-
Molecules (Basel, Switzerland) Apr 2020Fluorescence in situ hybridization (FISH) is a standard technique used in routine diagnostics of genetic aberrations. Thanks to simple FISH procedure is possible to... (Review)
Review
Fluorescence in situ hybridization (FISH) is a standard technique used in routine diagnostics of genetic aberrations. Thanks to simple FISH procedure is possible to recognize tumor-specific abnormality. Its applications are limited to designed probe type. Gene rearrangements e.g., , reflecting numerous translocational partners, deletions of critical regions e.g., 1p and 19q, gene fusions e.g., , genomic imbalances e.g., 6p, 6q, 11q and amplifications e.g., are targets in personalized oncology. Confirmation of genetic marker is frequently a direct indication to start specific, targeted treatment. In other cases, detected aberration helps pathologists to better distinguish soft tissue sarcomas, or to state a final diagnosis. Our main goal is to show that applying FISH to formalin-fixed paraffin-embedded tissue sample (FFPE) enables assessing genomic status in the population of cells deriving from a primary tumor or metastasis. Although many more sophisticated techniques are available, like Real-Time PCR or new generation sequencing, FISH remains a commonly used method in many genetic laboratories.
Topics: Biomarkers, Tumor; Chromosome Painting; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Molecular Probes; Neoplasms; Precision Medicine; Reproducibility of Results
PubMed: 32316657
DOI: 10.3390/molecules25081864 -
Anatomical Record (Hoboken, N.J. : 2007) Aug 2014In situ hybridization is a technique that is used to detect nucleotide sequences in cells, tissue sections, and even whole tissue. This method is based on the... (Review)
Review
In situ hybridization is a technique that is used to detect nucleotide sequences in cells, tissue sections, and even whole tissue. This method is based on the complementary binding of a nucleotide probe to a specific target sequence of DNA or RNA. These probes can be labeled with either radio-, fluorescent-, or antigen-labeled bases. Depending on the probe used, autoradiography, fluorescence microscopy, or immunohistochemistry, respectively, are used for visualization. In situ hybridization is extensively used in research, as well as clinical applications, especially for diagnostic purposes. This review discusses the basic technique of in situ hybridization. The standard in situ hybridization process is reviewed, and different types of in situ hybridization, their applications, and advantages and disadvantages are discussed.
Topics: Autoradiography; Humans; In Situ Hybridization; Isotope Labeling; Microscopy, Fluorescence; Paraffin Embedding; RNA Probes; Tissue Fixation
PubMed: 24810158
DOI: 10.1002/ar.22944 -
PloS One 2015In situ hybridization (ISH) is an extremely useful tool for localizing gene expression and changes in expression to specific cell populations in tissue samples across...
In situ hybridization (ISH) is an extremely useful tool for localizing gene expression and changes in expression to specific cell populations in tissue samples across numerous research fields. Typically, a research group will put forth significant effort to design, generate, validate and then utilize in situ probes in thin or ultrathin paraffin embedded tissue sections. While combining ISH and IHC is an established technique, the combination of RNAscope ISH, a commercially available ISH assay with single transcript sensitivity, and IHC in thick free-floating tissue sections has not been described. Here, we provide a protocol that combines RNAscope ISH with IHC in thick free-floating tissue sections from the brain and allows simultaneous co-localization of genes and proteins in individual cells. This approach works well with a number of ISH probes (e.g. small proline-rich repeat 1a, βIII-tubulin, tau, and β-actin) and IHC antibody stains (e.g. tyrosine hydroxylase, βIII-tubulin, NeuN, and glial fibrillary acidic protein) in rat brain sections. In addition, we provide examples of combining ISH-IHC dual staining in primary neuron cultures and double-ISH labeling in thick free-floating tissue sections from the brain. Finally, we highlight the ability of RNAscope to detect ectopic DNA in neurons transduced with viral vectors. RNAscope ISH is a commercially available technology that utilizes a branched or "tree" in situ method to obtain ultrasensitive, single transcript detection. Immunohistochemistry is a tried and true method for identifying specific protein in cell populations. The combination of a sensitive and versatile oligonucleotide detection method with an established and versatile protein assay is a significant advancement in studies using free-floating tissue sections.
Topics: Animals; Brain; Immunohistochemistry; In Situ Hybridization; Male; Neurons; Primary Cell Culture; Rats
PubMed: 25794171
DOI: 10.1371/journal.pone.0120120 -
BioTechniques Dec 2023Whole-mount hybridization is a critical technique for analyzing gene expression in planarians. While robust protocols have been developed, these protocols are...
Whole-mount hybridization is a critical technique for analyzing gene expression in planarians. While robust protocols have been developed, these protocols are laborious, making them challenging to incorporate in an academic setting, reducing throughput and increasing time to results. Here, the authors systematically tested modifications to all phases of the protocol with the goal of eliminating steps and reducing time without impacting quality. This modified protocol allows for whole-mount colorimetric hybridization and multicolor fluorescence hybridization to be completed in two days with a significant reduction in steps and hands-on processing time.
