisons between chromogenic immunohistochemistry and multiplex immunofluorescence on consecutive sections from the same tissue microarray showed significant correlation (r > 0.9, P-value < 0.0001), validating both panels. We identified many factors that influenced the quality of the acquired fluorescent images, including biomarker co-expression, staining order, Opal-antibody pairing, sample thickness, multispectral unmixing and biomarker detection order during image analysis. Overall, we report the optimisation and validation of a multiplex immunofluorescence process, from staining to image analysis, ensuring assay robustness. Our multiplex immunofluorescence protocols permit the accurate detection of multiple immune markers in various tissue types, using a workflow that enables rapid processing of samples, above and beyond previous workflows.
Topics: Epitopes; Fluorescent Antibody Technique; Humans; Imaging, Three-Dimensional; Reproducibility of Results; Tumor Microenvironment; Workflow
PubMed: 32671911
DOI: 10.1002/1878-0261.12764 -
Italian Journal of Dermatology and... Apr 2021Pemphigus comprises a heterogeneous group of autoimmune blistering diseases, which can affect both skin and mucous membranes, especially oral mucosa. This group of... (Review)
Review
Pemphigus comprises a heterogeneous group of autoimmune blistering diseases, which can affect both skin and mucous membranes, especially oral mucosa. This group of diseases shows usually a chronic-relapsing course. Since pemphigus is a rare disease, the diagnosis is often delayed, because it is based upon the recognition of consistent clinical, histologic, and direct immunofluorescence findings, as well as indirect immunofluorescence, and/or enzyme-linked immunosorbent assay. Usually the patients are treated for multiple other conditions before starting the correct therapy, leading to a critical reduction of the patients' quality of life. This review is a succinct compilation of pearls gathered from clinical experience in pemphigus and the myths that may have influenced everyday practice but have been proven false. This review provided a selection of such dilemmas and controversies, focusing on myths and pearls that can help young dermatologist in the clinic, while also dispelling them.
Topics: Autoimmune Diseases; Blister; Fluorescent Antibody Technique, Indirect; Humans; Pemphigus; Quality of Life
PubMed: 33960749
DOI: 10.23736/S2784-8671.21.06810-3 -
Molecular Brain May 2023Profiling molecular expression in situ allows the integration of biomolecular and cellular features, enabling an in-depth understanding of biological systems.... (Review)
Review
Profiling molecular expression in situ allows the integration of biomolecular and cellular features, enabling an in-depth understanding of biological systems. Multiplexed immunofluorescence methods can visualize tens to hundreds of proteins from individual tissue samples, but their application is usually limited to thin tissue sections. Multiplexed immunofluorescence of thick tissues or intact organs will enable high-throughput profiling of cellular protein expression within 3D tissue architectures (e.g., blood vessels, neural projections, tumors), opening a new dimension in diverse biological research and medical applications. We will review current multiplexed immunofluorescence methods and discuss possible approaches and challenges to achieve 3D multiplexed immunofluorescence.
Topics: Proteins; Fluorescent Antibody Technique
PubMed: 37131224
DOI: 10.1186/s13041-023-01027-9