Von Willebrand Disease - VWD | Choose the Right Test

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Medical Experts

Contributor

Moser

Karen A. Moser, MD

Associate Professor of Pathology (Clinical), University of Utah

Medical Director, Hematopathology and Hemostasis/Thrombosis Laboratory, ARUP Laboratories

Contributor

Smock

Kristi J. Smock, MD

Professor of Pathology (Clinical), University of Utah

Vice President, ARUP Institute for Clinical and Experimental Pathology^®

Medical Director, Hemostasis/Thrombosis Laboratory, ARUP Laboratories

Von Willebrand disease (VWD) is the most common inherited bleeding disorder and is classified into three major types. Types 1 and 3 VWD involve quantitative deficiencies in von Willebrand factor (VWF); these deficiencies are less severe in type 1 (the most common type, which includes the 1C subtype) and more severe in type 3. Type 2 VWD involves qualitative abnormalities in VWF and includes subtypes 2A, 2B, 2M, and 2N. Acquired VWD (known as acquired von Willebrand syndrome [aVWS]) can also occur, although it is rare. A broad range of clinical bleeding phenotypes are associated with VWD. No single laboratory test can be used to diagnose VWD. Initial testing involves assays that measure VWF antigen (VWF:Ag), VWF activity, and factor VIII (FVIII). VWF activity is measured by assessing VWF binding to glycoprotein IB (GPIb). Traditionally, platelet-based ristocetin-dependent assays (VWF:RCo) were used. Alternative assays using wild-type recombinant GPIb and ristocetin (VWF:GPIbR) or gain-of-function GPIb recombinant without ristocetin (VWF:GPIbM) can be used and may be preferred over VWF:RCo.^, Additional testing may be indicated for subtyping or genetic counseling.

Quick Answers for Clinicians

Which tests are most useful for the diagnosis of von Willebrand disease?

The initial workup for von Willebrand disease (VWD) involves evaluation of von Willebrand factor (VWF) quantity and function and measurement of factor VIII (FVIII).^, VWF quantity and function are measured via VWF antigen assays and VWF activity assays. Multiple clinical practice guidelines designate levels of either VWF antigen or VWF activity <30 IU/dL (30%) as diagnostic for VWD, regardless of bleeding,^,^, and a level of <50 IU/dL with abnormal bleeding as confirmatory for the diagnosis of VWD. Additional tests can be used to distinguish subtypes, and genetic testing may be used to diagnose and subtype difficult cases, aid in genetic counseling, or assist with treatment decision-making. Refer to the Laboratory Testing section for more information.

What issues can affect testing for von Willebrand disease?

Von Willebrand factor (VWF) values fluctuate over time, mild disease can yield normal lab test results, and VWF levels that are only mildly decreased are not diagnostic for von Willebrand disease (VWD). VWF is an acute phase reactant,^, and VWF levels are affected by stress, recent exercise, illness, estrogen, inflammatory states, and some medications. For those reasons, repeat testing on a new plasma specimen collected at a different time may be necessary for conclusive results.^, In addition, blood type influences test results. Patients with blood type O have 25% lower VWF levels than non-O individuals. However, blood group-specific reference intervals are not currently recommended.^, VWF and factor VIII (FVIII) results can also be affected by preanalytic variables such as collection technique and specimen handling, transportation, and processing.

When is genetic testing recommended for von Willebrand disease?

Genetic testing is not typically included in the initial diagnostic workup for von Willebrand disease (VWD), but it can be valuable for confirming phenotypic findings, classifying subtypes, evaluating at-risk relatives of individuals with known pathogenic variants, and informing family planning decisions. Genetic testing is recommended for patients with suspected type 2B or platelet-type VWD,^, patients with suspected type 2N VWD,^, or for cases in which von Willebrand factor (VWF) antigen assays are unable to differentiate type 3 VWD from severe type 1 VWD. For patients diagnosed with type 1 VWD based on clinical history and laboratory tests, genetic analysis may not be indicated.

Which tests are useful for monitoring patients with von Willebrand disease?

Von Willebrand disease (VWD) assays, specifically von Willebrand factor (VWF) activity and factor VIII (FVIII) activity, are recommended for monitoring patients with VWD who are being treated with medications such as desmopressin or VWF concentrates. These tests are also recommended before surgery or during pregnancy in patients with VWD. Refer to the Monitoring section for more information.

Indications for Testing

Laboratory testing for VWD is appropriate in the following circumstances:

Diagnosis of VWD in individuals with a personal history and physical examination suggestive of a mucocutaneous bleeding disorder (e.g., epistaxis, frequent gum bleeding, menorrhagia, surgery-related bleeding, bleeding associated with dental work, or postpartum hemorrhage)
Diagnosis of individuals with a family history of bleeding symptoms
Monitoring patients with an established diagnosis of VWD who are undergoing treatment or have a planned surgery

For individuals with a low probability of VWD, disease-specific testing should only be pursued after a positive validated bleeding assessment tool (BAT) result.

