Diagnosis of von Willebrand disease type 2N: A simplified method for measurement of factor VIII binding to von Willebrand factor

Abstract

Diagnosis of von Willebrand disease Type 2N (vWD 2N), which mimics hemophilia A and its carrier state, is important for accurate genetic counseling and appropriate therapy. To make testing for the disorder more clinically applicable, we developed a simplified method for measurement of factor VIII (FVIII) binding to von Willebrand factor (vWF) using commercially available reagents and standard clinical assays. FVIII binding to vWF was measured by capture of patient vWF by polyclonal antibodies on cyanogen bromide-activated Sepharose beads, reaction with recombinant FVIII, and assay of unbound FVIII by clotting methods. Unbound vWF was measured in patient plasma after capture by the Laurell method. The ratio of bound FVIII/bound vWF was normal in hemophilia A, vWD Type 1, and vWD Type 3 patients, and abnormal in 5 subjects from two families, all of whom had vWD 2N mutations. Patient 1, with FVIII 8 U/dl, vWF: Ag 61 U/dl, vWF:RC 74 U/dl, and FVIII binding nil, was homozygous for the Arg91 Gln mutation. She was followed during pregnancy and delivered an unaffected heterozygous son. Patient 2 had FVIII 8 U/dl, vWF:Ag 73 U/dl, and vWF:RC 71 U/dl, and very low FVIII binding. She was heterozygous for Arg91Gln, as were her mother and sister; no second vWD 2N mutation was found. Her brother, with FVIII 14 U/dl, vWF:Ag 113 U/dl, and vWF:RC 72 U/dl, has no evidence of vWD 2N. With an X-linked inheritance pattern of bleeding tendency, this family is the first reported with combined hemophilia A and vWD 2N.

Inheritance of von Willebrand's disease in a colony of Doberman Pinschers

“…Type 1 vWD, the most common of the 3 types, is characterized by low vWF concentrations, with a proportionate reduction of all vWF multimers. 7 Type 2 vWD is characterized by a selective decrease in the large vWF multimers, which are the most biologically active for hemostasis 7 ; there may or may not be qualitative abnormalities in these large multimers. 7 Type 3 vWD, the least prevalent type of vWD, is characterized by undetectable concentrations of vWF.…”

“…7 Type 2 vWD is characterized by a selective decrease in the large vWF multimers, which are the most biologically active for hemostasis 7 ; there may or may not be qualitative abnormalities in these large multimers. 7 Type 3 vWD, the least prevalent type of vWD, is characterized by undetectable concentrations of vWF. 7 The severity of vWD in humans and dogs varies with each affected individual.…”

Life-threatening bleeding in a patient with mild hemophilia A and heterozygosity for von Willebrand disease Type 2N

“…Two families have been reported previously with co-segregating F8 and VWF (p.R854Q) mutations. In these reports, the probands were both females and heterozygotes with mild bleeding phenotypes [5,6]. Recently, a pediatric male patient was reported with mild hemophilia and Type 2N VWD [7].…”

Use of a new enzyme-linked immunosorbent assay for the detection of type 2N von Willebrand disease and its prevalence in an Indian population

“…In contrast to the previous time-consuming and technically difficult chromogenic methods and other ELISA assays [4][5][6][7][8][9], this assay involves fewer steps and hence is simple, less time consuming, is reproducible and can be used in a routine hospital coagulation laboratory having access to an ELISA reader. All nine patients (VWF:Ag/FVIII ratio > 1.4) were confirmed as type 2N VWD, utilizing both the 'in-house' ELISA technique and the reported technique for the FVIII:VWF binding activity.…”

“…Various assays for measuring the binding of VWF to FVIII have been reported [6][7][8][9][10][11]; most of these assays are complex, involving multiple steps and expensive reagents. As a result, most of these assays have not been available to all the laboratories in developing countries.…”