HAEMOSTASIS
Classification of von Willebrand disease. Type
Inheritance Type 1 (partial
quantitative deficiency) Type 2 (qualitative variant) Type 2A Type 2B Type 2M Type 2N
Type 3 (severe) Autosomal dominant
vWF activity Decreased
Autosomal dominant or recessive Decreased Autosomal dominant
Autosomal dominant or recessive Decreased Autosomal recessive Autosomal recessive
Normal
Markedly decreased or absent
RIPA Decreased Decreased
Decreased Increased Decreased Normal
Multimer pattern Uniform decrease; all multimers present
Decreased large multimers Decreased large multimers
Uniform decrease all multimers present Normal
Markedly decreased Undetectable; usually cannot visualise or absent
vWF activity: ristocetin cofactor; RIPA: ristocetin-induced platelet aggregation. Adapted from The National Heart, Lung and Blood Institute. The diagnosis, evaluation and management of von Willebrand disease. Bethesda, MD: National Institutes of Health Publication 08-5832, 2007.
was unworkable. In 1994 the classification was restricted to six types (types 1, 2 and 3, with type 2 further subdivided), based on the results of routine laboratory tests. Also included in the 1994 classification was the condition that there had to be a mutation in the vWF gene, but this was removed in the 2006 classification. Subsequently, the von Willebrand ristocetin cofactor to antigen ratio was introduced to distinguish between types 1 and 2. Looking at classification in more detail,
type 1 is a partial deficiency of vWF, and this is found in the large majority of cases. Type 3 is rarer, with a prevalence of 1–5 per million, and virtual absence of vWF. This form is inherited from both parents. Type 2 vWD represent around a quarter of all cases, and these are further subdivided into those having problems with platelet binding (types 2A and 2M). Type 2A shows increased proteolytic degradation, with loss of the multimers and decreased platelet binding. Its counterpart is type 2M, where there is decreased binding of platelets but not through a loss of multimers. Another strange form, type 2B, is
characterised by increased binding of platelets which leads to the removal of multimers. A fourth type, 2N (Normandy), has nothing to do with platelets, but is a problem in binding factor VIII, which appears rather like a haemophilia-type disease. When the classification was simplified in 1994 a number of different variants were ‘lumped together’ as type 2A and in Antwerp we like to further subdivide 2A into those different types. When there is a defect in the propeptide the classification is type 2A/IIC. There is a multimeric problem in domain D3, resulting in a type 2A/IIE and this is of particular interest to the researchers in Antwerp. At the other end of the protein there are multimerisation problems giving rise to a type 2A/IID Together, they are officially classified as type 2A but there are large or semi-large differences between these entities. So, what can be said about the classic classification? It is based on laboratory tests
THE BIOMEDICAL SCIENTIST AUGUST 2016
‘The first step in the diagnosis of vWD, the determination of the quantity of the protein, is straightforward and performed by immunoassay’
performed in routine laboratories, but variations in individual coefficients of variation (CVs) introduce problems. In addition, important variations arise depending on when the patient analysis is performed. Most importantly, there is no correlation or no adequate correlation with severity of bleeding. Type 1 and type 2 show enormous variations, while type 3 has a severe bleeding phenotype, so there is a correlation here. Another ongoing debate is about exactly
when vWD is regarded as a disease. Currently, there is a proposition to reserve the diagnosis of ‘disease’ to vWF below 30 IU/dL, as in such cases there is a higher association with a mutation. However, the 30–50 IU/dL group can also bleed. Diagnosis itself is made based on the
patient’s personal bleeding history, which can be measured through standardised bleeding score. A familial bleeding history is also a requirement, and then laboratory- detected abnormalities are taken into account.
Laboratory testing The first step in the diagnosis of vWD, the determination of the quantity of the protein, is straightforward and performed by immunoassay. As with any test there is the question of variation, and it is important to
use reference values according to the blood group because there is a large difference (up to 25%) between the non-O and O blood groups. Results in group O patients are lower because of an increased elimination of vWF. So, if group A normal values are used for group O patients then more diagnoses of vWD will be made. Of particular interest to workers in
Antwerp are the important factors that influence vWF and factor VIII when blood is sampled. The first, adrenergic stimulation, means that values are increased by stress, by exercise, even by crying in a child. This makes sampling very difficult. Thus, it is impossible to say whether the values measured are really the lowest values the patient can produce. Other factors include inflammation and the menstrual cycle. In pregnancy a type 1 vWD can disappear, and be totally corrected by week 30. Then there is blood group, as mentioned previously (increased clearance in blood group O), and the effect of race and age. The other main test is for functional
activity which measures the binding to platelets in the presence of ristocetin. To interact with the platelets the vWF in the body undergoes a conformational change due to shear stress conditions, going from its globular form to an elongated form. This exposes the A1 loop in the A1 domain, which promotes binding to platelets. In vitro, the effect of shear stress is reproduced by ristocetin, which binds at either end of the A1 loop, pulling it together. Thus, the effect of ristocetin is to promote binding to platelets. Previously, the test was performed by manual aggregometer, but this has now been automated; however, it still suffers from an enormous variation and issues of reproducibility. Despite its limitations it remains a cornerstone in the diagnosis
‘The largest protein in the blood, von Willebrand factor undergoes conformational change under shear stress, going from a globular form to an elongated form in which the functional areas are exposed’
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