Topics: Animals; Planarians; In Situ Hybridization, Fluorescence
PubMed: 37851365
DOI: 10.2144/btn-2023-0074 -
Archives of Pathology & Laboratory... Dec 2020Accurate HER2 testing in breast cancer is crucial for appropriate precision therapy. HER2 testing is most commonly accomplished by a combination of immunohistochemistry...
CONTEXT.—
Accurate HER2 testing in breast cancer is crucial for appropriate precision therapy. HER2 testing is most commonly accomplished by a combination of immunohistochemistry and in situ hybridization techniques, as gene amplification is closely tied to protein overexpression. During the last 5+ years, brightfield dual in situ hybridization (DISH) has replaced fluorescence methods (fluorescence in situ hybridization [FISH]) in some laboratories.
OBJECTIVE.—
To analyze routine HER2 DISH performance in the field.
DESIGN.—
We reviewed our experience with HER2 DISH performed at outside laboratories and referred for patient care.
RESULTS.—
Of 273 identified retrospective DISH results, 55 had repeated FISH testing at our institution; 7 (13%) were discordant. Additional cases had technical flaws hampering appropriate scoring. In 23 cases (42%), HER2 DISH was performed without immunohistochemistry. Slide review of a prospective cohort of 42 consecutive DISH cases revealed 14 (33%) with technical or interpretative limitations potentially jeopardizing results. Commonly identified problems include lack of or weak signals in most tumor cells, and silver precipitate or red signals outside of nuclei, resulting in false-negative or false-positive interpretations, respectively. Further, 44% (24 of 55) of DISH reports lacked complete data, specifically average HER2 signals/cell.
CONCLUSIONS.—
While HER2 DISH can be an efficient and effective alternative to FISH, we illustrate pitfalls and reinforce that careful attention to slide quality and technical parameters are critically important. HER2 DISH cotesting with immunohistochemistry could help minimize false-negative or false-positive HER2 results.
Topics: Biomarkers, Tumor; Breast Neoplasms; Cohort Studies; False Negative Reactions; False Positive Reactions; Humans; Immunohistochemistry; In Situ Hybridization; In Situ Hybridization, Fluorescence; Prospective Studies; Retrospective Studies
PubMed: 32101450
DOI: 10.5858/arpa.2019-0510-OA -
Head and Neck Pathology Sep 2019Head and neck pathology present a unique set of challenges including the morphological diversity of the neoplasms and presentation of metastases of unknown primary... (Review)
Review
Head and neck pathology present a unique set of challenges including the morphological diversity of the neoplasms and presentation of metastases of unknown primary origin. The detection of human papillomavirus and Epstein-Barr virus associated with squamous cell carcinoma and newer entities like HPV-related carcinoma with adenoid cystic like features have critical prognostic and management implications. In salivary gland neoplasms, differential diagnoses can be broad and include non-neoplastic conditions as well as benign and malignant neoplasms. The detection of specific gene rearrangements can be immensely helpful in reaching the diagnosis in pleomorphic adenoma, mucoepidermoid carcinoma, secretory carcinoma, hyalinizing clear cell carcinoma and adenoid cystic carcinoma. Furthermore, molecular techniques are essential in diagnosis of small round blue cell neoplasms and spindle cell neoplasms including Ewing sarcoma, rhabdomyosarcoma, synovial sarcoma, biphenotypic sinonasal sarcoma, dermatofibrosarcoma protuberans, nodular fasciitis and inflammatory myofibroblastic tumor. The detection of genetic rearrangements is also important in lymphomas particularly in identifying 'double-hit' and 'triple-hit' lymphomas in diffuse large B cell lymphoma. This article reviews the use of in situ hybridization in the diagnosis of these neoplasms.
Topics: Biomarkers, Tumor; Head and Neck Neoplasms; Humans; In Situ Hybridization
PubMed: 30467669
DOI: 10.1007/s12105-018-0988-1 -
Methods in Molecular Biology (Clifton,... 2020During development, the mouse brain is progressively divided into functionally distinct compartments. Numerous neuronal and glial cell types are subsequently generated...
During development, the mouse brain is progressively divided into functionally distinct compartments. Numerous neuronal and glial cell types are subsequently generated in response to various inductive signals. Each cell expresses a unique combination of genes encoding proteins from transcription factors to neurotransmitters that define its role in brain function. To understand these important and highly sophisticated processes, it is critical to accurately locate the various proteins and cells that produce them. In this chapter, we introduce the techniques of Immunohistochemistry, which detects the localization of specific proteins, and RNA in situ hybridization, which enables the visualization of specific mRNAs.
