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A Practical Guide to Laboratory Haemostasis

 

Von Willebrand Factor: Immunological Assay



Introduction

Immunological assays of von Willebrand factors [VWF:Ag] are immunoassays that measure the concentration of the VWF protein in plasma. They give no indication as to VWF function. A number of methods exist for measuring VWF:Ag and these include both enzyme-linked immunosorbent assay (ELISA) or automated latex immunoassays [LIA.] Many laboratories now use a fully automated latex immunoassay. Historically laboratories used a variety of techniques including Laurell electroimmunoassay 'Laurell Rockets' but these are rarely used in most labs today.

Principles & Method

1. Latex Immunoassays: This assay uses latex micro-particles coated with an antibody to human VWF. in the presence of VWF the latex particles agglutinate and in proportion to the concentration of VWF:Ag in the plasma sample.

2. ELISA Assays: The sandwich ELISA for measuring VWF:Ag assay involves the use of antibody that is immobilised onto a microtitre plate and which is specific for human VWF. Patient plasma, controls and a series of diluted reference samples are added to the wells and the VWF in the samples is bound by the antibody. The plates are rinsed to remove any unbound VWF and then a second antibody is added conjugated to horseradish peroxidase [HRP]. A second wash step is performed and a substrate for the HRP is then added [tetramethylbenzidine - TMB] followed by hydrogen peroxide to develop the colour. The absorbance at 450 nm is measured and from the reference curve the concentration of VWF:Ag in the patient plasma sample can be derived. The use of controls of known VWF concentration ensures that the assay is precise.
Some monoclonal antibodies [binding to the GpIb binding region of VWF] have been shown to be able to discriminate between Type 2A and 2B VWD.

Interpretation

The interpretation of VWF:Ag assays must be made in conjunction with other assays [FVIII and VWF activity], the blood group and bleeding history. Furthermore, VWF is an acute phase protein and will rise e.g. in pregnancy or following surgery.

Reference Ranges

The reference range for VWF:Ag is 50-150 IU/dL.

What Test Next

In patients with suspected VWD the next most useful test is based on trying to establish the subtype as this can affect treatment options. Click HERE to access an Excel spreadsheet which summarises the various subtypes of VWD and can help guide further investigations.

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.