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.
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.
Zhukov
O
O
Quest Diagnostics Nichols Institute, San Juan Capistrano, CA 92675-2042, USA.
Popov
J
J
Ramos
R
R
Vause
C
C
Ruden
S
S
Sferruzza
A
A
Dlott
J
J
Sahud
M
M
D001780:Blood Coagulation Tests / Q000379:methods*; D019411:Clinical Laboratory Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D008297:Male; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D015203:Reproducibility of Results; D014842:von Willebrand Diseases / Q000235:genetics / Q000378:metabolism*; D014841:von Willebrand Factor / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D014841:von Willebrand Factor