Laboratory Testing

Diagnosis

Initial Hemostasis Tests

Initial tests in those with suspected bleeding disorders typically include CBC with platelet count, prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, and thrombin time (TT). However, basic clotting times and CBCs have limited use in the diagnosis of VWD because they are normal in most VWD subtypes.

Platelet function and aPTT tests are neither sensitive nor specific for VWD, but individuals with severe VWD will have abnormal results and those with mild/moderate VWD will often have normal results.

If the patient has no abnormalities detected by initial hemostasis tests or has an isolated prolonged aPTT result that corrects with a 1:1 mixing study, an initial VWD assay panel is indicated for further evaluation.

Initial Von Willebrand Disease Assays

No single test is used for diagnosis; it is necessary to use VWF antigen assays, VWF activity assays, and FVIII assays in combination.^,^,

A variety of factors, such as stress, recent exercise, estrogen treatment, pregnancy, inflammatory conditions, illness, and some medications can affect VWF levels, so it may be necessary to repeat the tests on a new plasma specimen collected at a different time for definitive results.

The Expected Laboratory Values in VWD by Type table describes typical results for these tests. If initial results are not diagnostic of VWD but there is high clinical suspicion for VWD, consider repeat testing in 1-3 months.

Von Willebrand Factor Antigen

The VWF antigen concentration, or amount of the protein present in the patient’s plasma, is generally determined using an immunoassay.^,

VWF antigen levels usually range from 50-200 IU/dL (50-200%). Levels <50 IU/dL are considered low and may be diagnostic of VWD in a patient with abnormal bleeding, and levels <30 IU/dL (30%) are considered diagnostic for VWD regardless of bleeding history. However, some patients with type 1 or 2 VWD may have levels in the range of 30-50 IU/dLcie9076; these patients may be asymptomatic or have minimal symptoms.

Von Willebrand Factor Activity Assays

The VWF:RCo assay is used to measure how effectively the VWF in plasma is able to bind platelets when ristocetin is present. Certain variants in the VWF gene (e.g., p.D1472H) can affect the ability of VWF to bind to ristocetin in the test while not interfering with VWF activity in vivo, potentially leading to diagnostic misclassification.^,^, Other types of VWF activity tests do not use ristocetin, such as collagen binding assays or VWF:GPIbM.

The VWF:GPIbM assay, or other newer activity assays, can be used as an alternative to VWF:RCo. Unlike the VWF:RCo assay, the VWF:GPIbM assay does not use ristocetin and thus does not produce decreased results due to genetic variants such as p.D1472H. There are also other newer activity assays that continue to use ristocetin as a reagent (e.g., VWF:GPIbR). Newer assays tend to have improved precision as compared with VWF:RCo since they don’t use intact platelets.

Factor VIII Activity

Laboratory testing of FVIII primarily focuses on measuring its coagulant activity using FVIII:C assays. FVIII:C assays should be included in the initial workup for all cases of suspected VWD. One-stage clotting assays are commonly used.^,

VWF is a carrier protein for FVIII, and individuals with type 1 or 2 VWD may have reduced levels of FVIII, whereas concentrations of FVIII in type 2N and type 3 VWD are markedly reduced.

Von Willebrand Factor Activity/Antigen Ratio

The ratio of VWF activity to VWF antigen is used for subtyping VWD, helping to distinguish between quantitative and qualitative deficiencies. In type 1 VWD, this ratio is typically >0.7, reflecting a proportional decrease in both activity and antigen levels. In contrast, type 2 VWD is usually characterized by a ratio ≤0.7, indicating disproportionately reduced VWF activity relative to antigen levels. An exception is type 2N VWD, which may present with activity/antigen ratios >0.7 but decreased FVIII ratios.

Second-Line Tests

Multimeric Analysis

Analysis of VWF multimers using a qualitative assay is helpful in distinguishing type 1 and type 2 VWD and is therefore useful as a second-line test if type 2 VWD is suspected following antigen and activity testing. However, multimeric analysis can be a useful initial test if aVWS is suspected. The assay results indicate the presence and pattern of the different sizes of multimers. VWF multimers are considered normal if there is a full spectrum of sizes from large to small; the absence of high- and/or intermediate-molecular-weight multimers (large multimers) is an abnormal result. Types 2A, 2B, and platelet type (also called pseudo VWD) usually demonstrate abnormal multimeric distributions, whereas types 1, 2N, and 2M VWD are expected to show a normal pattern of multimers. Type 3 VWD shows a marked decrease in multimers of all sizes or a complete absence of multimers.

Ristocetin-Induced Platelet Aggregation

The low-dose ristocetin-induced platelet aggregation (RIPA) test, or low-dose RIPA, is designed to detect the VWF hyperactivity associated with type 2B VWD and platelet-type VWD at low concentrations of ristocetin. It can be useful for subtype evaluation, particularly to distinguish type 2B and platelet-type VWD from other VWD types. However, genetic testing is often preferred to confirm a diagnosis in patients with suspected type 2B or platelet-type VWD.^,

RIPA testing must be performed by a local laboratory because of limited specimen stability.

