Challenges in diagnosing von Willebrand disease

Introduction

Von Willebrand disease (VWD) is the most common, although underdiagnosed, bleeding disorder caused by inherited defects in the concentration, structure, or function of VW factor (VWF). The prevalence of the disease in the general population, based on the presence of bleeding, is 1 in 1000. However, based on laboratory test results, it is 1 in 100.^1,2 Overall, its prevalence is estimated at 0.6%–1.3%.^1-3

VWD is primarily inherited in an autosomal dominant manner, although in subtypes 2N and type 3—in an autosomal recessive manner. Autosomal dominant inheritance is characterized by variable penetrance and expression, which is why individuals from the same family with the same mutation may experience bleeding of various severity.²

VWF is a protein composed of varying sizes of multimers synthesized in bone marrow megakaryocytes and vascular endothelial cells, and stored in the Weibel–Palade bodies of endothelial cells and the α granules of platelets.²

Von Willebrand factor

The VWF gene is located on the short arm of chromosome 12 (12p13.2). The structure of the gene, as well as the pseudogene located on chromosome 22, was first described in 1992.^1,2 The pseudogene structurally corresponds to exons 23 and 24 of the gene encoding VWF. Mutations in the gene encoding VWF cause dysfunction or deficiency of the factor, but their absence does not exclude the diagnosis of the disease.¹ It is estimated that in approximately 30% of the patients, none of the mutations identified so far are detected. Due to inheritance with incomplete penetrance, affected individuals may also not present clinical symptoms. Expression of the gene encoding VWF produces a prepropolypeptide, a precursor of VWF, composed of a signal peptide, a propeptide (domains D1 and D2), and a monomer. Each monomer is composed of 4 repeating domains (A, B, C, and D) from the N-terminus to the C-terminus, including the propeptides: D1, D2, D′, D3, A1, A2, A3, D4, B1, B2, B3, C1, C2, and CK (Figure 1).^4,5

Each monomer domain is responsible for the biological functions of VWF. Domains A1 and A3 are responsible for binding to collagen. Additionally, domain A1 binds to ristocetin and participates in platelet adhesion by binding to glycoprotein Ib (GPIb). Domain C1 binds to platelet GPIIb/IIIa during the aggregation process, while domains D3 and D′ bind to factor VIII (FVIII) and heparin.

The signal peptide is located upstream of the propeptide, and is responsible for transporting the precursor to the endoplasmic reticulum. In the endoplasmic reticulum, the signal peptide is cleaved, forming propolypeptides, which dimerize to form disulfide bridges between the CK domains. The dimers are then transported to the Golgi apparatus. Domains D1 and D2 constitute the propeptide involved in multimerization in the Golgi apparatus, and are cleaved at this stage. Hence, the sequence of the mature VWF monomer (subunit) is: D′, D3, A1, A2, A3, D4, B1, B2, B3, C1, C2, and CK. The molecular weight of the monomer is approximately 250 kDa. The acidic environment and high calcium ion concentration in the Golgi apparatus stimulate multimer synthesis by forming disulfide bridges between D3 domains, resulting in the formation of ultra large high-molecular-weight multimers (HMWMs). The resulting multimers are continuously released into the circulation, and some of them are stored in the Weibel–Palade bodies of endothelial cells or platelet granules. Despite detachment, the propeptide forms a noncovalent bond with VWF, and dissociation of the complex occurs after the factor is released into the bloodstream. Disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13 (ADAMTS-13) is responsible for the size variation of multimers released from endothelial cells.^4,5

The multimers presented in plasma may contain from 1 to 40 dimers, and their mass ranges from 500 to 10 000 kDa. The size of multimers stored in the endothelium and thrombocytes usually exceeds 10 000 kDa (ultra-large multimers). Physiologically, VWF occurs in plasma in the form of low-MWMs (LMWMs), intermediate-MWMs (IMWMs) and HMWMs. HMWMs are crucial for proper platelet adhesion and aggregation in vessels characterized by high shear forces (arteries, small vessels, and pathologically stenotic vessels). The half-life of VWF is 12 hours (9–15 h). Approximately 10%–15% of the total amount of VWF in the circulation is found in platelets.⁷

