Von Willebrand Disease Testing Research Articles

Laboratory Testing for von Willebrand Disease: The Past, Present, and Future State of Play for von Willebrand Factor Assays that Measure Platelet Binding Activity, with or without Ristocetin.

von Willebrand disease (VWD) was first described nearly a century ago in 1924 by Erik Adolf von Willebrand. Diagnostic testing at the time was very limited and it was not until the mid to late 1900s that more tests became available to assist with the diagnosis and classification of VWD. Two of these tests are based on ristocetin, one being ristocetin-induced platelet aggregation (RIPA) and the other the von Willebrand factor (VWF) ristocetin cofactor assay (VWF:RCo). The VWF:RCo assay provides functional assessment of in vitro VWF binding to the platelet glycoprotein (Gp) complex, GPIb-IX-V. Despite some advancements and newer technologies utilizing the principles of the original VWF:RCo assay, the original assay is still referred to as the gold standard for measurement of VWF activity. This article will review the history of VWD diagnostic assays, including RIPA and VWF:RCo over the past 40 years, as well as the newer assays that measure platelet binding with or without ristocetin, and which have been developed with the aim to potentially replace platelet-based ristocetin-dependent assays.

Utility of Thromboelastography for the Diagnosis of Von Willebrand Disease

Background: Von Willebrand Disease (VWD) is the most common bleeding disorder. Current gold standard diagnostic testing includes: VWF Activity (VWF:RCo), VWF Antigen (VWF:Ag) and Factor VIII Activity (FVIII). There are many difficulties associated with the current diagnostic methods. Thromboelastography (TEG) is a viscoelastic method of measuring coagulation function. The standard TEG assay has not been thought to be of use in VWD because of the lack of shear stress which is essential for the activation of VWF. Modified TEG using Ristocetin activation has been found to be useful in the diagnosis of VWD. The aims of this study were to evaluate the different parameters of Tissue factor (TF) initiated TEG in patients with VWD, to determine if this assay is sensitive to dysfunctional/low levels of VWF, as this does not require any significant change in procedure except for the use of TF as the activator instead of Kaolin.Methods: A retrospective chart review of patients who presented for a bleeding disorder workup that had TF initiated TEG analysis and Von Willebrand laboratory tests completed between January 2007 and December 2015 was performed. IRB approval was obtained, and current diagnostic tests for Von Willebrand Disease (CBC with platelet count, VWF:RCo and VWF:Ag, FVIII, ABO blood type; PT, PTT, Fibrinogen) and TF initiated TEG parameters, specifically K-Time and MRTG (Maximum rate of thrombin generation), were compared. To perform the TEG analysis, the citrated whole blood samples were activated using 20mL of 1:10,000 dilution of recombinant human tissue factor (Innovin, Dade Behring) and CaCl2.Results: A total sample size of 160 patients (ages ranging 2 weeks to 18 years) who had a workup for a bleeding disorder that included Von Willebrand studies and TEG were reviewed. Of these 160 patients, 75 patients had a VWF:RCo <50 IU/dL. These patients were categorized into two categories for the purposes of this study: Low VWF:RCo (30-50 IU/dL) and Abnormal VWF:RCo (<30 IU/dL). The clinical and lab characteristics of patients studied are presented in Table 1, including FVIII, platelet count, and fibrinogen levels. Fibrinogen levels on 15 patients with VWF:RCo <50 IU/dL were not reported, and are not included in the means as noted in Table 1. The TEG parameter, K-Time, (time for increase in amplitude from 2mm to 20mm representing the dynamics of clot formation, normal <2min) was determined abnormal for values greater than 2.2 (>10% above the normal). Of the patients with VWF:RCo <30 IU/dL, 23/30 (77%) had an abnormal K-Time of >2.2, with a p-value of ≤0.001 (Table 2); whereas patients with low VWF:RCo 30-50 IU/dL, only 13/45 (29%) had an abnormal K-Time of >2.2, which was not statistically significant. A ROC curve for patients with VWF:RCo <30 IU/dL and abnormal K-Time showed an area under the curve of 0.675, with a p-value of 0.003 (Fig 1). An analysis of the MRTG showed a mean of 9.45 in patients with VWF:RCo <30 IU/dL and a mean of 11.26 in patients with normal VWF:RCo, which was statistically significant (p=0.05). There was a moderate correlation (0.33) seen between patients with abnormal K-Time and FVIII, however, when analyzing regression data the correlation only accounts for 11% of the explained variance in abnormal K-Time values. Patients with abnormal K-Time and abnormal fibrinogen levels and platelet counts were also compared, and showed no significant correlation, indicating that these were not the determinants that influenced the K time.Conclusions: The TF activated TEG reflects impaired clot formation in patients with VWF <30% as reflected by the prolongation in the K-Time and the low MRTG. The TEG unlike VWF:RCo can be done in real time and results are available to the clinician within an hour. This will definitely be beneficial in acute situations like evaluation of and management of acute bleeding in patients with acquired deficiencies of VWF and may play an important role in the surgical management of patients with VWD. It will also help physicians monitor response to treatment, frequency of treatment, and the need for prophylactic dosing in patients with VWD. The next step will be to evaluate if this difference is also seen with Kaolin, which is the standard TEG assay. We anticipate that Kaolin being a stronger agonist may make the test less sensitive to VWF related changes. [Display omitted] DisclosuresChitlur:Novo Nordisk: Consultancy; Bayer Pharmaceuticals: Honoraria; Baxalta: Honoraria; Biogen Idec: Honoraria; Pfizer: Honoraria.

