Differences in the Composition of Activated Partial Thromboplastin Time (APTT) Reagents Affect Clot Waveform Analysis

Background Clot waveform analysis (CWA) based on activated partial thromboplastin time (APTT) is a useful assay for hemostasis. However, the effects of activators and phospholipid conditions on CWA have not been adequately investigated. Therefore, we characterized CWA using four different APTT reagents. Methods: We used 39 archived plasma samples from patients with hemophilia A (HA), 16 samples from patients with HA under emicizumab treatment, and 10 samples from healthy individuals for CWA with four different types of APTT reagents (reagents A, B, C, and D). We then compared Ad|min1|, Ad|min2|, and Ad|max2| from the CWA, which reflect the maximum velocity, maximum acceleration, and maximum deceleration, respectively, among the four reagents. Results: Similar clot waveform shapes were observed for each reagent in the healthy donor group, HA group, and HA under emicizumab group, and the waveform was different for each target group. Significant changes were found in clotting time (CT) (s), Ad|min1| (%/s), Ad|min2| (%/s2), and Ad|max2| (%/s2). The waveform pattern for the coagulation reaction by reagent D, comprising silica and synthetic phospholipids, was the fastest among the reagents examined. Further, the difference in Ad|min1| (%/s) and Ad|min2| (%/s2) was larger than that in CT depending on the reagent used(s), indicating that the measured value of CWA was affected by the reagent composition. Conclusion: Our results showed a significant difference among reagents with varying composition and concentration; this was found to affect the parameters obtained from CWA. Thus, the differences between reagents hinder standardization of quantitative evaluation using these parameters; further, this highlights the necessity of understanding the characteristics of APTT reagents and determining the reference range in individual facilities.