Approaches for Performance Verification Toward Standardization of Peripheral Blood Regulatory T-Cell Detection by...

Area of Science:

• Immunology
• Clinical Diagnostics
• Flow Cytometry

Background:

• Regulatory T-cell (Treg) detection via flow cytometry is crucial for diagnosing and managing immune-mediated diseases.
• Standardization and reliable performance verification of Treg assays are currently lacking.

Purpose of the Study:

• To standardize Treg assays by evaluating the performance of three commercial reagent sets.
• To assess Treg detection agreement across different reagent sets using flow cytometry.

Main Methods:

• Analytical performance of Treg assays was evaluated, including gating strategy, antibody optimization, sample stability, repeatability, reproducibility, reportable range, linearity, and assay carryover.
• Agreement between assays was analyzed using Bland-Altman plots and linear regression.
• The correlation between CD4+CD25+CD127low/- Tregs and CD4+CD25+Foxp3+ Tregs was assessed.

Main Results:

• Postcollection sample stability was confirmed up to 72 hours at room temperature.
• Assay accuracy, repeatability, and reproducibility met clinical analysis requirements, with excellent linearity (R2 ≥0.9) and no carryover.
• Results from three manufacturers showed good agreement, and CD4+CD25+CD127low/- Treg frequency correlated well with CD4+CD25+Foxp3+ Tregs.

Conclusions:

• This study provides the first systematic evaluation of Treg measurement performance in peripheral blood using flow cytometry.
• The findings offer a practical method for verifying the performance of flow cytometry-based immune monitoring assays in clinical settings.
• The validated methods contribute to the standardization of Treg detection for improved clinical practice.