Validation Study of the Composite Score to Identify Von Willebrand Disease in Children.

The diagnosis of type 1 von Willebrand disease (VWD) presents a diagnostic challenge in children. In fact, 25% or more of children with VWD may be diagnosed only after they experience postoperative bleeding. We previously described a 4-variable composite score that has 92.5% sensitivity and 95% specificity for diagnosing VWD in children with known VWD when 2 of 4 criteria are positive: (1) Tosetto bleeding score ≥ 1; (2) family history of VWD; (3) personal history of iron deficiency anemia; and/or (4) positive James early bleeding score. The purpose of this study was to prospectively validate a composite score of ≥ 2 for identifying children with VWD. Children without a previously diagnosed bleeding disorder presenting for hematology evaluation were enrolled. Sensitivity, specificity, positive, and negative predictive value of the composite score was determined. A total of 193 subjects were enrolled from 12 participating centers were included in the analysis. Forty-seven children had type 1 VWD, including 11 with von Willebrand Ristocetin Cofactor (VWF):RCo < 30 IU/dL, 14 subjects with a VWF:RCo 30 to 39 IU/dL, and 22 with a VWF:RCo 40 to 49 IU/dL. Including all 4 variables, a composite score of ≥ 2 had a sensitivity of 63.6% to 76.0%, specificity of 33.5% to 35.1%, negative predictive value of 76.9% to 93.8%, and positive predictive value of 5.5% to 25%. The negative predictive value of the composite score was robust, especially at lower VWF:RCo suggesting that VWD testing could be eliminated in nearly a third of children referred for VWD testing.

The Platelet Function Analyser (PFA)-100 and von Willebrand disease: a story well over 16 years in the making.

The Platelet Function Analyser (PFA)-100 and von Willebrand disease: a story well over 16 years in the making.

Changes in von Willebrand factor level and von Willebrand activity with age in type 1 von Willebrand disease.

C). VWF multimer analysis was normal in 28/31 cases performed. Mean age at diagnosis was 33 (range 16-60 years), and duration of follow-up ranged from 5 to 26 years (mean 11 years). Patients had 2-10 time points of VWD testing (mean of 5.2). The mean VWF:Ag, VWF:RCo and C at time of diagnosis were 0.44 IU mL(-1) 0.34 IU mL(-1) and 0.75 IU mL(-1) . At last follow-up, the mean VWF:Ag, VWF:RCo and C were significantly increased to 0.71 IU L(-1) , 0.56 IU mL(-1) and 0.90 IU mL(-1) (P ≤ 0.001, <0.001, and 0.0081 respectively). Here 18/31 patients had VWF:Ag, VWF:RCo and C levels that increased into the normal range. The rate of change in VWF:Ag, VWF:RCo and FVIII was 0.30 IU mL(-1) (0.21-0.39, CI 95%, P < 0.0001), 0.20 IU mL(-1) per decade (0.13-0.27, CI 95%, P = 0.0001) and 0.20 IU mL(-1) (0.11-0.29, CI 95%, P = 0.0011). Patients with type 1 VWD experience age-related increases to VWF:Ag and VWF:RCo which can result in normalization of VWF levels. Further studies are required to determine if the bleeding phenotype resolves with the increases in VWF:Ag and VWF:RCo levels.

Technological advances in diagnostic testing for von Willebrand disease: new approaches and challenges.

Diagnostic tests for von Willebrand disease (VWD) are important for the assessment of VWD, which is a commonly encountered bleeding disorder worldwide. Technical innovations have been applied to improve the precision and lower limit of detection of von Willebrand factor (VWF) assays, including the ristocetin cofactor activity assay (VWF:RCo) that uses the antibiotic ristocetin to induce plasma VWF binding to glycoprotein (GP) IbIXV on target platelets. VWF-collagen-binding assays, depending on the type of collagen used, can improve the detection of forms of VWD with high molecular weight VWF multimer loss, although the best method is debatable. A number of innovations have been applied to VWF:RCo (which is commonly performed on an aggregometer), including replacing the target platelets with immobilized GPIbα, and quantification by an enzyme-linked immunosorbent assay (ELISA), immunoturbidimetric, or chemiluminescent end-point. Some common polymorphisms in the VWF gene that do not cause bleeding are associated with falsely low VWF activity by ristocetin-dependent methods. To overcome the need for ristocetin, some new VWF activity assays use gain-of-function GPIbα mutants that bind VWF without the need for ristocetin, with an improved precision and lower limit of detection than measuring VWF:RCo by aggregometry. ELISA of VWF binding to mutated GPIbα shows promise as a method to identify gain-of-function defects from type 2B VWD. The performance characteristics of many new VWF activity assays suggest that the detection of VWD, and monitoring of VWD therapy, by clinical laboratories could be improved through adopting newer generation VWF assays.

