Summary
von Willebrand disease (VWD) is caused by a quantitative and/or qualitative deficiency of the von Willebrand factor (VWF). The laboratory diagnosis of VWD is dependent on the measurement of ...VWF antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo). The aim of this study was to undertake a two‐centre evaluation of two new automated VWF:Ag and VWF:RCo assays systems from Instrumentation Laboratory (Bedford, USA). Using the two new analytical systems that operated with different detection principles: immunoturbidimetric (TOP500 analyser) and chemiluminescent (AcuStar analyser), VWF:Ag and VWF:RCo levels were determined in samples from 171 healthy normal subjects, 80 VWD patients (16 type 1, 58 type 2 and 6 type 3) and 7 acquired von Willebrand syndrome patients. With commercial lyophilized normal and pathological plasmas VWF: Ag and VWF:RCo assays performed on both analysers exhibited low levels of inter‐assay imprecision (AcuStar: CV% range 3.3–6.9; TOP500: CV% range 2.6–6.3). Samples from normal healthy subjects (range: VWF:Ag 44.6–173.9 IU dL−1; VWF:RCo 43.1–191.5 IU dL−1) and patients (range: VWF:Ag <0.3–115.1 IU dL−1; VWF:RCo <0.5–57.2 IU dL−1) showed a good correlation between the two VWF:Ag and VWF:RCo methods (rs = 0.92 and 0.82 respectively), with only a few inconsistent cases among the patients' samples evaluated. The chemiluminescent assays had a lower limit of detection for both VWF:Ag and VWF:RCo compared to immunoturbidimetric tests (0.3 IU dL−1 vs. 2.2 IU dL−1 and 0.5 IU dL−1 vs. 4.4 IU dL−1 respectively). The TOP500 and AcuStar VWF:Ag and VWF:RCo assays were precise and compare well between centres, making these systems suitable for the diagnosis of VWD in non‐specialized and reference laboratories.
Essentials
New VWF activity assays are increasingly used but information on their comparability is limited.
This is an ISTH SSC‐organized study (expert labs, 5 countries) to compare all available ...assays.
VWF activity by six assays correlated well with each other.
The new assays show improved characteristics ‐ minor differences are noted.
Summary
Background
Several new assays have become available to measure von Willebrand factor (VWF) activity. The new assays appear to have improved performance characteristics compared with the old reference standard, ristocetin cofactor activity (VWF:RCo), but information is limited about how they compare with VWF:RCo and each other.
Methods
The von Willebrand factor Subcommittee of the International Society for Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee (SSC) designed a collaborative study involving expert laboratories from several countries to compare available tests with each other and with VWF:RCo. Eight laboratories from five countries were provided with blinded samples from normal healthy individuals and well‐characterized clinical cases. Laboratories measured VWF activity using all tests available to them; data from six laboratories, not affected by thawing during transportation, are included in this study.
Results
All tests correlated well with VWF:RCo activity (r‐values ranged from 0.963 to 0.989). Slightly steeper regression lines for VWF:Ab and VWF:GPIbM were clinically insignificant. The new assays showed improved performance characteristics. Of the commercially available assays, the VWF:GPIbR using the AcuStar system was the most sensitive and could reliably detect VWF activity below 1 IU dL−1. The lower limit of the measuring interval for the VWF:GPIbM and the VWF:GPIbR assays was in the 3–4 and 3–6 IU dL−1 range, respectively. Inter‐laboratory variation was also improved for most new assays.
Conclusion
All VWF activity assays correlated well with each other and the VWF:RCo assay. The slight differences in characteristics found in the COMPASS‐VWF study will assist the VWF community in interpreting and comparing activity results.
Introduction
Laboratory diagnosis of von Willebrand disease (VWD) is made by the measurement of von Willebrand factor (VWF) protein level and its activities. Current VWF activity tests include ...ristocetin cofactor and collagen binding (VWF:CB) assays.
Aim
We have undertaken an evaluation of a new fully automated VWF:CB assay relative to an established enzyme‐linked immunosorbent assay (ELISA) method.
Methods
The two analytical systems operate with different detection principles: a chemiluminescent method performed on ACL AcuStar Analyzer (the former) and a colorimetric ELISA by Asserachrom Stago (the latter) (type III collagen from human placenta). The HemosIL AcuStar VWF:CB assay is a chemiluminescent 2‐step immunoassay that uses magnetic particles coated with a type III collagen triple‐helical peptide. VWF:CB levels were determined in 50 healthy subjects and 100 VWD patients (22 type 1, 73 type 2 and 5 type 3).
Results
Eleven VWD samples reported VWF:CB values below the lower detection limit of one or both methods. The new method showed a good correlation with the ELISA method (r > .9, mean bias 3.85 IU/dL) in both healthy and VWD samples. One of 150 samples gave inconsistent results using the two assays, leading to an uncertain diagnosis of VWD type 1 (ELISA method) or type 2 MCB (fully automated method).
Conclusion
The new assay is rapid and simple to use, with its ready‐to‐use reagent cartridges. This VWF:CB assay, in addition to the measurement of VWF:Ag and VWF:RCo made on the same platform, gives additional information for the diagnosis of VWD in both nonspecialized and reference laboratories.
Introduction
We characterized five patients affected with von Willebrand disease (VWD) carrying the p.Arg1379Cys mutation. One was diagnosed as VWD type 1 and four as type 2M. The 2M patients also ...have the variant p.Ala1377Val in cis with p.Arg1379Cys.