VWF plays a dual role in hemostasis, participates in the process of platelet adhesion at the site of vessel damage, and stabilizes FVIII, with which it forms a complex in the plasma. This double role of VWF determines the classification of the disease as mixed platelet–plasma disease. In plasma, VWF is inactive and does not react with platelets.⁴ Primary hemostasis is initiated by damage to the vessel wall and the exposure of collagen in the subendothelial connective tissue. Released by thrombin, VWF binds to collagen via the A3 domain. The A1 domain binds to the platelet receptor GPIb, also forming connections between GPIb and collagen, thus enabling platelet adhesion to the endothelium. Following adhesion, platelets are activated, changing their shape and releasing VWF, among other substances, from their granules. Then, during the aggregation process, the platelet GPIIb/IIIa receptor binds to the C1 domain of VWF and fibrinogen. Connections are also formed between adjacent platelets, which, in turn, leads to the formation of a platelet plug. Large multimers (HMWMs) are primarily responsible for effective platelet adhesion and aggregation.⁸ Physiologically, in the circulating blood, VWF is noncovalently bound to FVIII, protecting it from proteolysis by the activated protein C system and faster elimination from the body. This function is performed by all multimer fractions (LMWMs, IMWMs, and HMWMs). The half-life of FVIII in the absence of VWF is shortened from 8–12 to 1–2 hours.^4,7

VWF is characterized by significant heterogeneity, and its plasma activity depends on age, genetic, and pathophysiological factors.⁸ Increased activity is typical of diabetes, cancer, hyperthyroidism, and inflammation. It also occurs after physical exercise, in women taking oral contraceptives, in stressful situations, during pregnancy, and as a result of exposure to tobacco smoke and other air pollutants.^8-10 The estimated physiological increase in VWF activity in healthy individuals is 15%–17% every 10 years, and in type 1 VWD, 3.5%. Despite the increase in factor activity with age, the tendency to bleed does not change. Increased activity is observed neither in type 2 due to a functional defect in VWF, nor type 3.^1,10,11 In healthy women and those with type 1 disease, VWF activity and concentration during pregnancy reach levels ​​up to 5 times higher than initial values.¹ A correlation between VWF activity and blood type has also been documented. In individuals with blood type 0, activity is up to 25% lower than in other blood types. Nevertheless, modifying reference ranges based on blood type is not recommended due to the correlation between the risk of bleeding and the factor activity in plasma, regardless of the patient’s blood type.^1,12

The activity might be reduced by medications, such as ciprofloxacin or valproic acid, as well as cancer, hypothyroidism, autoimmune, and cardiovascular diseases, which may cause acquired VW syndrome (AVWS).¹

Classification of von Willebrand disease

According to the 2006 International Society on Thrombosis and Haemostasis criteria^13, VWD is classified into 3 main types: partial quantitative deficiency of VWF (type 1; Mendelian Inheritance in Man [MIM], 193400), qualitative defects of VWF (type 2; MIM, 613554), and complete deficiency of VWF (type 3; MIM, 277480). Type 2 VWD is divided into 4 secondary categories differing in the nature of VWF dysfunction (Table 1).

Table 1. Characteristics of different types and subtypes of von Willebrand disease in terms of inheritance and bleeding severity¹

Type / subtype		Characteristics	Inheritance	Bleeding severity
Type 1 (MIM, 193400)		Partial deficiency of VWF	Autosomal dominant	Mild or moderate
Type 2 (MIM, 613554)		Dysfunction of VWF	Autosomal dominant or recessive	Usually moderate
Type 2 subtypes	2A	Decreased platelet adhesion and deficiency of large multimers	Autosomal dominant or recessive	Usually moderate
	2B	Increased VWF affinity to GPIb, deficiency of large multimers	Autosomal dominant	Usually moderate
	2M	Decreased platelet adhesion	Autosomal dominant or recessive	Usually moderate
	2N	Decreased binding VWF to FVIII, decreased FVIII activity	Autosomal recessive	Usually moderate
Type 3 (MIM, 277480)		Complete deficiency of VWF	Autosomal recessive	Severe
Abbreviations: FVIII, factor VIII; GPIb, glycoprotein Ib; MIM, Mendelian Inheritance in Man; VWF, von Willebrand factor

