Measurement of von Willebrand factor‐FVIII binding activity in patients with suspected von Willebrand disease type 2N: application of an ELISA‐based assay in a reference laboratory

Abstract

Laboratory diagnosis of von Willebrand disease type 2N (VWD2N) is based on costly mutation analysis or in vitro measurement of the ability of plasma von Willebrand factor (VWF) to bind exogenous factor VIII (FVIII); however, the VWF-FVIII binding activity assay is complex and not widely used. Our aim was to assess the utility of the in-house VWF-FVIII binding assay in the investigation of patients with suspected VWD2N. A previously described ELISA-based FVIII binding method was simplified and adapted for the clinical laboratory use by optimizing incubation time, reagent concentrations and assay standardization. The assay was validated using samples from eight individuals with known homozygous or heterozygous VWD2N mutations, and 100 healthy adults. An additional 392 patient samples were tested, including 314 with FVIII activity <50% of normal and 78 received for routine VWF-FVIII binding activity testing. Intra- and inter-assay variations were less than 10% and 17%, respectively, and the limit of quantification was estimated as 0.12. The reference range for healthy adults was 0.73-1.42. VWF:FVIII binding activity was consistent with the genotype in subjects with available genetic data, being low in three individuals with homozygous mutation (<0.12) and intermediate in five heterozygous individuals (0.44-0.61). Screening of the 392 clinical samples identified reduced VWF:FVIII binding in 19 subjects. This assay provides accurate measurement of VWF:FVIII binding activity and successfully identifies homozygous VWD2N patients and heterozygous carriers. Use of this ELISA-based assay may help avoid the need for mutation analysis in patients with unexplained low FVIII activity.

Structure and function of von Willebrand factor

“…The VWF and collagen binding (VWF:CB assay) can significantly reduce the diagnostic error rate as compared to the VWF:RCo assay [136][137][138]. Later, VWF-FVIII binding assay has been used for the investigation of patients with suspected VWDIIN [139]. Interestingly, such an assay provides an accurate measurement of VWF:FVIII binding activity and helps in the identification of homozygous patients with VWDIIN and heterozygous carriers.…”

The diagnosis and management of von Willebrand disease: a United Kingdom Haemophilia Centre Doctors Organization guideline approved by the British Committee for Standards in Haematology

“…These mutations may also complicate a type 1 phenotype causing a disproportionate reduction in FVIII level. The ability of patient VWF to bind added exogenous normal FVIII can be assessed using an immunosorbent plate-binding assay (Zhukov, et al 2009) but these assays are technically difficult and genetic analysis provides a practical alternative.…”

von Willebrand disease

“…This test which determines the ability of VWF to bind FVIII 26–27 is useful in the identification and differential diagnosis of type 2N VWD. 28 There are no standard units for the output of this test.…”