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Von Willebrands Disease Panel

Von Willebrand’s disease (vWD) is the most common inherited abnormality of hemostasis and is usually inherited in an autosomal dominant fashion. Currently, vWD is divided into 3 types (type 1,2 and 3) and type 2 vWD is further divided into 4 subclasses (2A, 2B, 2M, and 2N). Estimates of prevalence vary from 1 per 10,000 persons to 1 per 100 persons, with Type 1 vWD being the most common type. Prevalence of Type 3 vWD is 1.4 cases per million in the United States.

vWD Characteristics

vWD Type

Prevalence

Inheritance

vWF Defect

Type 1

75% vWD cases

Autosomal Dominant

Partial quantitative deficiency

Type 2

25% vWD cases

Qualitative defect

Type 2A

75% Type 2 cases

Autosomal Dominant

Decreased platelet adhesion with selective deficiency of high MW multimers

Type 2B

20% Type 2 cases

Autosomal Dominant

Increased affinity for platelet GP1b

Type 2M

<5% Type 2 cases

Autosomal Dominant

Decreased platelet adhesion w/o selective deficiency of high MW multimers

Type 2N

<5% Type 2 cases

Autosomal Recessive

Markedly decreased I binding affinity for FVIII

Type 3

Rare

Autosomal Recessive

Virtually complete quantitative deficiency

VWD is caused by a qualitative or quantitative abnormality of von Willebrand factor (vWF), leading to a disruption of primary hemostasis. Normally, vWF mediates the adhesion and aggregation of platelets to subendothelium in blood vessels with high shear force, such as arteries, by binding to platelet glycoproteins GPIb and GPIIb-IIIa. In addition, vWF binds to factor VIIIc and protects it against degradation by activated Protein C. Substantial deficiency of vWF (levels <30%) results in a primary hemostatic defect and most patients present with mild mucocutaneous bleeding including easy bruising, epistaxis, menorrhagia or excessive bleeding during surgery or trauma. Cases of Type 3 vWD with very low vWF also have very low factor VIII levels and may experience soft tissue and joint hemorrhages.

An initial hemostasis workup often includes a complete blood count and routine coagulation studies including APTT, PT, and fibrinogen. PTT is abnormal only in those types of vWD that have significantly decreased Factor VIII levels, such as type 2N and type 3. Factor VIII levels are often normal in types 1, 2A, 2B, and 2M. This testing neither rules in nor rules out vWD, but can suggest whether a coagulation factor deficiency or thrombocytopenia is a potential cause of bleeding. Platelet function tests, such as the PFA-100, may also be ordered. Persons who have severe type 1 vWD or type 3 vWD usually have abnormal PFA-100 values, whereas persons with mild or moderate type 1 and some who have type 2 vWD may not have abnormal results.

A panel of tests is required to screen adequately for the disorder, because routine screening tests such as the aPTT may be normal. The aPTT is abnormal only in those cases of vWD with low levels of factor VIIIc. The initial panel includes factor VIIIc assay, von Willebrand factor antigen (vWF Ag), and ristocetin cofactor (RcoF). These tests measure the (1) quantity of vWF antigen, (2) determine its ability to agglutinate platelets, (3) elucidate its multimeric structure and (4) measure factor VIII level.

VWF antigen is determined by an immunoassay that measures the quantity of vWF in plasma. VWF Ag levels span a wide range in the healthy population. The mean level of plasma vWF is 100 IU/dL and the reference range is 50 - 200 IU/dL. Persons with very low vWF levels of <20 IU/dL are likely to have vWF gene mutations, significant bleeding symptoms and a strongly positive family history. VWF Ag levels between 30 and 50 IU/dL often are not associated with a history of excessive bleeding or a strong family history of vWD. Approximately 80% of these patients have blood group O. The mean vWF Ag level for blood group O is 75 IU/dL and the normal range is 36-157 IU/dL, which is 25 – 35 IU/dL lower than other ABO groups. The diagnosis of type 1 vWD occurs more frequently in individuals who have blood group O.

vWF activity is determined by an automated immunoassay using latex particles coated with an antibody against the platelet binding site of vWF. It is much easier to perform than RcoF and serves as an excellent screening test for qualitative assessment of vWF (Haematologica 2007;92:712-713 and Am J Clin Pathol 2007;127:730-735). In conjunction with the vWF:Ag assay, a ratio of vWF activity to vWF antigen is provided. If vWF activity is decreased or the ratio is less than 0.7, the ristocetin cofactor assay is then performed as a reflex test, for further evaluation and classification of the type of vWD.