Von Willebrand Factor-Platelet Binding

The VWF-platelet binding test is designed to detect the abnormally increased platelet binding seen in type 2B disease and helps to differentiate type 2B from platelet-type VWD. Ordering should be limited to specialists.

Von Willebrand Factor-Factor VIII Binding

The VWF-FVIII binding test measures the degree to which VWF can bind recombinant FVIII and can be used to characterize type 2N VWD (FVIII binding defects) and distinguish it from mild hemophilia A. Ordering should be limited to specialists.

Von Willebrand Factor-Collagen Binding

The VWF-collagen binding assay (VWF:CB) is sometimes used for VWD diagnosis and subtyping and is helpful to distinguish type 1 VWD from type 2 VWD, similar to the way VWF:RCo or other activity tests can be used for type differentiation. The VWF:CB/VWF antigen (VWF:Ag) ratio may also be useful to distinguish VWD types 2A, 2B, and 2M.

Genetic Tests

Genetic testing is recommended for specific situations, such as subtype confirmation when results might affect therapeutic decisions. Genetic testing is most useful for type 2 diagnosis but may also be beneficial in confirming cases of type 1 or type 3 VWD.^,

Genetic/molecular testing can help distinguish subtype 2B from platelet-type VWD, distinguish subtype 2N from mild hemophilia A, and can also be useful in the evaluation of family members of individuals with known variants.

Expected Laboratory Values in VWD by Type
VWD Type/Subtype
Lab Test	1	1C	2A	2B and Platelet Type	2M	2N	3
VWF Ag (IU/dL)	<30^a	<30^a	<30-200 (commonly <50)	<30-200 (commonly <50)	<30-200 (commonly <50)	30-200	Absent
VWF:RCo or other activity assay (IU/dL)	<30^a	<30^a	<30^a	<30^a	<30^a	30-200	Absent
FVIII	Low or normal	Low or normal	Low or normal	Low or normal	Low or normal	Mildly to markedly low	Severely low (<10)
RCo:VWF Ag ratio^b	>0.5-0.7	>0.5-0.7	<0.5-0.7^c	Typically <0.5-0.7^c	<0.5-0.7	>0.5-0.7	n/a
Multimer pattern	Normal pattern but reduced intensity	Normal pattern but reduced intensity	Abnormal; loss of HMW and IMW multimers	Abnormal; loss of HMW multimers	Normal pattern, may have reduced intensity	Normal	No VWF present
RIPA^d	Usually normal	Usually normal	Often reduced aggregation at high ristocetin concentrations but no enhanced aggregation at low ristocetin concentrations	Abnormal; enhanced aggregation at low ristocetin concentrations	Reduced at high ristocetin concentrations	Normal	Absent
VWF:FVIII binding	Normal	Normal	Normal	Normal	Normal	Low	n/a
Desmopressin trial	≤30% decrease from peak VWF 4 hrs after infusion	≤30% decrease from peak VWF 4 hrs after infusion	n/a	n/a	n/a	n/a	n/a
^aThe NHLBI and ASH designate levels of either VWF antigen or VWF:RCo <30 IU/dL as diagnostic for VWD; however, some patients with subtype 1 or 2 will have values of 30-50 IU/dL. ^bThe cutoff ratio used to distinguish between type 1 and type 2 VWD varies across published guidelines with recommended values ranging from 0.5-0.7. ^cPatients with VWF gene variants affecting collagen binding may have decreased VWF:CB/VWF:Ag ratios but normal VWF:RCo/VWF:Ag ratios. ^dRIPA must be performed by a local laboratory due to limited stability of specimen. Ag, antigen; ASH, American Society of Hematology; HMW, high molecular weight; IMW, intermediate molecular weight; ISTH, International Society on Thrombosis and Haemostasis; n/a, not applicable; NHF, National Hemophilia Foundation, NHLBI, National Heart, Lung and Blood Institute; WFH, World Federation of Hemophilia Sources: James, 2021; Nichols, 2008; Favaloro, 2020

Monitoring

VWD assays are recommended for monitoring patients receiving medications such as desmopressin or VWF concentrates for VWD (appropriate treatment depends on subtype). Use of desmopressin often requires a desmopressin trial in a controlled setting to determine efficacy of the therapy. A desmopressin trial involves measuring VWF activity and FVIII activity at baseline and at one or more time points after administration to determine adequate response and clearance characteristics.^, VWF activity and FVIII activity tests are indicated before surgery in patients with VWD to assess whether target activity levels have been achieved with prophylaxis. These tests may also be used for monitoring pregnant individuals with VWD.

Patients who experience heavy menstrual bleeding should also be monitored for iron deficiency and anemia.