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A Practical Guide to Laboratory Haemostasis

 

Von Willebrand Factor Ristocetin Cofactor [VWF:RCo] Assay



Introduction

The Von Willebrand Ristocetin Cofactor [vWF:RCo] assay measures the ability of a patient’s plasma to agglutinate platelets in the presence of the antibiotic Ristocetin.  The rate of Ristocetin induced agglutination is related to the concentration and functional activity of the plasma von Willebrand factor.  Ristocetin is thought to bind to VWF at Glu1239-Pro-Gly Gly1242.
Ristocetin induced platelet agglutination (RIPA) is similar to the Ristocetin Cofactor Assay but RIPA measures platelet agglutination induced by Ristocetin-mediated VWF binding to the platelet Gp1b receptor but in this case the ristocetin is added directly to the patient’s platelet rich plasma and there are no serial dilutions of the plasma sample.
A number of methods exist for measuring VWF:RCo. Originally, fresh washed platelets were used in the VWF:RCO assay but now frozen or formaldehyde-fixed platelets are commonly used.  However there are other approaches and these are listed below.

Method

Platelet Agglutination method: The method is similar to a factor assay.

Step  
1 Fresh or fixed platelets are mixed with dilutions of patient plasma in a standard platelet
2 Patient samples are normally performed in duplicate at a single dilution
3 Agglutination is allowed to proceed and from the traces the slope of the curve is derived. 
A standard curve is established by using a similar method but replacing patient plasma with serial dilutions of normal plasma. 
4 The slope of the curve is plotted against dilution on double-log paper and a straight line obtained.  From this the Ristocetin cofactor activity in the patient’s plasma samples can be derived.

The following traces demonstrate this more clearly:



If you click HERE you can see a larger version of this image/data.

Remember - the slope is calculated by plotting the tangent of the slope and them measuring the distance from the origin to where the tangent crosses the X or Y axis. The distance on the Y axis is then divided by the distance on the X-axis and the slope derived. For example if for the normal plasma sample, the distance from the origin to where the tangent crosses intercepts the X-axis is 12mm and the distance on the Y axis is 44mm then the slope is 44/12 = 4.
5 If we calculate the slopes (S) of each of these traces (Y/X) derived from serial dilutions of normal plasma and plot these against dilution on double-log paper - then we end up with a straight line from which the VWF:RCo activity of an unknown plasma sample can be calculated - see below:


6 In Plasma Sample - 1, there is no aggregation [more correctly no agglutination] and so the VWF:RCo activity is <1%.
In Plasma Sample - 2, the slope is 0.65 and so from our standard curve we can calculate that this equates to a VWF:RCo activity of ~8.5%. We assume that our plasma standard has a concentration before dilution of 100%.

We can check that this is correct because the slope of Plasma Sample 2 lies between that of the 1/8 and 1/16 dilution of our normal plasma sample.

Finally - we are using neat patient plasma in Plasma Samples 1 and 2 and so there is no need to make any corrections for dilution.

It is possible to replace the aggregometer with a microtitre plate or an ELISA plate reader but the principles remain the same.

2. ELISA assays that assess direct binding of plasma VWF to platelet GpIb: In these assays the GpIb can be of one of two sources:
a. A recombinant GpIb fragment. Briefly, an ELISA plate is coated with an anti-GpIb monoclonal antibody after which the recombinant GpIb fragment is added. VWF is then added [either the reference plasma or the test sample] to the GpIb-coated wells in the presence of a fixed concentration of ristocetin. VWF in the plasma sample binds to the immobilised GpIb fragment and the bound vWF is detected with an anti-human VWF horseradish peroxidase [HRP] labelled antibody. The bound antibody is quantified in a colorimetric reaction by adding the HRP substrate OPD [1,2 ortho-phenylendiamine dihydrochloride], and measuring the absorbance at 492 nm. The absorbance is proportional to the concentration of VWF. A calibration curve is constructed by using serial dilutions of a reference plasma against which the absorbance of the test plasma is measured and from which the VWF:RCo activity can be derived.
b. A proteolytic fragment termed 'glycocalicin' consisting of the extracellular part of the α-chain of the platelet GpIb receptor, which can be removed from the platelet surface by treatment with various proteases. interestingly glycocalicin is also found in normal plasma and can be purified to be used in the assay and was the source of the glycocalicin in the original assay [see reference 17.]
In the glycocalicin VWF:RCo assay, microtitre plates are incubated with an anti-GpIbα monoclonal antibody and after washing the plates are saturated with glycocalicin. Plasma [controls, tests and standard] and a fixed concentration of ristocetin are added and the bound vWF is detected with an anti-human VWF horseradish peroxidase [HRP] labelled antibody. The bound antibody is quantified in a colorimetric reaction by adding the HRP substrate OPD [1,2 ortho-phenylendiamine dihydrochloride] and measuring the absorbance at 492 nm.

3. Flow Cytometry: VWF:RCo can also be measured by flow cytometry. In this method a mixture of green and red fluorochrome-labeled and formalin-fixed platelets are incubated with plasma samples in the presence of ristocetin. Each VWF multimer has multiple binding sites for the platelet GpIb receptor. When both green and red fluorescently-labeled platelets bind to a single VWF molecule, a microaggregate forms. The extent of platelet microaggregates, reflected by the double positive (green and red) events, correlates with plasma VWF:RCo activity. The extent of the microaggregate formation is affected by the binding affinity of VWF to platelets and the molecular weight of the VWF multimer. There are no double positive events if the VWF multimer molecular weight is too low to bind at least two platelets.

Interpretation

The VWF:RCo assay has high intra-laboratory and interlaboratory variation, and it does not actually measure physiological function. However despite the limitations of the VWF:RCo assay it remains one of the most frequently requested tests of VWF function.

In individuals with normal VWF structure and function, the results of VWF:RCo and VWF:Ag assays are similar.

Reference Ranges

The reference range for VWF:RCo is usually in the region of 50-150 IU/dL. Von Willebrand factor is an acute phase protein and so the levels may rise at times of stress e.g. in pregnancy.

What Test Next

Measurement of VWF:RCo levels is usually undertaken as part of a screen to establish or exclude a diagnosis of von Willebrand disease. These assays are also used to monitor replacement therapy in patients with VWD.

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.