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A Practical Guide to Laboratory Haemostasis

 

Factor VIII Binding Assay



Introduction

Type 2N [Normandy] VWD is a recessively inherited disorder due to a mutation in von Willebrand Factor that impairs its ability to bind to and transport FVIII in the plasma. As a result the T½ of FVIII is significantly reduced as it is no longer protected from proteolytic degradation within the plasma. Individuals with Type 2N VWD were and occasionally still are, misdiagnosed as having mild Haemophilia A.
Diagnosis of 2N VWD involves:
      - Pedigree analysis [2N VWD is recessively inherited]
      - Measuring both FVIII and VWF activity [FVIII is reduced but VWF activity is normal]
      - FVIII binding studies
      - Genetic analysis

Principles & Method

There are a number of methods available to measure the binding of FVIII to VWF.
The ELISA method is commonly used:

1. Standard 96-well plates are coated with an anti-human VWF antibody.
2. The plates are incubated with plasma samples [allowing the anti-VWF antibody that has been immobilised onto the microtitre plate to bind to the plasma VWF-FVIII complex].
3. The plates are washed and then incubated with calcium chloride to strip endogenous factor VIII bound to VWF [this ensures that only the VWF is bound to the plate.]
4. A standard concentration of recombinant factor VIII is added which binds to the immobilised VWF in all samples.
5. After further washing the plate is exposed to either a peroxidase-conjugated rabbit anti-VWF or peroxidase-conjugated sheep–antihuman factor VIII. After a final washing step tetramethylbenzidine dihydrochloride substrate is added and colour generation is detected as for a standard VWF:Ag assay.
6. A standard curve is generated using serial dilutions of pooled normal plasma (using a pool from >60 persons).
7. For each plasma dilution, the values of bound rFVIII are plotted against the amount of immobilised VWF. The VWF:FVIII values are expressed in percentages compared with normal plasma.

Interpretation

Individuals with normal VWF will show normal binding to FVIII in step 4 whereas individuals with 2N VWD will show defective binding of FVIII.



What Test Next

Most cases of 2N VWD are/were misdiagnosed as mild haemophilia A. Any individual with mild haemophilia A should be screened for 2N VWD. One should also consider the possibility of combined FV and FVIII deficiency in mild cases of haemophilia A.

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.