Comprehensive von Willebrand Disease Evaluation: von Willebrand Disease Panel With and Without Collagen Binding Assay

von Willebrand factor (VWF) has 3 important hemostatic functions: (1) mediating platelet-subendothelium adhesion and platelet aggregation at sites of vascular injury, (2) binding to collagen at sites of vascular injury, and (3) binding and stabilitizing circulating blood clotting factor VIII (FVIII).¹

von Willebrand disease (VWD) is a bleeding disorder in which there is a quantitative or qualitative abnormality of von Willbrand factor (VWF). It may be inherited or acquired. The acquired form is usually secondary to valvular stenosis, hypothyroidism, or monoclonal gammopathies of unknown origin or associated with use of a left-ventricular assist device.

The disease is classified into 3 types. Type 1 is characterized by a partial quantitative deficiency of VWF with type 2 is characterized by a qualitative defect in VWF function, and type 3 is characterized by an almost complete deficiency of VWF. In addition, there is a so-called “platelet-type” VWD, where there is an increased ability of the patient’s platelet glycoprotein 1b alpha protein to bind to VWF, mimicking a type 2 qualitative defect (specifically type 2b VWD).  

Lastly, type 1C is characterized by accelerated clearance of VWF, which can be seen in a 4-hour post-infusion challenge with desmopressin. Patients with type 1C VWD generally cannot sustain VWF levels greater than 50% after 4 hours, while those with type 1 VWD increase VWF levels 2- to 3-fold over baseline and maintain levels well above 50%.^2,3

The qualitative defects are further divided into 4 categories: types 2A, 2B, 2M, and 2N. Type 2A is characterized by decreased platelet adhesion resulting from a deficiency of high– and intermediate–molecular–weight multimers. Type 2B includes variants with increased affinity for platelet glycoprotein I and deficiency of high–molecular–weight multimers. Type 2M is characterized by decreased VWF-dependent platelet adhesion without a deficiency of high–molecular–weight multimers. Lastly, type 2N is characterized by a decreased binding affinity for FVIII. Platelet type VWD is similar to type 2B with a gain-of-function mutation in the platelet glycoprotein 1b alpha gene. 

Multiple factors affect the plasma concentration of VWF. A major contributor is blood group. On average, levels are lower in blood group O individuals compared to non–blood group O individuals. Why the difference? The blood group A and B alleles encode glycosyltransferases that add carbohydrate moieties to the precursor side chains, converting them to the A or B antigens. The O alleles do not encode this transferase and thus express the unmodified precursor. VWF undergoes glycosylation, and the ABO carbohydrate structures have been identified within the A1 domain of the protein. Although the exact mechanism is unknown, one hypothesis is that the reduced level of glycosylated VWF in individuals with blood group O is associated with decreased survival of the protein.⁴

Factors associated with higher VWF levels include race (15% higher in African Americans)*, chronic inflammation, acute infection/trauma, pregnancy, oral estrogen replacement, use of oral contraceptives, age (higher in neonates), diabetes, malignancy, stress, surgery, and exercise. A factor associated with reduced VWF level is hypothyroidism. Furthermore, female patients experiencing heavy menstrual bleeding will also have higher VWF levels which can mask the diagnosis of VWD.⁵ A recent ASH publication on when and how to test for VWD patients with heavy menstrual bleeding has been published.⁶ The key recommendation is that testing be repeated at baseline during a period of wellness when heavy menstrual bleeding is not occurring if VWF values are between 50% and 100%.⁶

In addition, VWF levels may be decreased with the use of ristocetin-based VWF activity assays in patients with the nonpathological VWF polymorphism DH1472H. Studies have shown that patients with this polymorphism can have decreased ristocetin-based VWF activity by approximately 10% to 30%, with the decrease dependent on whether the patient is heterozygous or homozygous for this polymorphism. This polymorphism is seen in 60% to 70% of patients of African origin and 15% to 20% in the Caucasian population.⁷ This can lead to misclassification as to whether a patient has VWD and/or the type of VWD.⁶ Currently, the International Society for Thrombosis and Haemostasis, suggests that VWF assays use non–ristocetin-based activity assays for the evaluation of VWD such as von Willebrand Factor GP1bM activity (VWF:GPIbM, test code 13995).²

*There is insufficient research evidence to support disease associations by race; these differences are more likely due to social determinants.

