Von Willebrand Screen

The von Willebrand screen measures von Willebrand factor (VWF) antigen, VWF activity and factor VIII activity, with further assays to define the type. VWF is a large multimeric glycoprotein made by endothelial cells and megakaryocytes. It anchors platelets to damaged vessel wall at high shear, and carries and protects factor VIII in the circulation. Deficiency or dysfunction causes mucocutaneous bleeding and, in severe forms, joint and muscle bleeding.

Von Willebrand disease is the most common inherited bleeding disorder, with symptomatic disease in about 1 in 1000 people, and laboratory abnormalities in up to 1%. About 70 to 80% of cases are type 1, and 20 to 30% are type 2. Type 3 is rare. Acquired von Willebrand syndrome arises with other conditions and is far less common.

Indications.

• Menorrhagia, easy bruising or epistaxis.
• Bleeding after dental extraction, surgery or childbirth.
• A family history of von Willebrand disease.
• A prolonged aPTT with low factor VIII.
• Unexplained mucocutaneous bleeding with a normal platelet count.
• Aortic stenosis or a left ventricular assist device with bleeding.
• Before invasive procedures when the bleeding history is positive.

Specimen. Citrated platelet poor plasma, centrifuged twice and tested promptly or frozen at minus 70 °C. Avoid testing during acute illness, pregnancy, exercise, stress or oestrogen use, because VWF and factor VIII rise, which can hide disease. A repeat test is often needed. Collect at least 2 occasions before excluding disease.

Adult reference ranges are given. Local ranges take precedence.

VWF antigen (VWF:Ag). The concentration of VWF protein, measured by immunoassay. Range: about 50 to 150 IU/dL, with lower levels in blood group O (mean about 75 IU/dL, range about 36 to 157) than in non-O groups (mean about 100 IU/dL, range about 55 to 200). Raised: acute phase response, pregnancy, oestrogen, exercise, stress, liver disease, diabetes, malignancy, older age, African ancestry. Lowered: von Willebrand disease types 1, 2 and 3, blood group O, hypothyroidism, acquired von Willebrand syndrome.

A level under 30 IU/dL is diagnostic of von Willebrand disease even without a bleeding history, and 30 to 50 IU/dL is low VWF, which causes bleeding in some patients and is treated by symptoms.

VWF activity. The ability of VWF to bind platelet glycoprotein Ib. It is measured by the ristocetin cofactor assay (VWF:RCo, an agglutination assay with ristocetin and fixed platelets), or by ristocetin independent gain-of-function glycoprotein Ib binding assays (VWF:GPIbM, VWF:GPIbR) and the immunoturbidimetric VWF:Ab assay. Range: about 50 to 150 IU/dL. Raised: acute phase response, pregnancy, oestrogen, exercise, stress, liver disease. Lowered: von Willebrand disease, particularly types 2A, 2B and 2M, and acquired disease.

The ristocetin cofactor assay is insensitive at low levels and has a high coefficient of variation (over 20%). Modern activity assays are more reproducible. A common variant (D1472H) can lower VWF:RCo without disease.

VWF activity to antigen ratio. Calculated from the two assays. A ratio of 0.6 to 0.7 or more is normal. A ratio under 0.6 suggests a type 2 variant with loss of high-molecular-weight multimers (2A, 2B) or dysfunction (2M). In type 1 and type 3, the ratio is normal because both fall together.

Factor VIII activity. Range: about 50 to 150 IU/dL. Raised: acute phase response, pregnancy, liver disease. Lowered: types 1 and 3 (secondary to low VWF, with levels in type 3 of about 1 to 9 IU/dL), type 2N, haemophilia A, and acquired haemophilia.

In type 2N, factor VIII is disproportionately low compared with VWF:Ag, which resembles mild haemophilia A in male patients, and a normal VWF:Ag and activity with low factor VIII needs the VWF:FVIII binding assay or genotyping.

Collagen binding (VWF:CB). Measures binding to collagen, which depends on large multimers. A ratio of VWF:CB to VWF:Ag under 0.6 suggests loss of high-molecular-weight multimers.

Multimer analysis. Gel electrophoresis of VWF multimers. Type 1 shows a full complement at reduced intensity. Type 2A and 2B show loss of the large multimers. Type 2M shows a normal distribution. Type 3 shows no multimers.

