Overview
Polymerase Chain Reaction (PCR) in Pathology is a molecular technique used to amplify specific DNA sequences for diagnostic, prognostic, and research purposes. This technique allows detection of genetic mutations, infectious agents, and gene expression patterns in tissue samples.
PCR is essential for molecular diagnosis, detecting genetic mutations in cancer, identifying infectious agents, and determining treatment response. It enables personalized medicine approaches and provides critical information for patient management decisions.
Learn more in our comprehensive RxDx Pathology Course
Indications
Detection of genetic mutations in tumors
Identification of viral, bacterial, fungal agents
Detection of inherited genetic disorders
Monitoring response to targeted therapies
Gene expression studies and research
Validation of molecular diagnostic tests
Contraindications
Absolute Contraindications
Relative Contraindications
📋 Equipment Checklist
Check off items as you gather them:
Pre-procedure Preparation
Laboratory preparation includes ensuring PCR equipment is calibrated, checking reagent quality, verifying proper sample preparation, and calibrating thermal cyclers. All reagents should be properly stored and contamination prevention measures in place.Step-by-Step Procedure
Step 1: Sample Preparation
Extract DNA/RNA from tissue samples using appropriate extraction methods. Quantify nucleic acid concentration and assess quality. Prepare samples in appropriate buffer solutions.
⚠️ Common Mistakes to Avoid:
- Poor quality nucleic acid extraction
- Inadequate quantification
- Contamination during extraction
💡 Pro Tip:
Always use fresh reagents and sterile techniques to prevent contamination.
Step 2: Reaction Setup
Prepare PCR reaction mix with appropriate concentrations of primers, dNTPs, Taq polymerase, and buffer. Include positive and negative controls. Set up reactions in sterile conditions.
⚠️ Common Mistakes to Avoid:
- Incorrect reagent concentrations
- Missing controls
- Contamination during setup
💡 Pro Tip:
Include positive and negative controls with each PCR run.
Step 3: Thermal Cycling
Program thermal cycler with appropriate temperature cycles: denaturation (94°C), annealing (50-65°C), extension (72°C). Run appropriate number of cycles based on target detection.
⚠️ Common Mistakes to Avoid:
- Incorrect temperature settings
- Wrong number of cycles
- Poor temperature control
💡 Pro Tip:
Optimize annealing temperature for specific primer pairs.
Step 4: Product Analysis
Analyze PCR products using gel electrophoresis or real-time PCR. Visualize bands on agarose gel or analyze fluorescence curves. Document results and assess quality.
⚠️ Common Mistakes to Avoid:
- Poor gel quality
- Incorrect interpretation
- Inadequate documentation
💡 Pro Tip:
Use appropriate molecular weight markers for size determination.
Step 5: Quality Control
Assess PCR quality by checking positive and negative controls, evaluating band intensity, and verifying expected product sizes. Document any deviations or problems.
⚠️ Common Mistakes to Avoid:
- Inadequate quality control
- Missing control evaluation
- Poor documentation
💡 Pro Tip:
Always evaluate controls before interpreting results.
Step 6: Result Interpretation
Interpret PCR results based on expected product sizes, control reactions, and clinical context. Document findings and prepare report with appropriate clinical correlation.
⚠️ Common Mistakes to Avoid:
- Incorrect interpretation
- Missing clinical correlation
- Poor documentation
💡 Pro Tip:
Correlate molecular findings with histopathological features.
Step 7: Documentation and Reporting
Document all PCR parameters, results, and interpretations. Prepare comprehensive report including methodology, results, and clinical significance. Maintain quality control records.
⚠️ Common Mistakes to Avoid:
- Incomplete documentation
- Poor reporting
- Missing quality control records
💡 Pro Tip:
Maintain detailed records of all PCR parameters and results.
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Post-procedure Care
Post-PCR 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 |
|---|---|---|---|---|
| Contamination | 5-10% | False positive results, unexpected bands | Decontamination, repeat with fresh reagents | Sterile technique, separate work areas |
| Poor amplification | 10-15% | Weak or absent bands | Optimize conditions, check reagents | Quality control, proper optimization |
| Non-specific amplification | 5-10% | Multiple unexpected bands | Optimize annealing temperature | Proper primer design, optimization |
| Equipment failure | 1-3% | Incomplete cycling, temperature errors | Equipment repair, repeat reactions | Regular maintenance, backup equipment |
| Sample degradation | 3-7% | Poor quality results, failed reactions | Repeat with fresh samples | Proper storage, quality assessment |
Clinical Pearls
Always use sterile technique and separate work areas to prevent contamination.
Include positive and negative controls with each PCR run for quality control.
Optimize annealing temperature for specific primer pairs to improve specificity.
Always evaluate controls before interpreting results.
Maintain detailed records of all PCR parameters and results.
Correlate molecular findings with histopathological features for accurate diagnosis.
Be aware of the specific requirements and limitations of different PCR applications.