PDF Fluorescence In-Situ Hybridisation (FISH) Techniques

Fluorescence In-Situ Hybridisation (FISH) Dr Temba Mudariki Learning Outcomes Introduction to FISH Basics of FISH Technique Applications of FISH in Clinical Diagnostics Interpretation of FISH Results Laboratory Demonstration and Practice Ethical and Regulatory Considerations Fut...

Fluorescence In-Situ Hybridisation (FISH) Dr Temba Mudariki Learning Outcomes Introduction to FISH Basics of FISH Technique Applications of FISH in Clinical Diagnostics Interpretation of FISH Results Laboratory Demonstration and Practice Ethical and Regulatory Considerations Future Directions and Research Trends in FISH Summary Definition and Principles Fluorescence In-Situ Hybridisation (FISH) o Molecular cytogenetic technique o Detect and localize specific DNA sequences. Fundamental Principle o Use of fluorescently labelled DNA probes o Binding to complementary target sequences o Visualization of genetic loci and chromosomal abnormalities  Historical Background and Development of FISH Origins in the 1980s Conceptualized as a molecular biology technique Study of DNA sequence organization and distribution within chromosomes Prominence in Cytogenetics and Molecular Diagnostics Accurate identification of genetic aberrations and rearrangements Detection of gene copy number variations at the cellular level Evolution and Advancements Improvements in probe design and fluorescence detection methods Integration with advanced imaging technologies Indispensable role in genetic research, clinical diagnostics, and cancer biology Importance of FISH in Medical Science Crucial Role in Medical Science Precise detection and characterization of genetic abnormalities Association with congenital and acquired diseases Clinical Diagnostics Identification of chromosomal aberrations in prenatal and postnatal genetic testing Diagnosis and prognosis of genetic disorders (e.g., Down syndrome, Turner syndrome, leukaemia) Importance of FISH in Medical Science Implications in Cancer Genetics Visualization of gene amplifications, deletions, and translocations in tumour cells Aids in cancer subtype classification, treatment selection, and prognostic evaluation Significance in Genetics Research Mapping DNA sequences to chromosomes Studying chromosomal architecture Elucidating the genetic basis of inherited diseases  Basics of FISH Technique DNA Probe Selection and Design o Critical for FISH success o Short, single-stranded DNA sequences labelled with fluorescent dyes Selection Considerations o Specificity to target DNA sequences o Considerations: probe length, GC content, avoidance of repetitive DNA elements Types of Probes o Whole chromosome painting probes o Locus-specific probes o Centromere-specific probes o Utilized in various FISH applications for visualizing specific genetic loci or chromosomal regions. Sample Preparation and Fixation for FISH Essentiality of Sample Preparation Critical for successful FISH analysis Involves collection and fixation of biological specimens (cells, tissues, chromosomes) Fixation Methods Chemical fixation with fixatives (e.g., formaldehyde, methanol) Immobilization and stabilization of cellular components Enables subsequent probe hybridization and imaging. Hybridization Process and Detection Methods in FISH Hybridization Process Denaturation of target DNA Probe binding, annealing, and incubation under controlled conditions. Detection Methods Direct detection using fluorescently labelled probes. Indirect detection through amplification techniques (e.g., tyramide signal amplification - TSA) Enhances signal and improves detection sensitivity.  Imaging and Microscopy Techniques for FISH Analysis Critical Role of Imaging and Microscopy Enable visualization and analysis of fluorescently labelled DNA probes bound to target DNA sequences Equipment Used High-resolution fluorescence microscopes Appropriate filter sets Imaging software Functionality Capture fluorescent signals emitted by probes Visualization and analysis of chromosomal or genetic abnormalities at the subcellular level Application of FISH in Clinical Diagnostics Genetic Disease Diagnosis and Prognosis Enables detection of chromosomal abnormalities, gene deletions, or duplications Identification of genetic aberrations related to conditions such as Down syndrome, Prader-Willi syndrome, and Angelman syndrome Provides insights for patient management and genetic counselling Application of FISH in Clinical Diagnostics Prenatal Screening and Diagnosis Extensively used in prenatal screening to detect common aneuploidies (e.g., trisomy 21, trisomy 18, trisomy 13) in foetal cells obtained through procedures like amniocentesis and chorionic villus sampling (CVS) Allows for timely and accurate prenatal diagnosis, aiding informed decision-making and appropriate prenatal care Application of FISH in Clinical Diagnostics Detection of Chromosomal Abnormalities and Genetic Markers in