Establishment and clinical application of flow cytometric bead assay for detecting vWF activity based on mutant GPⅠbα
10.3760/cma.j.cn114452-20241208-00667
- VernacularTitle:基于突变型GPⅠbα检测vWF活性的流式微球方法的建立及评价
- Author:
Yunxiao ZHAO
1
;
Yang HE
1
;
Changgeng RUAN
1
;
Fei SHEN
1
Author Information
1. 苏州大学附属第一医院江苏省血液研究所，苏州215006
- Publication Type:Journal Article
- Keywords:
von Willebrand Diseases;
Flow cytometry;
von Willebrand factor activity
- From:
Chinese Journal of Laboratory Medicine
2025;48(10):1310-1316
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a flow cytometric bead assay (FCBA) for detecting von Willebrand factor (vWF) activity based on mutant GPⅠbα and to perform its clinical validation.Methods:Recombinant GPⅠbα protein with M239V, D235Y, and C65A point mutations was constructed and bound to flow cytometric beads. vWF in samples bound to the recombinant protein, and FITC-labeled rabbit anti-human vWF polyclonal antibody was added for detection and analysis using flow cytometry. A total of 32 patients with von Willebrand disease (10 males and 22 females, aged 26.81±6.96 years) admitted to the Department of Hematology at the First Affiliated Hospital of Soochow University from January 1, 2022, to November 30, 2023, were enrolled. Additionally, 51 healthy controls (17 males and 34 females, aged 31.56±8.98 years) were included. Plasma from 20 healthy controls was pooled in equal proportions to create standard plasma. Fourteen dilutions were prepared, ranging from the original plasma to a 1∶8, 192 dilution, and a curve was plotted according to the FCBA protocol to determine the optimal dilution. Plasma from one healthy control was aliquoted and frozen, and FCBA was performed on days 1, 5, 15, 30, 45, and 60 after bead coating to evaluate stability. The same healthy control plasma sample was tested 20 times using both FCBA and vWF enzyme-linked immunesorbent assay (ELISA) to calculate within-batch precision. The same sample was tested daily for 10 consecutive days using both FCBA and vWF-ELISA to calculate between-batch precision. All samples were tested using FCBA, and the same samples were also tested using vWF-ELISA. The χ2 test was used to compare the positive rates, negative rates, and accuracy of the two methods. One sample was used as the base sample, and another sample with a known activity of 100% was diluted to high, medium, and low activities and added to the base sample. Recovery rates were calculated using both FCBA and vWF-ELISA. Re-collected 11 cases of vWD patient samples and 10 cases of healthy subjects, and detected vWF activity using the FCBA method to validate the concordance of FCBA in clinical applications. Results:The optimal dilution for standard plasma was determined to be 1∶64. The coated beads remained stable for detection up to 60 days, with correlation coefficients of the standard curve on days 1, 5, 15, 30, 45, and 60 being 0.98, 0.98, 0.97, 0.95, 0.95, and 0.95, respectively. The within-batch coefficients of variation for FCBA and vWF-ELISA were 5.35% and 6.08%, respectively, while the between-batch coefficients of variation were 7.02% and 7.98%, respectively. The correlation coefficient between FCBA and vWF-ELISA was R2=0.798 ( P0.01). The positive rates were 90.63% and 84.38% ( χ2=0.571, P0.05), the negative rates were 92.16% and 94.12% ( χ2=0.153, P0.05), and the accuracy rates were 91.57% and 90.36% ( χ2=0.073, P0.05) for FCBA and vWF-ELISA, respectively. In the recovery experiment, the high-value recovery rates for FCBA and ELISA were 102.11% and 99.32%, respectively, the medium-value recovery rates were 98.50% and 95.66%, and the low-value recovery rates were 95.34% and 88.51%. The FCBA method achieved a 100% concordance rate in detecting type 1, type 2, and type 3 vWD, as well as healthy subjects. Conclusion:A ristocetin-independent FCBA method for detecting vWF activity based on mutant GPⅠbα is successfully established.
