Abstract Number: PB0350
Meeting: ISTH 2020 Congress
Background: The one stage coagulation assay is widely used in diagnostic laboratories to monitor Factor IX levels in patients with Hemophilia B despite poor precision at low levels of Factor IX. We report here the performance characteristics of a newly developed chromogenic assay for Factor IX on an automated analyzer platform (Siemens BCS XP).
Aims: Summarize the assay format and performance of an automated chromogenic assay using a single calibration curve for the measurement of Factor IX in human plasma samples
Methods: The assay principle is based on the activation of Factor FIX in the sample, mediated by Factor XIa in the reagent. The activated Factor IX then potentiates the activation of Factor X in the presence of Factor VIIIa, Calcium and Phospholipids. A chromogenic substrate is used to measure the activity of the Factor Xa generated. This assay uses Thrombin to improve the efficiency of activation of Factor FVIII. The assay uses a single calibration curve from 100% down to 1% FIX using a sample dilution of 1:40.
Results: The assay reagents show excellent reproducibility of calibration curves verified across multiple runs per day and multiple days which showed pooled CV of 3.2% across the calibration interval. Preliminary estimation of the assay sensitivity showed LOB and LOD of 0.1% and 0.3% respectively. Upon reconstitution, the assay reagents are stable for up to 24 hours under refrigerated storage. The reagents also show on-board stability of 8 hours, consistent with other commercial kits. Recovery studies were performed with sample Factor IX levels across the calibration curve (88%, 5%, 2% & 1%) and showed good recovery and precision (≤ 12% CV) across the measured levels.
Conclusions: With a single calibration curve, this chromogenic assay offers a simplified and cost-effective approach towards the measurement of Factor IX in Hemophilia B patients
To cite this abstract in AMA style:Ramamurthy N, Duffin S, Kucharski C, Vandertuin Z, Kaushik S, Morris M. Development and Performance Characterization of an Automated Chromogenic Assay for Human Factor IX Activity [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/development-and-performance-characterization-of-an-automated-chromogenic-assay-for-human-factor-ix-activity/. Accessed January 28, 2022.
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