Toward standardized antithrombin activity assays: multicenter evaluation and optimization

Antithrombin (AT) is a critical anticoagulant whose deficiency, which is common in many clinical conditions, requires precise management, although accurate laboratory measurements of low AT levels are challenging. This study aims to evaluate the precision and comparability of AT activity assays across different analytical systems, particularly at low activity levels, with the goal of developing validated optimization strategies to improve performance and interlaboratory harmonization. Standardized lyophilized plasma materials (with AT activities of 10%, 15%, 35% and 100% of normal) were distributed to nine clinical laboratories via Systems A and B, with bovine thrombin as the reagent, and System C, with bovine activated coagulation factor Ⅹ as the reagent. Baseline performance was assessed through a laboratory evaluation and external quality assessment style comparison. Optimization measures (standardized reagent reconstitution, calibration curves at low levels, enhanced quality control protocols) were then applied in laboratories with better baseline performance, followed by a re-evaluation study to assess improvements. The initial evaluation confirmed significant variability at low AT activity levels: Systems A and B demonstrated closer agreement with target levels, whereas System C exhibited significant bias and poor reproducibility and was consequently excluded from optimization. After implementation of the optimization protocols, low-level AT activity measurements showed markedly reduced bias and improved precision. At the 10% and 15% levels, both the interlaboratory coefficients of variation and the recovery rates significantly improved. In the re-evaluation study, Systems A and B demonstrated enhanced performance across all participating laboratories. Conclusions : This multicenter evaluation established the feasibility of significantly improving AT activity assay performance through targeted methodological optimization. Through the use of quality control materials and harmonized procedures, the method achieved reliable low-level activity measurements, thereby paving the way for the reliable clinical application of AT activity measurements.