RcoF is a functional assay of vWF that measures its ability to interact with normal platelets. The antibiotic, ristocetin, causes vWF to bind to GP1b on platelets, resulting in agglutination. RcoF activity does not actually measure physiological function and has poor precision.

Factor VIII coagulant activity provides information about the ability of vWF to function as a carrier protein for factor VIII. Factor VIII is always decreased in patients with Type 2N and Type 3 vWD. In type 3 vWD, plasma factor VIII level is usually <10 IU/dL. Factor VIII levels do not always parallel vWF antigen levels. Factor VIII levels are often normal in types 1, 2A, 2B and 2M vWD. In type 1 vWD, factor VIII is often slightly higher than vWF Ag and may fall within the normal range. In persons with type 2 vWD, except for 2N, factor VIII is often 2 to 3 times higher than RcoF.

If one or more of the initial tests are abnormal, additional testing may performed to further classify vWD. Ristocetin induced platelet aggregation (RIPA) is particularly useful to distinguish type 2A from type 2B vWD, because it can demonstrate the higher than normal affinity of vWF for the platelet GP1b/IX complex that occurs in Type 2a. In this assay, varying concentrations of ristocetin (0.5, 1.0, & 1.5 mg/mL) are mixed together with platelet rich plasma. The minimal concentration of ristocetin able to cause 30% platelet aggregation is recorded. Platelets from unaffected individuals require 1.0 – 1.5 mg/dL of ristocetin for aggregation. Platelets from patients with type 2B aggregate with 0.5 mg/mL ristocetin, but platelets from patients with type 2A do not. Patients with type 3 vWD do not aggregate at higher concentrations of ristocetin (1.1-1.3 mg/mL).

VWF multimer analysis involves the separation of vWF molecules by protein electrophoresis and detection of all molecular weight forms by Western Blot. This test is used to subclassify Type 2 vWD into types 2A, 2B and 2M. Types 2A and 2B have a decrease in high molecular weight multimers, while type 2M has a normal distribution.

von willebrands disease panel

Based on the levels of RcoF, vWF antigen, factor VIII activity and distribution of vWF multimers, vWD can be classified into quantitative and qualitative abnormalities. Types 1 and 3 represent partial and severe quantitative deficiencies respectively, whereas type 2 vWD has a normal quantity of vWF that is functionally and structurally defective. In types 1 and 3 vWD, the vWF antigen and RcoF activity values are both diminished to a similar extent. In contrast, type 2 vWD is characterized by RcoF activity that is reduced more than vWF antigen. Panel results are interpreted as follows:

Laboratory Classification of von Willebrand’s Disease

vWD Type

RcoF

IU/dL

vWF Ag

FVIIIc

RcoF/Ag

Ratio

Platelet count

Low Dose RIPA

Multimers

1

Decreased

<30-50

Decreased

<30-50

Normal/ Decreased

>0.5-0.7

Normal

Absent

Normal

2A

Decreased

<30-50

Normal

Borderline

<50-200

Normal/ Decreased

<0.5-0.7

Normal

Absent

Loss of high & intermediate

2B

Decreased

<30-50

Normal

Borderline

<30-200

Normal/ Decreased

Usually

<0.5-0.7

Decreased

Present

Loss of High

2M

Decreased

<30-50

Normal

Borderline

<30-200

Normal

<0.5-0.7

Normal

Absent

Normal

2N

Normal

Borderline

<30-200

Normal

Borderline

30-200

Marked

Decreased

>0.5-0.7

Normal

Absent

Normal

3

Absent

<3

Absent

<3

Marked Decrease

Not applicable

Normal

Absent

Absent

Type I vWD is the mildest form of the disease and is characterized by a concordant mild quantitative decrease in vWF level and ristocetin cofactor activity (30-50%). Factor VIIIc may be normal or slightly decreased, and is not decreased as much as vWF Ag. The FVIII:vWF Ag ratio is 1.5 – 2.0. There is no decrease in large vWF multimers. A diagnosis of Type 1 vWD is harder to establish when the vWF level is near the lower end of the reference range.

Type 2 vWD is caused by a qualitative defect in vWF that may be associated with either deficient multimer formation or increased susceptibility of vWF to proteolytic degradation. VWF antigen level and RcoF activities are often discordant; RcoF is usually low and vWF and FVIII are usually borderline. Usually, the RcoF:vWF antigen ratio is less than or equal to 0.7. Approximately 20% of patients with type II vWD have a normal vWF antigen level.

Type 2 vWD can be further subclassified into type 2A, 2B, 2M, or 2N. Type 2A is characterized by decreased vWF dependent platelet adhesion due a decrease of high molecular weight multimers. Levels of vWF Ag and FVii may be normal or modestly decreased, but vWF function is abnormal as shown by markedly decreased RcoF. Platelet rich plasma from patients with Type 2A does not aggregate in the presence of dilute ristocetin.

Type 2B vWD is caused by mutations that pathologically increase vWF binding to platelets, which leads to proteolytic degradation and depletion of large vWF multimers. Patients with Type 2B often have mild thrombocytopenia that is exacerbated by surgery, pregnancy or other stress. Te diagnosis of type IIb depends on the demonstration of abnormally increased ristocetin induced platelet aggregation (RIPA) at low concentrations of ristocetin.

Type 2M (M for multimers) vWD is caused by mutations that decrease the interaction of vWF with platelet GP1b or with connective tissue and do not impair multimer formation. VWF dependent platelet adhesion is decreased. Type 2M is distinguished from Type 2A by multimers analysis.

Type 2N (N for Normandy) vWD is caused by mutations in vWF that impair its binding to factor VIII, causing increased clearance of Factor VIIIc. Because Factor VIIIc levels are decreased, Type 2N vWD resembles an autosomal recessive form of hemophilia A. In typical cases, FVIII level is <10% with a normal vWF Ag and RcoF.

Type III vWD is the most severe form of vWD. There is almost total absence of vWF and RcoF activity and markedly decreased factor VIIIc. Approximately 5% of patients with Type III vWD develop antibodies to vWF after transfusion of plasma products and are at higher risk of uncontrollable bleeding and severe allergic reactions.

Mild vWD may be difficult to detect because assay values in a patient may vary at different times. Repeat testing after an interval of 2 to 4 weeks may be required to confirm or exclude the diagnosis. Inflammation, stress, pregnancy, estrogen therapy, and combined oral contraceptive pills may increase vWF levels above baseline and potentially mask diagnosis of mild vWD. Repeat testing should be undertaken 4 to 6 weeks after discontinuation of estrogen or oral contraceptives.

Acquired von Willebrand Syndrome (AVWS) is caused by other medical disorders that produce autoantibodies, increase proteolysis of vWF or increase binding of vWF to platelets or other cells. Autoimmune and lymphoproliferative disorders may produce autoantibodies that increase the clearance of vWF. The high shear stress associated with aortic stenosis causes proteolysis of high molecular weight multimers. The elevated platelet counts associated with myeloproliferative disorders promote increased cleavage of vWF by ADAMTS13. AVWS has also been associated with hypothyroidism and several drugs including valproic acid, ciprofloxacin, griseofulvin and hydroxyethyl starch. Laboratory findings in AVWS may include decreased values for vWF Ag, RcoF or FVIII. Multimer analysis may be normal or show a decrease in large multimers similar to type 2A vWD.

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