Overview
Fluorescence In Situ Hybridization (FISH) is a molecular cytogenetic technique used to detect and localize specific DNA sequences on chromosomes or in cells. This technique uses fluorescent probes that bind to specific DNA sequences, allowing visualization of genetic abnormalities, gene amplifications, and chromosomal rearrangements.
FISH is essential for molecular diagnosis, detecting chromosomal abnormalities, gene amplifications, and genetic rearrangements in cancer and genetic disorders. It provides critical information for diagnosis, prognosis, and treatment decisions. FISH is particularly valuable in hematological malignancies and solid tumors.
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Indications
Detection of chromosomal abnormalities in tumors
Detection of specific genetic rearrangements
Detection of inherited chromosomal abnormalities
Detection of gene copy number changes
Monitoring response to targeted therapies
Chromosomal studies and research
Contraindications
Absolute Contraindications
Relative Contraindications
📋 Equipment Checklist
Check off items as you gather them:
Pre-procedure Preparation
Laboratory preparation includes ensuring FISH equipment is calibrated, checking probe quality and specificity, verifying proper slide preparation, and calibrating fluorescence microscopes. All reagents should be properly stored and contamination prevention measures in place.Step-by-Step Procedure
Step 1: Slide Preparation
Prepare tissue sections on appropriate slides. Deparaffinize and rehydrate sections if using paraffin-embedded tissue. Ensure proper slide quality and labeling.
⚠️ Common Mistakes to Avoid:
- Poor slide quality
- Incomplete deparaffinization
- Poor labeling
💡 Pro Tip:
Always use high-quality slides and proper preparation techniques.
Step 2: Pretreatment
Apply pretreatment solutions to improve probe accessibility. Use appropriate pretreatment for tissue type (formalin-fixed, frozen, etc.). Optimize conditions for specific tissue types.
⚠️ Common Mistakes to Avoid:
- Inadequate pretreatment
- Over-treatment causing tissue damage
- Wrong pretreatment for tissue type
💡 Pro Tip:
Optimize pretreatment conditions for specific tissue types and fixation methods.
Step 3: Denaturation
Denature DNA in tissue sections and probes simultaneously. Use appropriate temperature and time for denaturation. Ensure proper temperature control.
⚠️ Common Mistakes to Avoid:
- Incorrect denaturation temperature
- Wrong denaturation time
- Poor temperature control
💡 Pro Tip:
Use precise temperature control for optimal denaturation.
Step 4: Hybridization
Apply fluorescent probes to denatured tissue sections. Incubate in humidified chamber at appropriate temperature. Allow sufficient time for hybridization.
⚠️ Common Mistakes to Avoid:
- Incorrect hybridization temperature
- Insufficient hybridization time
- Poor humidity control
💡 Pro Tip:
Maintain proper humidity and temperature during hybridization.
Step 5: Post-hybridization Washing
Wash slides to remove unbound probes. Use appropriate stringency conditions. Apply counterstain if needed for nuclear visualization.
⚠️ Common Mistakes to Avoid:
- Inadequate washing
- Wrong stringency conditions
- Poor counterstaining
💡 Pro Tip:
Optimize washing conditions for specific probe-target combinations.
Step 6: Mounting and Visualization
Mount slides with antifade mounting media. Examine under fluorescence microscope. Capture digital images for documentation and analysis.
⚠️ Common Mistakes to Avoid:
- Poor mounting
- Inadequate image capture
- Poor documentation
💡 Pro Tip:
Use antifade mounting media to preserve fluorescence signal.
Step 7: Analysis and Documentation
Analyze FISH signals for specific patterns. Count signals in appropriate number of cells. Document results and prepare comprehensive report.
⚠️ Common Mistakes to Avoid:
- Incorrect signal counting
- Poor documentation
- Inadequate analysis
💡 Pro Tip:
Count signals in sufficient number of cells for statistical validity.
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Post-procedure Care
Post-FISH care involves proper documentation of results, quality control assessment, result interpretation, and reporting. All data must be documented thoroughly and results properly validated.Complications & Management
Complication | Incidence | Signs | Management | Prevention |
---|---|---|---|---|
Poor signal quality | 10-15% | Weak or absent fluorescence signals | Optimize conditions, repeat with fresh reagents | Quality control, proper optimization |
High background | 5-10% | Excessive background fluorescence | Adjust washing conditions, optimize pretreatment | Proper pretreatment, optimal washing |
Non-specific binding | 5-10% | Unexpected fluorescence patterns | Optimize probe specificity, adjust conditions | Proper probe design, optimization |
Tissue damage | 3-7% | Tissue loss or damage | Repeat with gentler conditions | Gentle pretreatment, optimal conditions |
Equipment failure | 1-3% | Microscope or camera problems | Equipment repair, backup systems | Regular maintenance, backup equipment |
Clinical Pearls
Always optimize pretreatment conditions for specific tissue types and fixation methods.
Use precise temperature control for optimal denaturation and hybridization.
Count signals in sufficient number of cells for statistical validity.
Use antifade mounting media to preserve fluorescence signal quality.
Maintain detailed records of all FISH parameters and results.
Correlate FISH findings with histopathological features for accurate diagnosis.
Be aware of the specific requirements and limitations of different FISH applications.