Type 1 VWF is inherited in an autosomal dominant manner. It occurs in 75%–85% of the VWD patients, with clinical symptoms typically mild or moderate. This type is characterized by quantitative VWF deficiency (proportional reduction in VWF concentration and activity). The molecule of VWF is functionally normal and all multimer fractions are present.⁹

Subtype 1C is characterized by accelerated clearance of VWF from the circulation. To confirm type 1C, a desmopressin response test should be performed, with an assessment of VWF activity 1 and 4 hours after drug administration.¹²

Type 2 is diagnosed in 20%–35% of the patients with VWD.¹ This type is characterized by qualitative VWF dysfunction and is divided into 4 secondary categories: 2A, 2B, 2M, and 2N. The severity of symptoms is variable, but bleeding is usually moderate. Depending on the subtype, inheritance is either autosomal dominant or recessive.¹⁴

Subtype 2A includes variants with decreased platelet adhesion caused by selective deficiency of VWF HMWMs due to increased susceptibility to cleavage by ADAMTS-13 or impaired synthesis; there is a deficiency of HMWMs, which leads to impaired VWF-dependent platelet adhesion to the vascular endothelium. The activity of VWF is decreased but its level is normal or slightly reduced.

Subtype 2B is characterized by increased binding affinity for the primary thrombocyte receptor for VWF, GPIb, with secondary consumption of HMWMs and faster removal from the circulation.^1,14 The aggregates of platelets are associated with abnormal VWF, leading to impairment of platelet adhesion. The presence of aggregates may cause thrombocytopenia in this subtype, which is exacerbated by stress, pregnancy, and surgical procedures.^12,15

Platelet-type VWD is a congenital thrombocytopathy with autosomal dominant inheritance, and it presents similarly to VWD subtype 2B, with cutaneous and mucosal bleeding, thrombocytopenia (large platelets are present), and loss of HMWMs. The disease is caused by a mutation in the gene encoding the α subunit of platelet GPIb (GPIBA), leading to increased affinity of GPIb for VWF. Differentiating between platelet-type VWD and subtype 2B is important due to therapeutic options—in the cases of platelet-type VWD, platelet transfusions have been performed with positive effects.¹⁶

Subtype 2M features decreased binding affinity for GPIb without a selective reduction of HMWMs. All fractions of multimers are present, but platelet adhesion is impaired due to decreased binding of VWF to GPIb (mutation in A1 domain of VWF) and connective tissue components (collagen).

Subtype 2N is characterized by a decreased binding affinity for FVIII, which leads to decreased FVIII activity (approximately 5%–40%). The activity and level of VWF may remain within references ranges. Phenotypically, this subtype is close to hemophilia A but with autosomal recessive inheritance.¹⁷

In type 3 (severe), the activity and level of VWF are undeterminable, and FVIII activity is very low (<⁠10%). This type (approximately 1% of all VWD cases) is inherited in an autosomal recessive pattern.^18,19

AVWS is characterized by a lack of previous bleeding and negative family history, with clinical symptoms similar to those present in patients with inherited VWD. AVWS may occur in cancer (lymphoproliferative, myeloproliferative disorders, and nonhematological malignancies), cardiovascular, and autoimmune disorders, and it may be associated with the use of certain drugs (eg, valproic acid, ciprofloxacin, hydroxyethyl starch, and griseofulvin). Deficiency or impaired activity of VWF can result from the presence of specific autoantibodies shortening the half-life of VWF, its adsorption onto the surfaces of neoplastic cells, mechanic injury (cardiovascular disorders), or increased proteolysis by ADAMTS-13. Diagnosis is based on the measurements of plasma concentration and activity of VWF, as well as multimeric analysis (with normal multimer pattern or a pattern typical of subtype 2A).²⁰

