Diagnosis of VWD and its subtypes

The diagnosis of VWD is based on three main criteria:

  1. The patient should have significant bleeding symptoms,

  2. There should be a family history of appropriately diagnosed VWD or significant bleeding symptoms, except in recessively inherited subtypes, and

  3. VWF levels should be significantly and repeatedly decreased.

As VWD is a heterogeneous disease, its classification by best standards requires a quite extensive laboratory armamentarium (see Table 1).

A clinician with special knowledge of coagulation disorders should diagnose VWD. General practitioners should refer the patient to a hematologist or a specialist working at the unit of coagulation disorders. The diagnosis should be done carefully in children due to the developing coagulation system and reassessed regularly, e.g. every 10 years, during lifetime, as ageing populations with bleeding disorders may be subject to overdoses of replacement therapy and, possibly, to the risk of thrombosis.

Initial hemostasis screening tests

The activated partial thromboplastin time (APTT) and the Owren and/or Quick prothrombin time (PT) test are often normal, but the APTT may be prolonged in cases where FVIII level are below 30-40 IU/dL. The complete platelet count is usually normal with exception of a mild to moderate thrombocytopenia found in patients with VWD type 2B or the platelet-type VWD. The Platelet Function Analyzer (PFA)-100, and the upgraded version PFA-200, may be utilized as a primary hemostasis-screening test device to identify moderately or severely low VWF and some platelet function disorders. However, it has low sensitivity for mild defects in primary hemostasis and lacks specificity since it does not differentiate VWD from other platelet defects. Its strengths include whole blood environment and interaction with collagen under high-shear blood flow conditions. Irrespective if PFA is used or not, it is important that samples from patients with suspected VWD is sent to a specialized coagulation laboratory for further evaluation.

Specific tests for VWD diagnosis

As shown by the algorithm in Figure 1, the diagnosis of VWD is dependent on a significantly reduced platelet-dependent VWF activity (VWF:GIbR or VWF:GPIbM), with the exception of type 2N.

The Nordic Hemophilia Council recommends that the diagnosis of VWD usually is made by repeated VWF activity levels below 35 IU/dL to confirm VWD diagnosis. However, the exact level can be flexible, and some individuals will be diagnosed having VWD with somewhat higher VWF activity levels, dependent on symptoms and family history after exclusion of platelet defects.

In patients with a bleeding phenotype and mildly reduced VWF activity levels of 35 – 50 IU/dL, “low VWF” is preferred (after exclusion of platelet defects) rather than “VWD”.

Measuring FVIII:C level is important as a prerequisite to approach the subtype 2N diagnosis. Additionally, FVIII plays an important role in assessment of the bleeding and likely thrombosis risks in VWD. However, in all subclasses of VWD the level of FVIII may be decreased, and it is particularly low (< 5 IU/dL) in type 3.

The VWF antigen and activity tests, as well as tests for VWD subtyping, are described in Table 1 and discussed in more detail in the section below.

Table 1: Recommended laboratory assays used for diagnosis and subclassification in VWD
Method Diagnostic and monitoring purposes
VWF antigen, VWF:Ag Quantitation of VWF antigen (protein). Used to differentiate between VWD type 1 and 3 or between type 1 and 2.

Platelet-dependent VWF activity

a) VWF:GPIbR

b) VWF:GPIbM

The platelet-dependent VWF activity is the main functional method. The different assays have in common the capacity to capture VWF binding to the platelet receptor GPIb.

The original assay, denoted VWF:RCo, utilizes reconstituted lyophilized platelets and ristocetin is not longer recommended to use. The VWF:RCo assay should be replaced by the VWF:GPIbR and VWF:GPIbM assays that utilizes recombinant GPIb fragments, with or without ristocetin, respectively.
VWF collagen binding, VWF:CB Measures the capacity of VWF to bind to collagen. The assay is sensitive to detect multimer impairment. The discriminatory power is dependent on type of collagen used. Reduced VWF activity but normal multimeric pattern is indicative of VWD type 2M.
VWF multimer analysis, VWF:Multimers

Electrophoretic procedure essential for detection of multimeric size distribution (band pattern). The faster moving bands are denoted low-molecular weight multimers (LMWM) and the slower migrating bands are indicated as high-molecular weight multimers (HMWM). The protein bands in between are often called intermediate-molecular weight multimers (IMWM).

The test can be performed with different gel concentrations that allows different resolutions of the bands. With high gel concentration it is possible to examine the oligomeric structure of the VWF bands (known as triplet) for further subclassification of VWD type 2 A variants (i.e., IIA, IIB, IIC, IID etc.).
FVIII binding capacity, VWF:FVIIIB Determines the capacity of VWF to bind FVIII. Specific test for VWD type 2N.
Ristocetin induced platelet aggregation, VWF:RIPA Determination of the ristocetin sensitivity using the patient’s platetet-rich plasma. Increased sensitivity (0.5 mg/mL or lower) is indicative of VWD type 2B. VWF:RIPA is absent in VWD type 3 and generally decreased in VWD type 2A.
VWF propeptide, VWFpp The propeptide is released from VWF after synthesis. As it is synthesized in the same molar ratio of the mature subunit, but circulates in plasma independently from VWF, with different half-lives it is possible to estimate a VWFpp/VWF:Ag ratio. A reduced VWF:Ag and an increased VWFpp/VWF:Ag ratio are indicative of a VWD type 1 with increased clearance (e.g. VWD type 1C).
FVIII coagulation activity, FVIII:C Determination of FVIII coagulation activity. A disproportionally reduced FVIII (compared to VWF) is typical for VWD type 2N.