Our guide to vWD diagnosis

Von Willebrand Disease Subtypes for the Exam

Diagnosis of subtypes of von Willebrand disease are an almost certainty for the exam, often in EMQ format. We suggest that you follow this stepwise approach to determining the diagnosis:

Remember

• Type 1 – partial quantitative defect
• Type 2 – qualitative defects
  • Type 2A – A for Absent multimers
  • Type 2B – B for Bonkers platelets
  • Type 2M – M for Maintained multimers
  • Type 2N – N for No Number 8
• Type 3 – severe quantitative defect

1) Can VWD be excluded (apart from type 2N)? What’s the vWF:RCo
A normal vWF:RCo (>0.5 iu/ml) can pretty much exclude VWD. The exception is type 2N (covered later)

2) Is it type 1 or 3? What’s the absolute level of VWF (VWF:AG)?
Low in the subtypes where there is a quantitative defect in vWF – type 1 (partial deficiency) and type 3 (severe deficiency). If VWF:Ag and VWF:RCo are undetectable, it’s probably type 3.

3) Is it type 2? What’s the VWF:RCo/VWF:Ag ratio?
If there is a quantitative defect, the VWF activity is proportional to the amount there is i.e. 1 – type 1. Qualitative defects that impair VWF activity will yield a low ratio i.e. <0.6-0.7.

4) Is it type 2N? What’s the factor VIII level?
VWF is the carrier of factor VIII so it can be low in all forms but is very low in type 2N and type 3

5) Is it 2M? What’s the multimer pattern / collagen binding activity?
If the multimer pattern shows intact high molecular weight multimers (HMWMs) and/or the VWF:CB (collagen binding) is not reduced, think type 2M.

6) Is it type 2B or type 2A? What’s the response to low-dose RIPA?
If you’ve gotten this far and have no answer, look to the low-dose RIPA. In type 2B or platelet type vWD, there is an accentuated response to low-dose RIPA.

7) If it’s 2B, could it be platelet type?
Differentiate between type 2B and platelet type VWD

• Cryoprecipitate challenge
  Type 2B: Cryoprecipitate + platelet rich patient plasma leads to aggregation in platelet-type vWD but not in type 2 vWD.
• RIPA mixing studies
  Patient plasma + control platelets + low dose ristocetin = agglutination in type 2B, not in platelet-type
  Control plasma + patient platelets + low dose ristocetin = agglutination in platelet type, not in type 2

Can you understand why this is the case? Don’t move on until you do!
In type 2B, the activating mutation is in vWF, in platelet type, it’s in the VWF platelet receptor, Gp1b.

• Cryoprecipitate challenge
  As cryoprecipitate from healthy donors contains VWF, adding cryo to patient platelet-rich plasma will cause platelet agglutination in platelet type but not in type 2B. This is because the patient’s platelets are super sensitive to the added vWF.
• RIPA mixing studies
  a) In type 2B, patient plasma has vWF that is too active. Combining this with control platelets will lead to agglutination
  b) In platelet-type, control plasma has normal vWF but the patient’s platelets are hyper-responsive so this will lead to agglutination

8) Is it Vicenza?
Type 1c (Vicenza) is a subtype of type 1 VWD caused by accelerated clearance of VWF from the plasma causing a quantitative defect. There are three hallmarks of this to be aware of: 1) short response to desmopressin, 2) high VWF propeptide to VWF:Ag ratio, 3) presence of ultra-high molecular weight multimers

What’s the difference between the vWF ristocetin cofactor assay (VWF:RCo) and RIPA?

This is something that confused the hell out of me, and it’s not very well explained in any of the resources I used for revision, including Practical Haemostasis. Firstly, ristocetin is a failed antibiotic because it induced thrombocytopaenia. Through an unknown mechanism, it increases the affinity for VWF-platelet binding (through the Gp1b receptor). In normal people, if you add enough ristocetin to platelets, you get agglutination.

vWF:RCo vs. RIPA
The von Willebrand Ristocetin Cofactor Assay is also known as the RICOF, or the vWF:RCo. It is a quantitative test of von Willebrand factor activity. Ristocetin is a failed antibiotic that caused thrombocytopaenia and was thus taken off the market. In the assay, patient platelet poor plasma is mixed with formalin fixed (dead) platelets and ristocetin. Ristocetin potentiates binding of vWF to the platelet Gp1b receptor thus inducing platelet agglutination. Agglutination leads to increased light passing through the mixture and the rate of change in light transmission is recorded. The steepness of the curve corresponds to the amount of vWF present.

It is different to the Ristocetin Induced Platelet Agglutination (RIPA assay). The RIPA is not used to quantify von Willebrand factor activity, rather it is used in the work-up of patients with bleeding disorders and is one of the components of platelet aggregometry testing. The RIPA uses ristocetin which is added directly to the patient platelet rich plasma and there are no serial dilutions of the plasma sample. Platelet activity is then measured by lumi-aggregometry and often reported as a % maximal agglutination. It is done with low dose ristocetin (~0.5μg/ml) and high dose ristocetin (~1.5μg/ml). With normal plasma, low dose ristocetin does not have an effect but in patients with mutations that potentiate the Gp1b-vWF interaction, agglutination is seen. The two conditions that can cause this are type 2B von Willebrand disease, and platelet-type von Willebrand disease.

Reagents for the von Willebrand Cofactor Assay

• Formalin fixed platelets
• Ristocetin
• Serial dilutions of platelet poor patient plasma containing von Willebrand factor

The steepness of the slope is then used to determine the von Willebrand factor content

Reagents for the Ristocetin Induced Platelet Agglutionation (RIPA) assay

• Platelet rich plasma
• High dose or low dose ristocetin

Semi-quantitative readout. This is the old way of doing a VWF:RCo. Now, ELISA is used whereby Gp1b fragments are bound to a plate, plasma is added and any vWF that is present can bind. An anti-vWF antibody is then used to bind to the bound vWF. This antibody has an enzyme such as Horse Radish Peroxidase bound to it. The enzyme is able to catalyse the conversion of a substrate in the solution to a coloured product. The intensity of the colour that is produced is proportional to the amount of vWF bound to the plate.

Here’s a link to a video recap on ELISA:  but bare in mind that instead of looking for an antibody, we are looking for VWF.