Improved performance characteristics of the von Willebrand factor ristocetin cofactor activity assay using a novel automated assay protocol

Abstract

Willebrand factor ristocetin cofactor activity assay using a novel automated assay protocol. J Thromb Haemost 2010; 8: 2216-23.Summary. Background, objectives and methods: An accurate, sensitive and precise assay for reliable determination of the ristocetin cofactor activity of von Willebrand factor (VWF:RCo) in plasma and von Willebrand Factor (VWF)-containing concentrates has been evaluated. The assay is based on a commercially available automated protocol with modifications including a combination of adding additional ristocetin and the use of two calibration curves for the high and low measuring ranges. Results: Addition of extra ristocetin resulted in improved measurement of VWF recoveries from various VWF-containing concentrates that were underestimated using the standard automated protocol. The modifications resulted in improved assay performance over an extended measuring range (2.00-0.03 IU mL )1). Accuracy was tested using VWF deficiency plasma spiked with the 1st international standard (IS) for VWF concentrate. Seven dilutions, ranging from 1.80 to 0.05 IU mL )1, were analyzed and resulted in measured concentrations between 80% and 100% of the assigned potency of the standard. Linearity was determined from the regression plot of the same concentrate dilutions and resulted in a correlation coefficient of 0.998. The repeatability, expressed as coefficient of variation, was 2% in the normal range (0.90 IU mL . The corresponding reproducibility results were 2% and 15% at the normal and low measuring ranges, respectively. Conclusions: Analysis of patients with von Willebrand disease (VWD) indicates that the modified automated BCS Ò protocol has a superior discrimination power compared with the standard protocol. This is especially true in samples with low VWF, as in patients with type 3 VWD.

von Willebrand Factor Assay Proficiency Testing Continued

“…Here, there is at least a potential solution, given recently reported vWF:RCo assay improvements. 8,9 Although lower limit of detection is notably better using vWF:antigen, vWF:CB, and vWF:Act assays, laboratories will still have difficulty accurately identifying vWF values between 0 and 10 U/dL. 7 Fourth, although the vWF:CB was found to provide the best discrimination of types 1 and 2 vWD by Chandler et al, 1 in support of previous findings, it was interesting that this was achieved using commercial kits that have been found to be less optimal in direct vWF:CB comparison assessments.…”

Rapid discrimination of the phenotypic variants of von Willebrand disease

“…34 A few assays have taken advantage of GPIb binding independent of ristocetin through using recombinant human GPIb with 2 gain-of-function mutations inserted. 12,13,35 Flood et al have described the VWF:IbCo assay, which demonstrated a CV of 7% and lower limit of detection of 3 U/dL.…”

Diagnosis and classification of von Willebrand disease

“…Automation of VWF:RCo appears to improve precision, but in general does not improve LOD issues [24], and does not seem to protect laboratories against VWD identification errors [26,27]. However, a recently reported modification to automated VWF:RCo testing would appear to have largely overcome the LOD problem, with this now reported to be as low as 2 U/dl in recent testing [47,48].…”

“…Historically, VWF:RCo was first utilized as an agglutination assay primarily in platelet aggregometers or using a manual visual slide agglutination procedure; both of these methods are laborious. VWF:RCo by platelet agglutination has also been successfully automated on many modern haemostasis analysers, including the Siemen's BCS, IL ACL instrumentation, Stago STAR and most recently the Sysmex CA-2000i/CA2100i instruments [41][42][43][44][45][46][47][48]. Methods for VWF:RCo based on flow cytometry have also been described [49,50], as have ELISA-based methods [51][52][53].…”

“…For most assays, this is usually within the 20-100 U/dl VWF detection region. For VWF: RCo, an improved automated assay with low detection limits has recently been described [47,48].…”