von Willebrand Comprehensive Panel

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

von Willebrand disease (VWD) is a bleeding disorder in which there is a quantitative or qualitative abnormality of VWF. It may be inherited or acquired. The acquired form is usually secondary to valvular stenosis, hypothroidism, or monoclonal gammapathies 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, type 2 by a qualitative defect in VWF function, and type 3 by an almost complete deficiency of VWF.

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. Last, with type 2N VWD, there is a decreased binding affinity for FVIII.

There are multiple factors that affect the plasma concentration of VWF. A major contributor is blood group. On average, levels are lower in blood group O individuals in comparison 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.¹

Factors associated with higher vWF levels include race (15% higher in African Americans), chronic inflammation, acute infection/trauma, pregnancy, oral estrogen replacement, birth control pill use, age (higher in neonates), diabetes, malignancy, stress, surgery, and exercise. A factor associated with reduced VWF level is hypothyroidism.

The major determinant of bleeding symptoms or risk is low VWF. As recommended by the National Heart, Lung and Blood Institute VWD expert panel, population-based reference ranges, rather than blood group-specific ranges, may be more useful clinically.²

The VWF:RCo assay is the most commonly 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 is used to assist in the discrimination of the quantitative (types 1 and 3) from the qualitative (type 2 VWD) defects. With type 1 and type 3 VWD, the VWF antigen and VWF:RCo will be proportionally decreased. With type 2 VWD, the VWF antigen may be normal to reduced, whereas the VWF:RCo will be greatly reduced in comparison 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 is useful to 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; this necessitates near-patient testing. If local testing is not an option, an alternative is VWD mutation analysis (test code 19837[X]), which is useful for subtyping the qualitative defects.

The VWF:CB assay is a functional assay that detects VWF adhesive activity to collagen. In vivo, collagen within the subendothelial matrix is exposed subsequent to 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 assists in the identification and discrimination of 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 VWD.^3,4

VWF and factor VIII belong to a class of proteins whose levels increase in response to stress and inflammation (positive acute phase proteins). Since an acute phase response may mask the presence of VWD, it may be advisable to repeat testing at a time more representative of baseline, if clinical suspicion is high.

VWF and factor VIII are unique proteins in that both are susceptible to cold-activation. Storage of the whole-blood citrate tubes at cold temperatures (2-4°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.⁵ Therefore, it is critical that whole-blood samples be stored at room temperature prior to centrifugation. The following illustration outlines the recommended procedure for preparation of platelet poor plasma.

In the absence of a factor VIII inhibitor, the differential diagnosis for low factor VIII in a female patient includes hemophilia A carrier, 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 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 (test code 70068) is used to discriminate type 2N VWD from hemophilia A: patients with type 2N VWD will exhibit defective binding in the assay, whereas patients with hemophilia A will exhibit normal binding.