von Willebrand disease profile (VWF:RCo, VWF:Ag, VWF:CBA, FVIII)

Description: 
von Willebrand disease (VWD) is the most common hereditary bleeding disorder and the deficiency can be quantitative, involving reduced levels of normally functioning von Willebrand factor (VWF), or qualitative, involving dysfunctional molecules. Laboratory investigation of VWD encompasses a battery of assays that assess different aspects of the molecule which inform sub-classification and clinical management. First line VWD screening employs a coagulation screen, VWF:RCo assay, VWF:Ag assay, VWF:CB assay and a FVIII assay.

The VWF ristocetin co-factor (VWF:RCo) assay is performed with a new generation automated assay employing latex beads coated with a recombinant fragment of GpIbα through a highly specific monoclonal antibody. VWF in the sample binds to the GpIbα fragment via the antibiotic ristocetin and the degree of agglutination is directly proportional to VWF (GPIb-binding) activity, determined by decreased light transmittance due to the agglutination.

Concentration of VWF irrespective of function is ascertained with a latex immunoassay which employs latex particles coated with antibodies to VWF to capture VWF in the test plasma. The VWF acts as a bridge between latex particles that leads to a degree of agglutination directly proportional to VWF concentration, which is measured turbidimetrically.

The VWF collagen binding assays (VWF:CB) assay is performed with an indirect ELISA. VWF in patient plasma is captured in microtitre plates coated with type III human collagen. Unbound material is then washed away and a solution of antibody to human VWF that is conjugated to an enzyme is added to ‘tag’ onto any captured VWF. Unbound conjugate is washed off and a substrate for the enzyme is added, the product of the enzyme-substrate reaction being coloured. Colour intensity is in direct proportion to the degree of conjugate-binding, itself proportional to the amount of VWF capture and thus, VWF collagen binding activity.

The two-stage FVIII activity assay involves first generating FXa in a reaction where the patient's FVIII is the rate limiting factor. The second stage involves reacting the FXa generated in the first stage with a chromogenic substrate generating a coloured product, the intensity of which is proportional the the FXa present, which itself is directly proportional to the FVIII actvity.
Clinical details: 
von Willebrand factor (VWF) is a large adhesive glycoprotein synthesised in endothelial cells and megakaryocytes. Unlike the activated coagulation factors of secondary haemostasis it is not an enzyme and its functions involve binding to cells and molecules. Upon vessel injury, VWF binds directly to exposed sub-endothelial collagen and remains anchored. Blood flow unravels anchored VWF to expose the binding site for the constitutively expressed platelet surface receptor glycoprotein Ib. VWF captures and tethers platelets arriving at the scene which promotes subsequent events of primary haemostasis towards formation of a platelet plug. VWF also serves as the plasma carrier of FVIII to protect it from proteolytic degradation and also to ‘deliver’ it to sites of injury and clot formation.

von Willebrand disease (VWD) is the most common hereditary bleeding disorder and the deficiency can be quantitative, involving reduced levels of normally functioning VWF, or qualitative, involving dysfunctional molecules. Laboratory investigation of VWD encompasses a battery of assays that assess different aspects of the molecule which inform sub-classification and clinical management:

VWF:RCo assay measures glycoprotein Ib binding
VWF:Ag assay measures total protein concentration irrespective of function
VWF:CB assay measures collagen binding
VWF:FVIIIB assay measures FVIII binding
Multimer analysis investigates VWF structure
FVIII activity is measured as levels can be reduced due to reduction of its carrier
Reference range: 

FVIII 50 - 150

Blood group O
VWF:RCo 41 - 107
VWF:Ag 50 - 124
VWF:CB 42 - 124

Non-O blood group
VWF:RCo 58 - 127
VWF:Ag 67 - 172
VWF:CB 59 - 147

Units: 
VWF:RCo IU/dl VWF:Ag IU/dl VWF:CB IU/dl FVIII IU/dl
Sample type and Volume required: 
External requests: Citrated platelet poor plasma
400µL x 4 aliquots
Internal requests: please refer to EPR label


Turnaround time: 
7 - 10 days
Special sample instructions: 

The samples should be analysed or manipulated & stored in the laboratory within 4 hours of venepuncture. Please ensure sample tubes are filled exactly to the fill-line as underfilling creates a dilution error and leads to inaccurate results.

Contacts:
Diagnostic Haemostasis and Thrombosis Department
020 7188 2797
St Thomas' Hospital
North Wing - 4th and 5th Floors
Westminster Bridge Road
London SE1 7EH

Laboratory opening times
24/7
For clinical advice or interpretation of results, please contact the laboratory in the first instance.

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Last updated: 09/03/2017