Clinical symptoms of von Willebrand disease

Characteristic symptoms VWD include: mucocutaneous bleeding, prolonged bleeding from small wounds, excessive and prolonged menstrual bleeding, nosebleeds, gingival bleeding, easy bruising, as well as hemorrhages following dental extraction, trauma, surgery, and postpartum hemorrhage.^2,6,13 Most episodes are mild or moderate and do not require hemostatic treatment.¹ In approximately 20% of the women with unexplained heavy menstrual bleeding, VWD is diagnosed. Heavy, prolonged menstrual bleeding may be the only symptom of the disease, and a diagnosis may be delayed by the occurrence of this form in several women in the family. Patients with a mild course of VWD may be asymptomatic. Life-threatening bleeding, including gastrointestinal or central nervous system bleeding, may only occur in type 3 of the disease, and it is very rare in types 1 and 2.^21-23

The 2010 standardized questionnaire from the International Society on Thrombosis and Haemostasis – Bleeding Assessment Tool Working Group²⁴ is currently used to assess bleeding symptoms in adults. It takes into account the frequency, severity, site, and cause of bleeding, the possibility of stopping it, and the course of treatment. A significant symptom in the classification is the one that requires medical attention or interferes with daily functioning.

Diagnosis of von Willebrand disease

The criteria for the final diagnosis of VWD include a history of hemorrhage, family history, and results of diagnostics test (screening and confirmatory). Given the heterogeneity of this disease, no single diagnostic test is available, and an algorithmic series of tests is recommended (Figure 2).

The laboratory tests necessary to diagnose VWD are divided into 3 groups: screening, initial, and specific tests (Table 2).

Table 2. Laboratory tests used in diagnosing von Willebrand disease

Test types	Tests
Screening tests	Blood count and platelet count
	Closure time (PFA-200)
	Basic coagulations tests (PT, APTT, fibrinogen, TT)
Initial tests	VWF activity: VWF ristocetin cofactor activity; VWF activity
	VWF collagen binding
	VWF antigen
	Factor VIII coagulant activity
Specific tests	Multimers analysis
	Low-dose ristocetin-induced platelet aggregation
	Test assessing VWF binding to factor VIII
	Genetic tests (NGS, CNV)
Abbreviations: CNV, copy number variation; NGS, next generation sequencing; others, see Figure 2

Screening tests

Screening tests include blood count, platelet count, activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen concentration, and closure time (CT).

In VWD, platelet counts are normal, except for type 2B, where thrombocytopenia may be present. PT, fibrinogen concentration, and TT are normal in most cases in all disease types. Prolonged APTT is observed in patients with VWD with FVIII activity below 30%, that is, in patients with type 3 disease, and rarely in types 2 and 1. CT, performed using the platelet function analyzer PFA 200 ( Siemens, Munich, Germany), assesses platelet adhesion and aggregation. In most patients with VWD, CT is prolonged (except for type 2N), but this test has low sensitivity and specificity, so results in VWD patients may be normal. Interpretation is complicated by the fact that the CT may be prolonged in patients with thrombocytopenia, thrombocytopathy, individuals on anticoagulants, and in the presence of anti-VWF antibodies (a complication of VWD concentrate treatment or AVWS). In the case of prolonged CT, the disease should be differentiated from thrombocytopathies.²⁵

Initial tests

Initial tests include examinations of VWF activity and concentration, and FVIII activity. Screening tests are normal in most patients with suspected VWD, so the decision to expand the diagnosis with initial tests depends on the presence of clinical symptoms. In the cases of very severe symptoms, such as a bleeding diathesis, such tests can be performed during the first medical visit.¹

VWF activity is measured using a functional ristocetin cofactor activity test (VWF:RCo) or newer tests that directly assess VWF binding to platelets receptor GPIbα. In both cases, the reference range is 50%–150% (50–150 IU/dl).^9,26

VWF:RCo allows the assessment of the binding capacity of VWF to the GPIb receptor of platelets in the presence of ristocetin. This antibiotic mediates the reaction leading to platelet agglutination.²³ This test is routinely used in the diagnosis and differentiation of VWD, despite its low sensitivity at measurement of activities below 10% and high 30% variability.²⁶