Cancer Enables detection of specific chromosomal abnormalities, gene amplifications, deletions, and translocations associated with various types of cancer. Visualization of genetic markers (e.g., HER2/neu amplification in breast cancer, BCR- ABL fusion in chronic myeloid leukaemia, MYC amplification in solid tumours) Aids in cancer subtype classification, treatment stratification, and prognostic evaluation Interpretation of FISH Results Understanding FISH Patterns and Signals Involves comprehending patterns and signals related to the number, size, and location of fluorescent signals corresponding to DNA probes bound to specific chromosomal or genetic loci. Patterns include normal diploid signals, monosomy, trisomy, deletion, duplication, translocation, split signals, or other aberrations, each with distinct implications for genetic and clinical analysis. Interpretation of FISH Results Case Studies and Examples of FISH Application Genetic Disorders Down Syndrome Diagnosis: In cases where an obstetrician suspects Down syndrome in a foetus, maternal blood testing or ultrasound findings may lead to genetic testing. Foetal DNA karyotyping or FISH can be used to detect the presence of three copies of chromosome 21, indicative of Down syndrome. Interpretation of FISH Results Prenatal Samples Detection of Chromosomal Aberrations: A study evaluated the feasibility of retrospective genetic testing for numerical chromosomal aberrations in formalin-fixed foetal tissue using FISH. It concurred with karyotype in all cases with sufficient cells and diagnosed numerical aberrations in cases with foetal malformations. Interpretation of FISH Results Cancer Specimens Retrospective Chromosome Analysis: The use of FISH on paraffin-embedded abortion material has been demonstrated as a means of retrospective chromosome analysis, indicating its potential applicability in cancer specimens as well. Clinical Significance and Implications Clinical Significance and Implications Contribution to accurate disease diagnosis, prognosis, and treatment decisions Informs patient management, genetic counselling, and personalized therapeutic interventions based on the genetic profile revealed by FISH analysis. Ethical and Regulatory Considerations Ethical Guidelines for FISH Research and Clinical Use Nuremberg Code o Emphasizes voluntary consent and minimizing risks in clinical research studies. Declaration of Helsinki o Provides ethical principles for biomedical research, emphasizing the protection of research participants. Belmont Report o Outlines principles of respect for persons, beneficence, and justice, serving as a foundation for ethical conduct in research practices Patient Confidentiality and Informed Consent Confidentiality o Laws such as HIPAA in the USA and similar regulations in the UK ensure the protection of patients' health information and require consent for disclosure. Informed Consent o The principles of voluntary consent and the provision of relevant information to patients are fundamental to the ethical conduct of FISH testing and research.  Regulatory Frameworks and Standards in FISH Testing and Reporting HIPAA o Sets limitations on the disclosure of protected health information and establishes requirements for consent in the USA State and Federal Regulations o Specific regulations and codes governing the handling of health information, reporting of abuse cases, and the requirements for informed consent in both the UK and USA, ensuring the ethical conduct of FISH testing and reporting Future Directions and Research Trends in FISH Advancements in FISH Technology and Applications High-Resolution Imaging o Integration of super resolution imaging systems for enhanced visualization of nuclear structures and gene functions Multiplex FISH o Advancements in probe labelling efficiency facilitating the development of multiplex FISH assays for simultaneous detection of multiple DNA or RNA sequences in single cells Microfluidic Platforms o Use of microfluidic devices for FISH analysis, streamlining the process and offering miniaturized and automated solutions for detecting chromosome abnormalities  Emerging Research Areas and Potential Impact on Medical Science Personalized Medicine o Increasing demand for FISH technology in biomarker research and personalized medicine, contributing to tailored therapies in cancer and other diseases Single-Cell Analysis o Single-molecule RNA FISH and simultaneous RNA-DNA FISH techniques opening new avenues for quantitative imaging of RNA molecules and simultaneous detection of mRNA and protein in single cells Expanded Disease Research o FISH playing a pivotal role in detecting expanded repeats in human diseases, analysing sperm aneuploidy frequencies, and contributing to the diagnosis and tailored therapies in solid tumours  Summary Fundamentals of FISH Interpretation of FISH Results Ethical and Regulatory Considerations Future Directions and Research Trends