Dihydrorhodamine-123 flow cytometry method: time for substantial revision in technical procedure.

Area of Science:

• Immunology
• Cellular Biology
• Diagnostic Assays

Background:

• The dihydrorhodamine 123 assay is a key method for measuring intracellular reactive oxygen species (ROS) in neutrophils.
• This assay is crucial for diagnosing chronic granulomatous disease (CGD).
• Numerous preanalytical and analytical variables can impact the assay's reliability.

Purpose of the Study:

• To identify optimal conditions for the dihydrorhodamine 123 assay.
• To establish standard operating procedures for reliable ROS measurement.
• To compare preanalytical and analytical phases influencing test outcomes.

Main Methods:

• Investigated the impact of different anticoagulants on assay results.
• Assessed the effect of sample transport and storage temperatures (4°C vs. 24°C).
• Evaluated the influence of test prime run time, solution concentrations, and incubation temperatures (24°C vs. 37°C).

Main Results:

• Specific anticoagulants and temperature conditions significantly affected ROS production measurements.
• Incubation temperature and sample handling protocols demonstrated a notable impact on assay sensitivity.
• Optimized parameters were identified to enhance assay precision and accuracy.

Conclusions:

• Standardizing preanalytical and analytical steps is essential for reproducible dihydrorhodamine 123 assay results.
• Implementing defined operating procedures based on these findings will improve diagnostic accuracy for CGD.
• Further research may refine these parameters for even greater reliability.