What is the procedure for performing a double dye test?

Double Dye Test Procedure

Overview

The double dye test refers to a technique using two different colored dyes simultaneously to improve diagnostic accuracy, most commonly applied in sentinel lymph node biopsy for breast cancer, where it combines blue dye (typically methylene blue or Patent Blue V) with a second dye (indocyanine green or a different colored dye) to enhance lymph node identification. 1, 2

Procedure for Sentinel Lymph Node Biopsy (Most Common Application)

Pre-Procedure Preparation

• Verify no contraindications: Exclude children, pregnant women, lactating women, and patients with known allergies to either dye 3
• Obtain informed consent discussing potential allergic reactions including rare anaphylaxis 3
• Prepare equipment: Sterile syringes, gloves to prevent staining, and gauze swabs 3

Dye Preparation and Injection Technique

For the dual dye method combining blue dye and indocyanine green:

• Blue dye (Patent Blue V or Methylene Blue): Inject subareolarly at the time of surgery under general anesthesia 3, 2

  • Patent Blue V: Use 2-mL vials at 2.5% concentration 3
  • Allow 10-15 minutes for dye migration from injection site to sentinel lymph node 3

• Second dye (typically Indocyanine Green): Inject peritumorally 2

  • This creates a two-site injection strategy that decreases false-negative rates compared to single-site injection 2

Timing Considerations

• Blue dye injection: Performed intraoperatively, approximately 10-15 minutes before node identification begins 3
• The interval between injection and visualization is critical for adequate lymphatic uptake 3

Node Identification

• Use both visual inspection and detection equipment:

  • Blue-stained nodes are identified visually 1, 2
  • Indocyanine green requires near-infrared fluorescence imaging 1
  • Mixed-color nodes (showing both dyes) should be counted 2

• Harvest all nodes showing either color or both colors 2

  • Nodes showing only blue dye 2
  • Nodes showing only the second dye 2
  • Nodes showing mixed coloration 2

Critical Technical Points

The double-mapping method with two injection sites (subareolar + peritumoral) achieves:

• Identification rate: 96.6% 2
• Sensitivity: 95.1% 2
• False-negative rate: 4.9% (compared to 10% with single-site injection) 2

Single-site injection has higher false-negative rates:

• Subareolar alone: 10.3% false-negative rate 2
• Peritumoral alone: 10.0% false-negative rate 2

Special Considerations for Tumor Location

• Upper-outer quadrant tumors require particular attention to the double-mapping technique, as lymphatic channel variations in the lateral breast may cause false-negative results with single-site injection 2
• All four patients with discordant node positivity (where only one color showed positive nodes) had upper-outer quadrant tumors 2

Safety Monitoring

• Monitor for anaphylaxis during and immediately after injection 3
• Have resuscitation equipment available 3
• If allergic reaction occurs: Discontinue injection immediately and initiate appropriate resuscitation; decision to continue procedure should involve surgeon and anesthesiologist 3

Documentation Requirements

• Record both injection sites used 2
• Document number of nodes identified by each dye color 2
• Note any nodes showing mixed coloration 2
• Record time interval between injection and node identification 3

Common Pitfalls to Avoid

• Do not rely on single-site injection alone for tumors in the upper-outer quadrant, as this increases false-negative rates to 10% 2
• Do not assume blue dye staining interferes with pathological interpretation of specimens, as this has not been demonstrated 3
• Avoid using fluorescein-anesthetic combination drops if performing ocular dye testing, as anesthetics may affect results 4