Validation of an Optimized DuraClone Phenotyping Kit Workflow for TBNK Subset Quantification.

Area of Science:

• Clinical immunology
• Flow cytometry
• Hematology

Background:

• Accurate quantification of T-cells, B-cells, and NK-cells (TBNK) is vital for diagnosing immune disorders and monitoring therapies.
• Standardization of TBNK subset analysis in peripheral blood is essential for reliable clinical results.

Purpose of the Study:

• To standardize and validate an optimized workflow for the Beckman Coulter DuraClone IM Phenotyping Basic Kit for TBNK subset quantification.
• To improve workflow efficiency and analytical performance of TBNK enumeration.

Main Methods:

• Modified the DuraClone IM kit protocol, including changes to lysis buffer, addition of counting beads, elimination of centrifugation, and increased acquisition volume.
• Validated the optimized method according to CLSI guidelines (H42-A2, H62) for accuracy, precision, linearity, and limit of quantification (LLOQ).
• Assessed accuracy using Immuno-Trol controls and Bland-Altman analysis, and evaluated external proficiency via the CAP TBNK program.

Main Results:

• The modified protocol reduced procedural steps by 50% and processing time by 38.6%.
• Demonstrated high accuracy with low bias, strong agreement in Bland-Altman analysis, and successful completion of CAP proficiency tests.
• Achieved excellent precision (CV < 10%), linearity (R² > 0.99), and clinically relevant LLOQ (< 10-50 cells/μL) for TBNK subsets.

Conclusions:

• The optimized DuraClone IM kit protocol offers a standardized, reliable, and time-efficient method for lymphocyte subset quantification.
• The validated workflow improves efficiency and analytical performance without compromising accuracy or reproducibility.
• This provides a valuable alternative for clinical TBNK enumeration in peripheral blood.