Ristocetin induced platelet aggregation (RIPA). Patient platelet rich plasma is tested with low dose ristocetin (0.5 to 0.7 mg/mL). Aggregation at a low dose indicates type 2B or platelet type von Willebrand disease, and a reduced response at high dose suggests types 1, 2A, 2M and 3.

VWF propeptide (VWFpp) to VWF:Ag ratio. A raised ratio indicates increased clearance of VWF, for example in type 1C (Vicenza).

Type 2B. Thrombocytopenia and large platelets are common, sometimes made worse by pregnancy, surgery or desmopressin. Platelet type (pseudo) von Willebrand disease is a platelet defect that gives an identical picture, and is separated by platelet glycoprotein Ib genotyping or mixing studies.

Acquired von Willebrand syndrome. Causes include aortic stenosis (Heyde syndrome), left ventricular assist devices, extracorporeal oxygenation, myeloproliferative neoplasms with very high platelet counts, monoclonal gammopathy, lymphoproliferative disorders, hypothyroidism, autoimmune disease and some drugs (valproate, ciprofloxacin, hydroxyethyl starch). The multimers are lost, with normal VWF:Ag in many.

Desmopressin challenge test. Used to predict response in types 1 and some type 2 disease. Desmopressin is given intravenously at 0.3 microg/kg in 50 to 100 mL of saline over 20 to 30 minutes, usually to a maximum of 20 microg. Factor VIII, VWF:Ag and VWF activity are measured before, and at 1 and 4 hours after the infusion.

A good response is a rise to at least 50 IU/dL and at least a 3-fold increase from baseline at 1 hour, sustained at 4 hours. Type 3 does not respond. Type 2B is a relative contraindication because of thrombocytopenia. Contraindications and cautions include hyponatraemia, children under 2 years, cardiovascular disease, uncontrolled hypertension, seizure disorder, psychogenic polydipsia and pregnancy at risk of fluid overload. Fluids are restricted for 24 hours, and sodium is checked.

Critical values and findings needing immediate action.

• Factor VIII and VWF under 10 IU/dL with active bleeding, or type 3 disease needing surgery or childbirth.
• Type 2B with thrombocytopenia before surgery or delivery, because desmopressin can worsen it.
• Severe bleeding in a patient with aortic stenosis and loss of high-molecular-weight multimers.
• Platelet count under 50 x10⁹/L with large platelets, which needs exclusion of type 2B and platelet type disease.

Interferences and pitfalls.

• A normal level in a single sample does not exclude disease, since VWF varies with acute phase, stress, exercise, pregnancy, oestrogen, ovulation and the menstrual cycle (lowest in the first days of the cycle). Repeat the test.
• Blood group O lowers the mean level by about 25%, so a level of 40 to 50 IU/dL in a person of group O may be physiological, and it still counts if bleeding is present.
• Low levels in a person of African ancestry are less common, since levels are higher on average.
• Delay in processing, cold storage and haemolysis lower VWF activity.
• Platelet contamination of plasma lowers VWF and alters RIPA results.
• A common D1472H variant, particularly in people of African ancestry, can lower VWF:RCo and cause a falsely low activity to antigen ratio.
• The coefficient of variation of the ristocetin cofactor assay limits the diagnosis of type 2 disease at low levels.

Special groups.

• Pregnancy: VWF and factor VIII rise 2 to 3 times by the third trimester, which can normalise type 1 disease. Levels fall rapidly in the first days and weeks after delivery, with a risk of delayed postpartum haemorrhage. Test early and again at 34 to 36 weeks, and plan delivery from the result.
• Neonates and infants: VWF is high at birth, so a normal level does not exclude disease, and testing is repeated after 6 months. A very low level can still be diagnosed.
• Children: venepuncture stress can raise levels.
• Older adults: levels rise with age and may normalise type 1 disease. A new bleeding disorder suggests an acquired cause.
• Menorrhagia: iron deficiency is common, and a bleeding score assessment helps interpretation.

Follow up tests.

• Platelet count and mean platelet volume, blood film and clotting screen.
• VWF multimer analysis, RIPA, VWF collagen binding, VWF:FVIII binding assay and VWF propeptide.
• VWF gene sequencing.
• Platelet function tests and platelet glycoprotein Ib genotyping.
• Ferritin, thyroid function tests and blood group.
• Echocardiography when acquired disease from valve disease is suspected.
• Serum protein electrophoresis.