Performance Characteristics for Flow Cytometric Enumeration of Peripheral Blood Lymphocyte Subsets under Harmonized Laboratory Conditions: A Multicenter Study.
Abstract
Background
Flow cytometric enumeration of peripheral blood lymphocyte subsets (EPBLS) is essential for disease management, yet standardized validation criteria are lacking. We defined key performance characteristics under harmonized conditions to support criteria development.
Methods
205 whole blood samples and 54 quality control samples at two levels from nine clinical laboratories across five regions in China were used to measure absolute counts (AC) and relative counts (RC) of lymphocyte subsets using flow cytometric single-platform. Performance characteristics were evaluated based on 2,865 measurements and an integrated statistical approach combining the concordance correlation coefficient, Passing-Bablok regression, and Bland-Altman plots.
Results
Performance characteristics for precision, comparability, and sample stability were established to cover the major clinically relevant lymphocyte subset distributions (RC: 1.32-97.53%; AC: 3.37-6,846.98 cells/μL). Intra-assay and inter-assay coefficients of variation were below 8% and 6% for RC, below 10% and 9% for AC, respectively. A rigorous dual-criteria framework (comparability criteria: consistency and correlation; deviation criteria: absolute maximum bias threshold of <20%), supported by a triple-statistical approach, confirmed agreement across reagent batches, laboratories, and storage time points, with comprehensive bias profiles further reported. Whole blood remained stable for 24 hours post-collection (RC bias <14%; AC bias ≤15%), and processed samples for 48 hours post-staining (RC bias <13%; AC bias <19%).
Conclusions
This study establishes a comprehensive performance threshold and dual-criteria evaluation system covering precision, comparability, and stability for EPBLS, based on a harmonized multicenter protocol using stratified routine clinical samples, providing a practical reference for laboratory validation and quality specification development.