The major determinant of bleeding symptoms or risk is low VWF. While it is recognized that blood group O individuals on average have lower VWF levels, this is not included in the 2021 diagnostic guidelines from the American Society of Hematology (ASH), the International Society on Thrombosis and Haemostasis (ISTH), the National Hemophilia Foundation (NHF), and the World Federation of Hemophilia (WFH).²

The VWF:RCo assay is the most performed automated test to assess VWF function. It measures the ability of VWF in the patient plasma to induce platelet agglutination in the presence of ristocetin. In combination with the VWF antigen assay, it helps discriminate the quantitative (types 1 and 3 VWD) from the qualitative (type 2 VWD) defects. With type 1 and type 3 VWD, both the VWF antigen and VWF:RCo will be decreased. With type 2 VWD, the VWF antigen may be normal to decreased, whereas the VWF:RCo will be greatly decreased compared to the antigen level.

In contrast, the RIPA assay measures the aggregation of the patient's platelet-rich plasma to various concentrations of ristocetin, thus providing an assessment of platelet−VWF interaction. It helps discriminate type 2B VWD from other functional defects. Type 2B is characterized by increased responsiveness, whereas types 2A and 2M yield reduced responsiveness (in type 1 VWD, the level is dependent on the plasma concentration of VWF). However, a major limitation of the RIPA assay is the requirement for fresh blood, which necessitates near-patient testing. Guidelines recommend obtaining VWD gene sequencing (test code 36494), which may help confirm type 2 VWD, such as types 2B and 2N, and severe type 1 and type 3 VWD.^2,8

The VWF:CB assay is a functional assay that detects VWF adhesive activity to collagen. In vivo, collagen within the subendothelial matrix is exposed after endothelial cell damage. VWF binding to exposed collagen is critical for platelet adhesion, aggregation, and eventual clot formation. Used in combination with the VWF:RCo and VWF:Ag assays, the CBA assay helps identify and discriminate most types of VWD (types 1, 2A, 2B, 2M, and 3).

These 3 tests make up a much more powerful test panel than the 2-test combination of VWF:Ag and VWF:RCo and can better distinguish type 2M from type 2A or 2B VWD.^9,10 When used in combination with VWF multimeric analysis, the collagen binding activity (when the VWF:RCo, VWF:Ag assays are normal/near normal and VWF multimer distribution is normal) may help identify rare qualitative VWF collagen binding defects as well as confirm type 2M VWD.¹¹ A VWF panel is available that includes collagen binding activity and encompasses all the important VWF functions is the von Willebrand Disease Panel with Collagen Binding Assay (test code 15540). 

An elevated VWF level suggests that an inflammatory process or an acute phase response is present.  It may also be elevated in response to stress. If clinical suspicion is high, repeat testing at a time more representative of baseline may be advisable.

VWF and factor VIII are both susceptible to cold activation. Storage of the whole-blood citrate tubes at cold temperatures (2 °C to 4 °C) before centrifugation may lead to subsequent activation and loss of VWF and, consequently, factor VIII activity. Depending on the tests ordered, the potential for misdiagnosis as hemophilia or VWD exists. The exact mechanism is unknown, though it is presumed that exposure of whole blood to cold temperatures activates VWF proteases and/or other enzymes, which in turn degrade VWF.¹² Centrifugation in a cold centrifuge may also lead to cryoprecipitation.¹³ Therefore, storage of whole-blood samples at room temperature prior to centrifugation is critical. The following illustration outlines the recommended procedure for preparation of platelet-poor plasma. 

Decreased factor VIII inhibitor in a female patient may be caused by being a hemophilia A carrier, having type 2N VWD, or a pre-analytic etiology (ie, improper sample collection). Type 2N VWD is characterized by defective VWF–factor VIII binding. As a result, factor VIII is not protected from proteolytic degradation in circulation and levels decrease. Patients with type 2N VWD may have normal VWF studies (antigen, ristocetin cofactor [or GPIbM activity], and multimers) yet have reduced levels of factor VIII activity. The same pattern of results is observed in hemophilia A carriers.

The VWF:factor VIII binding activity assay may discriminate type 2N VWD from hemophilia A: patients with type 2N VWD exhibit defective binding in the assay, whereas patients with hemophilia A exhibit normal binding. Genetic testing using von Willebrand Disease Gene Sequencing (test code 36494) may be an alternative test if the VWF:factor VIII binding activity assay is unavailable or equivocal.²