The assays that directly evaluate VWF binding to platelets include those that require the presence of ristocetin (VWF:GPIbR) or those that do not (VWF:GPIbM). The VWF:GPIbR assay evaluates the binding of VWF to recombinant GPIbα coated on latex beads in the presence of ristocetin. The VWF:GPIbM assay tests the binding of VWF to recombinant GPIb (altered by a gain-of-function mutation) in the presence of latex beads coated with anti-GPIb antibodies. In both assays, platelet agglutination occurs at the final stage, which is measured using the immunoturbidimetric method.^1,16,18 Due to the lower detection threshold, variability below 10%, and good correlation with ristocetin cofactor activity, these assays are used increasingly and / or interchangeably with VWF:RCo, and are suggested by the recent guidelines as the preferred method of assessment of VWF platelet-binding activity.^26-28

VWF VIII collagen binding (VWF:CB) is performed using immunoenzymatic or chemiluminescent methods.¹⁸ The type of collagen used significantly impacts the sensitivity and diagnostic value of the test.²⁴ There is a very good correlation between the decrease in the collagen-binding capacity of VWF and the loss of large multimers; therefore, it is important in differentiating types 1 and 2 (subtypes 2A, 2B, and 2M). There are documented cases of patients in whom the only defect of the VWF molecule is collagen-binding dysfunction, hence the VWF:RCo test results are normal, and the diagnosis depends on the VWF:CB assay. Currently, it is being considered for inclusion in routine initial testing but should not be used interchangeably with the VWF:RCo test, as they assess different biological properties of VWF.^29,30

VWF antigen (VWF:Ag) is most commonly detected using immunological methods, such as immunoenzymatic (enzyme-linked immunosorbent assay [ELISA], chemiluminescent, or immunolatex). The immunolatex method is not recommended for type 3 diagnosis due to its detection threshold of 10%.¹¹

The FVIII activity (FVIII:C) test is performed using a single-stage coagulometric method, as a modification of APTT. In rare situations, when distinguishing type 2N VWD from mild or moderate hemophilia A, a chromogenic FVIII assay may be used. Normal FVIII activity does not exclude VWD.

A proportional decrease in both VWF activity and concentration is characteristic of type 1 VWD. The activity of FVIII is normal or slightly decreased. Subtypes 2A, 2B, and 2M are characterized by a significant decrease in VWF activity and a slightly decreased or normal VWF and FVIII concentrations. Reduced FVIII activity (usually down to 5%–40%) in the presence of slightly decreased or normal activity and concentration of VWF may indicate subtype 2N. It should be differentiated from mild hemophilia A. Undetectable VWF concentration and activity occurs only in type 3 disease. FVIII activity is low and may reach values ​​below 10%.¹

The measurements of all 3 parameters—VWF:Ag, VWF:RCo, and FVIII:C—are usually expressed in international units per deciliter (IU/dl) or as percentage of normal. One IU expresses the activity of a coagulation factor in 1 ml of normal plasma prepared from blood mixed with 3.2% sodium citrate (9:1). For healthy individuals, plasma VWF:Ag, VWF:RCo, and FVIII:C levels are within 0.5–1.5 IU/ml (which corresponds to 50–150 IU/dl or 50%–150% of the reference range).¹

The results of the initial tests in patients with different types and subtypes of VWD are presented in Table 3.

Table 3. Results of initial tests in patients with different types and subtypes of von Willebrand disease¹

Type / subtype	VWF:Ag	VWF:RCo	FVIII:C	VWF:CB
Type 1	↓a	↓	Nb or ↓	↓
Subtype 2A	N or ↓	↓	N or ↓	↓↓
Subtype 2B	N or ↓	↓	N or ↓	↓↓
Subtype 2M	N or ↓	↓	N or ↓	N or ↓
Subtype 2N	N or ↓	N or ↓	↓/↓↓	N or ↓
Type 3	Undetectable	Undetectable	↓↓↓	Not applicable
a Decreased value b Normal value Abbreviations: see Figure 2

Specific tests

Specific tests are performed when it is difficult to distinguish between type 1 and 2, or type 2 its subtypes. Specific tests include: VWF multimer analysis, standard ristocetin-induced platelet aggregation (RIPA) or low-dose ristocetin-induced platelet aggregation (LD-RIPA) tests, test assessing binding VWF to FVIII (VWF:FVIIIB), and genetic tests. Additionally, VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios are calculated.¹³