Topics: Animals; Brain; Gene Expression Regulation, Developmental; Gene Regulatory Networks; Immunohistochemistry; In Situ Hybridization; Mice; RNA Probes; Tissue Distribution
PubMed: 31552672
DOI: 10.1007/978-1-4939-9732-9_27 -
Archives of Pathology & Laboratory... Nov 2018To update key recommendations of the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) human epidermal growth factor receptor 2 (HER2)...
Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Focused Update.
PURPOSE.—
To update key recommendations of the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) human epidermal growth factor receptor 2 (HER2) testing in breast cancer guideline.
METHODS.—
Based on the signals approach, an Expert Panel reviewed published literature and research survey results on the observed frequency of less common in situ hybridization (ISH) patterns to update the recommendations.
RECOMMENDATIONS.—
Two recommendations addressed via correspondence in 2015 are included. First, immunohistochemistry (IHC) 2+ is defined as invasive breast cancer with weak to moderate complete membrane staining observed in >10% of tumor cells. Second, if the initial HER2 test result in a core needle biopsy specimen of a primary breast cancer is negative, a new HER2 test may (not "must") be ordered on the excision specimen based on specific clinical criteria. The HER2 testing algorithm for breast cancer is updated to address the recommended workup for less common clinical scenarios (approximately 5% of cases) observed when using a dual-probe ISH assay. These scenarios are described as ISH group 2 ( HER2/chromosome enumeration probe 17 [CEP17] ratio ≥2.0; average HER2 copy number <4.0 signals per cell), ISH group 3 ( HER2/CEP17 ratio <2.0; average HER2 copy number ≥6.0 signals per cell), and ISH group 4 ( HER2/CEP17 ratio <2.0; average HER2 copy number ≥4.0 and <6.0 signals per cell). The diagnostic approach includes more rigorous interpretation criteria for ISH and requires concomitant IHC review for dual-probe ISH groups 2 to 4 to arrive at the most accurate HER2 status designation (positive or negative) based on combined interpretation of the ISH and IHC assays. The Expert Panel recommends that laboratories using single-probe ISH assays include concomitant IHC review as part of the interpretation of all single-probe ISH assay results.
Topics: Female; Humans; Biomarkers, Tumor; Breast Neoplasms; Immunohistochemistry; In Situ Hybridization; Medical Oncology; Receptor, ErbB-2; United States; Systematic Reviews as Topic
PubMed: 29846104
DOI: 10.5858/arpa.2018-0902-SA -
Journal of Veterinary Diagnostic... Jan 2019Rotavirus groups A, B, and C (RVA, RVB, and RVC, respectively) have been the most prevalent and pathogenic in pigs. To date, immunohistochemistry is only available for...
Rotavirus groups A, B, and C (RVA, RVB, and RVC, respectively) have been the most prevalent and pathogenic in pigs. To date, immunohistochemistry is only available for RVA because of the lack of commercial antibodies for RVB and RVC. We developed a novel in situ hybridization RNA-based chromogenic technique (ISH-RNA) to detect and subtype RVA, RVB, and RVC. We evaluated 33 samples that were reverse-transcription PCR positive for RVA, RVB, and/or RVC. ISH-RNA was able to detect as few as 10 RV RNA copies/mL. The new ISH-RNA test can be useful for routine investigation of rotavirus enteritis in order to guide strategies for control of the infection in pigs, but a full validation study needs to be completed. Pathogenesis studies may be conducted using ISH-RNA based on the identification of replicating virus.
Topics: Animals; In Situ Hybridization; Rotavirus; Rotavirus Infections; Sus scrofa; Swine; Swine Diseases
PubMed: 30541408
DOI: 10.1177/1040638718817502 -
Journal of Clinical Laboratory Analysis 1997In situ hybridization (ISH) combines molecular biological techniques with histological and cytological analysis of gene expression. RNA and DNA can be readily localized... (Review)
Review
In situ hybridization (ISH) combines molecular biological techniques with histological and cytological analysis of gene expression. RNA and DNA can be readily localized in specific cells with this method. ISH has been useful as a research tool, and recent studies have used this technique in the diagnostic pathology laboratory and in microbiology for the tissue localization in infectious agents. Other recent developments in the applications of ISH involve in situ polymerase chain reaction (PCR) and in situ reverse transcription (RT)-PCR, which can be used to detect very low levels of nucleic acids in tissues by taking advantage of the powerful amplification capacity of PCR. In situ PCR will contribute significantly to progress in this field because of the marked increase in the sensitivity of this method.
Topics: Cytogenetics; DNA; Fluorescent Antibody Technique; Gene Expression; Humans; In Situ Hybridization; Infections; Isotope Labeling; Nucleic Acid Probes; Polymerase Chain Reaction; RNA, Messenger
PubMed: 9021518
DOI: 10.1002/(SICI)1098-2825(1997)11:1<2::AID-JCLA2>3.0.CO;2-F