Aim
To evaluate the role of p.Ala1377Val and p.Arg1379Cys von Willebrand factor (VWF) variants to explain patients' phenotype.
Methods
Conventional phenotype tests were used to evaluate patients' plasma and platelets. Direct sequence analysis of exon 28 was carried out. The allele frequency of p.Ala1377Val was evaluated using online database. pcDNA3.1‐VWF‐WT and mutant (A1377V, R1379C and A1377V‐R1379C) expression vectors were transiently transfected in HEK293 cells. The capacity of WT and mutant recombinant (r)VWF (along with patients' plasma VWF) to bind glycoprotein Ibα (GpIbα) were evaluated, using two ELISA assays. One with a wild‐type (WT) recombinant (r)GpIbα at increasing ristocetin concentrations (from 0 to 1.50 mg mL−1) and the other with a gain‐of‐function mutant rGpIbα (VWF:GPIbM).
Results
The substitution c.4130C>T (p.Ala1377Val) was reported as rare variant in online databases. At 0.25 mg mL−1 of ristocetin, WT, A1377V and R1379C showed 6, 7.5 and 12‐fold increased binding to rGpIbα, respectively. A1377V‐R1379C rVWF showed no increased binding to rGpIbα at the same ristocetin concentration and reached the highest binding, of only 3‐fold increased, at 1.50 mg mL−1 of ristocetin. The VWF:GPIbM showed strongly reduced values for the A1377V‐R1379C rVWF and the 2M patients' plasma.
Conclusion
Our study showed that the presence of both p.Ala1377Val and p.Arg1379Cys mutations (synergistic effect) abolishes the binding of rVWF to rGpIbα, explaining patients' 2M phenotype.
Summary
Background
Diagnosis of von Willebrand disease (VWD) type 2 usually relies on the discrepancy between the von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) and VWF antigen ...(VWF:Ag). Type 2B patients can be discriminated from other qualitative VWD variants by using ristocetin‐induced platelet agglutination (RIPA) test. The major limitation of RIPA is the requirement of fresh blood sample.
Objectives
In this study, we evaluated the VWF gain‐of‐function mutant GPIb binding (VWF:GPIbM) and VWF:RCo assays to investigate whether the VWF:GPIbM/VWF:RCo ratio was able to identify the type 2B variant among an heterogeneous VWD population, previously characterized following the ISTH‐SSC guidelines.
Patients/methods
Seventy‐six VWD patients and 31 healthy subjects were evaluated by using VWF:Ag, VWF:RCo, and VWF:GPIbM assays.
Results
The mean (minimum–maximum values) VWF:GPIbM/VWF:RCo ratio was higher in type 2B patients (2.53, 0.84–6.11) than in healthy controls (1.05, 0.87–1.34), type 1 (0.85, 0.51–1.15), 2A (1.20, 0.36–2.82), and 2M (1.07, 0.91–1.38) (P < 0.0001). Type 2B variants were divided into four groups (A, B, C, and D) according to their different multimeric patterns. The mean value of the VWF:GPIbM/VWF:RCo ratio in the four groups showed an increasing trend from group A (1.08) to D (3.69), proportional to the loss of high molecular weight multimers. Among 32 type 2B patients, previously diagnosed with RIPA, 8 (mainly with a type I New York/Malmö phenotype) were not confirmed using the VWF:GPIbM/VWF:RCo ratio.
Conclusions
Whenever the RIPA test is not feasible, the VWF:GPIbM/VWF:RCo ratio might help to identify severe type 2B VWD patients.
Summary
Background
Shear stress triggers conformational stretching of von Willebrand factor (VWF), which is responsible for its self‐association and binding to the platelet receptor glycoprotein ...(GP)Ibα. This phenomenon supports primary hemostasis under flow. Type 2B VWF natural mutants are considered to have increased affinity for platelet GPIbα.
Objectives
To assess the mechanism responsible for the enhanced interaction of the p.R1306W VWF mutant with the platelet receptor.
Methods
The interaction of GPIbα with wild‐type (WT) and p.R1306W VWF multimers and A1–A2–A3 constructs was investigated with surface plasmon resonance spectroscopy. Analysis of the static VWF conformation in solution was performed with dynamic light scattering spectroscopy. The shear stress‐induced self‐association of VWF multimers was investigated with atomic force microscopy (AFM) over a 0–60 dyn cm−2 range.
Results
WT VWF did not interact with GPIbα under static conditions, whereas the mutant at ~ 2 μg mL−1 already bound to the receptor. By contrast, the WT and p.R1306W‐A1–A2–A3 constructs showed comparable affinities for GPIbα (Kd ~ 20 nm). The hydrodynamic diameter of resting R1306W VWF multimers was significantly greater than that of the wild type (210 ± 60 nm vs. 87 ± 22 nm). At shear forces of < 14 dyn cm−2, the p.R1306W multimers rapidly changed conformation, entering a regime of self‐aggregation, which, in contrast, was induced for WT VWF by shear forces of > 30 dyn cm−2. Mechanical stretching AFM experiments showed that p.R1306W multimers needed less energy per length unit (~ 10 pN) to be stretched than the WT protein.
Conclusions
The increased affinity of p.R1306W VWF for GPIbα arises mostly from higher sensitivity to shear stress, which facilitates exposure of GPIbα binding sites.
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