Multimers in plasma are present in 3 main fractions: LMWMs, IMWMs, and HMWMs (Figure 3). An analysis of VWF multimers is based on the qualitative assessment of the distribution of fractions, separated by size, using electrophoresis. Currently, semiautomated methods are used, including nonreducing agarose gel electrophoresis and immunofixation. This method is primarily used to distinguish subtypes 2A and 2B from 2M. Normal multimer distribution occurs in type 1, subtypes 2M, and 2N of VWD. In type 1, all fractions are visible, but there is a uniform decrease in band intensity due to an overall decrease in VWF concentration. In some cases, mild abnormalities, such as a slight proportional decrease in HMWMs, are present. Subtype 2A is characterized by loss of HMWM fraction and, less frequently, complete or partial loss of IMWM fraction. In subtype 2B and the platelet-type VWD, loss of large multimers is typical (Table 4). The electrophoretic pattern of type 3 shows an absence of individual multimer fractions due to undetectable VWF levels.^13,31

Table 4. Multimer pattern in different types and subtypes of von Willebrand disease³¹

Type / subtype	VWF defect	Multimer pattern
Type 1	Partial quantitative deficiency	Normal
Type 1C	Increased clearance from the blood	Normal or subtle loss of HMWMs or presence of ultra-large multimers
Type 2A	Decreased platelet adhesion	Loss of HMWMs +/–IMWMs
Type 2B	Increased VWF affinity to platelet GPIb	Loss of HMWMs
Type 2M	Decreased platelet adhesion	Normal
Type 2N	Decreased binding of VWF to FVIII	Normal
Type 3	Severe quantitative deficiency	Absent multimers
Abbreviations: see Table 1 and Figure 3

RIPA is the test assessing platelet agglutination in the presence of a standard concentration of ristocetin, and is only used in the diagnosis of type 3 VWD. In these cases, aggregation is impaired at a ristocetin concentration of 1.1–1.3 mg/ml.¹⁶

LD-RIPA is performed in platelet-rich plasma in the presence of a low concentration of ristocetin (<⁠0.6 mg/ml). In healthy individuals, platelet agglutination does not occur. The test is used to identify VWD subtype 2B, in which the result is positive and the increase in transmission at a ristocetin concentration of 0.6 mg/ml is greater than 30%. Similar results are present in platelet-type VWD. To differentiate between platelet- and 2B types, genetic tests are necessary.¹⁶

The VWF:FVIIIB test measures the amount of FVIII bound by VWF using the immunoenzymatic method (ELISA), and allows for the differentiation of hemophilia A from VWD subtype 2N.⁸

The calculation of the VWF:RCo/VWF:Ag ratio can be used to differentiate VWD type 1 from type 2. According to the 2022 recommendations of the Hemostasis Group of the Polish Society of Hematologists and Transfusionists,¹ the limit value of 0.6 is recommended. A ratio lower than 0.6 indicates abnormal VWF function, and therefore type 2, except for subtype 2N, for which the value is the same as for type 1 (>0.6).³²

The ratio of VWF:CB/VWF:Ag offers greater sensitivity in detecting subtypes 2A and 2B than VWF:RCo/VWF:Ag. If a collagen-binding test is available, it is recommended to calculate both ratios, if possible. In recent reports, Favaloro et al²⁹ and Kimiaei et al³³ suggest that this ratio is a very significant predictor of the total bleeding score in patients with type 2A and 2M VWD. The abnormal result of ratio equal to or below 0.6 requires a confirmatory multimers analysis; however, a normal result above 0.6 eliminates the need for this analysis. This is why the authors propose to add VWF:CB test to the initial panel testing in diagnosing VWD. The diagnosis should also be confirmed based on specialized tests, such as LD-RIPA, multimers analysis, or genetic tests. The results of specific tests are outlined in Table 5.

Table 5. Results of specific tests in patients with different types and subtypes of von Willebrand disease¹

Type / subtype	Distribution of VWF multimers	RIPA	LD-RIPA, %	VWF:RCo/VWF:Ag
Type 1	Normal	Na/↓b	<⁠30	>0.6
Subtype 2A	Abnormal	↓	<⁠30	<⁠0.6
Subtype 2B	Abnormal	N/↓	≥30	<⁠0.6
Subtype 2M	Normal	↓	<⁠30	<⁠0.6
Subtype 2N	Normal	N	<⁠30	>0.6
Type 3	No distribution	↓↓↓	Not applicable	Not applicable
a Normal value b Decreased value Abbreviations: Table 1 and Figure 2

Genetic diagnostics is recommended primarily in subtypes 2 and type 3 VWD.¹ However, it is not recommended for type 1, as the probability of detecting mutations is lower than 40%. Most mutations in subtype 2A occur in domains A1, A2, D2, and D3. In subtype 2B, these are domains A1, 2M–A1, A3, 2N–D3, and D′. Genetic testing for the VWF gene mutations, in parallel with routine testing for VWD diagnosis, is recommended only for subtypes 2B and 2N. For subtype 2B, point mutations in the GPIBA gene, encoding the glycoprotein GPIbα, are also tested to differentiate from the platelet-type VWD. However, type 2 mutations are diagnosed in less than 90% of the cases. The use of genetic testing in patients with type 3 is important for genetic counseling and determining the likelihood of rare complications—anti-VWF alloantibodies.^1,8,11

The final diagnosis of VWD might be difficult, especially when type 1 VWD is suspected and the activity of VWF is close to the lower range of the normal value (30%–50%). All 3 preliminary markers (VWF:Ag, VWF:RCo, and FVIII:C) present high variability (up to 30%); therefore, the tests should be performed several times (3 to 4 times).¹ The likelihood of the disease increases with decreasing VWF activity. Plasma VWF activity below 30% is considered a reliable diagnosis, regardless of the symptoms or assay method used. The authors of the Polish guidelines for the management of VWD recommend to diagnose the disease at VWF levels in plasma below 30 IU/dl.¹ For the activity levels between 30% and 50%, it is recommended to base the diagnosis on the hemorrhagic symptoms after excluding other causes of bleeding episodes. In children and adults who have not yet undergone procedures associated with a risk of bleeding, diagnosis based on close family members diagnosed with VWD is also acceptable. VWF activity range of 50%–60%, if clinical criteria are confirmed, constitutes borderline VWF activity, and is associated with an increased risk of bleeding. Negative family and hemorrhagic history as well as normal screening test results do not exclude VWD diagnosis.¹ The activity of VWF and FVIII increases under the influence of many factors, such as age, stress, physical exercises, use of oral contraceptives, surgery or invasive diagnostic procedures, inflammation, and comorbidities (atherosclerosis, cancer, diabetes, kidney disease, or liver disease).

The increase of both parameters is also observed during pregnancy, reaching ​​2 to 5 times the values recorded in ​the third trimester, as compared with initial values (also in women with type 1 VWD).¹

Differentiating between VWD types and subtypes is important due to different treatment and bleeding prophylaxis methods. Desmopressin is typically used in patients with type 1 disease, but it may also be effective in type 2. In type 2, it increases VWF concentrations, but it remains dysfunctional, so the effectiveness of decompression is observed only in some patients with subtypes 2A and 2M. In both types 1 and 2, a desmopressin response test is necessary before initiating treatment. The correct response is a 2- to 5-fold increase in VWF and FVIII activity approximately 40 minutes after administration (intravenously or subcutaneously). Patients with type 2 and 3 VWD are primarily treated with infusions of concentrates containing VWF.^1,34

Conclusions

Diagnosing VWD is a difficult and multistep process due to the limitations of the methods used, the heterogeneity of the factor, and the nature of VWD. In most patients with suspected VWD, screening test results are normal. Initial tests used in the diagnosis of the disease are characterized by high variability, which is one of the reasons for diagnostic difficulties. Differentiating the types and subtypes of VWD is crucial for implementing appropriate treatment. The goal of therapy is to increase the amount of functionally normal VWF as well as stimulate and stabilize hemostasis.