Perils, Problems, and Progress in Laboratory Diagnosis of von Willebrand Disease

Diagnosis of von Willebrand disease (VWD) merits consideration of personal and family history of bleeding symptoms along with confirmatory laboratory testing. As the latter yields quantifiable results, overreliance on a laboratory diagnosis may occur. However, existing tests for VWD contain potential sources for error. Both intrinsic and extrinsic factors affecting these assays can contribute to either falsely normal or falsely abnormal results. This article will discuss the present available assays as well as new developments in diagnostic testing. A clear understanding of the limitations of VWD testing is helpful for ensuring the correct diagnosis of affected patients.

Validation Study Of The Composite Score To Identify Von Willebrand Disease in Children

BackgroundThe diagnosis of type 1 von Willebrand disease (VWD), the most common inherited bleeding disorder, presents a diagnostic challenge in children. In the absence of a prior hemostatic challenge in children, a VWD diagnosis may be missed by the Tosetto bleeding score, as it is based, in part, on bleeding symptoms, i.e. with surgery, menses, or childbirth, that may not occur until after childhood. In fact, 25% or more of children with VWD may be diagnosed only after they experience postoperative bleeding. Other bleeding scores, such as the James score, which assesses early life bleeding, e.g. cephalohematoma, macroscopic hematuria, and umbilical stump, post-circumcision, post-venipuncture bleeding, has been validated in children with VWD, but early life bleeding events are uncommon in VWD. We previously described a 4-variable Composite Score that has 92.5% sensitivity and 95% specificity for diagnosing VWD in children < 11 years of age, when at least two of four criteria are positive: 1) Tosetto bleeding score ≥ 1; 2) family history of VWD or bleeding; 3) personal history of iron deficiency anemia; and/or 4) positive James early bleeding score. The purpose of this study was to validate a Composite Score of ≥ 2 (i.e. at least 2 of 4 variables positive) for identifying children with VWD. MethodsWe designed and conducted a prospective validation study to determine the accuracy of the Composite Score for identification of children (<11 years of age) with VWD. Following parental consent, children without a prior history of VWD undergoing VWD testing were enrolled, and survey data were collected including personal bleeding history (including Tosetto and James scores), history of iron deficiency anemia, and family history of VWD or bleeding. Results of VWD testing were collected, including VWF:RCo, VWF:Ag, and VIII:C in addition to VWF multimers, if performed. Survey data and bleeding scores were compared between those with and without VWD, defined by VWF:RCo <30 (NIH 2008 criteria), or <40, or <50 IU/dL. Prior to study, it was determined that a Composite Score ≥ 2 would have 90% power to identify VWD defined by VWF:RCo <0.30 IU/dL, and 80% power to identify VWD by VWF:RCo <40 IU/dL. Categorical variables were compared using chi-square or Fischer's exact test. Continuous variables were compared using two-sample t-test or Wilcoxon's rank sum test. Sensitivity, specificity, positive and negative predictive value of the Composite Score of ≥ 2 to diagnose VWD were determined. ResultsA total of 195 subjects were enrolled from 12 participating centers from 2010-2013, of whom 193 patients with no missing data were included in the analysis. A total of 81 (41.9%) children had type 1 VWD, including 11 (5.7%) with VWF:RCo <30 IU/dL (Group A), 24 (12.4%) with VWF:RCo <40 IU/dL (Group B), and 46 (23.8%) with VWF:RCo <50 IU/dL (Group C); the remainder with VWF:RCo ≥50 IU/dL were unaffected. There were no significant demographic differences between groups: overall 54.9% were male, 82.4% were Caucasian, and the mean age was 4.7 years. A composite score of ≥ 2 had 63.6%, 75%, and 67.4% sensitivity in diagnosing type 1 VWD in Groups A, B, and C respectively; 33.5%, 34.9%, and 34% specificity in diagnosing type 1 VWD, respectively; and 93.8%, 90.8%, and 76.9% negative predictive value, respectively. Based on the performance of the composite, with VWD being defined by a VWF:RCo of <30, <40, or <50 IU/dL, 31.6%, 30.6%, and 25.9% of subjects, respectively, had a negative composite score and therefore would not have required VWD testing. DiscussionThis is the first study to prospectively validate the Composite Score to identify children with type 1 VWD. Although the sensitivity and specificity of the Composite Score were lower than in the original study (above), the latter included only VWD subjects, while this validation study included all, VWD and non-VWD, undergoing testing. The negative predictive value of the Composite Score was robust, especially at lower VWF:RCo, and indicates that VWD testing could be eliminated in nearly a third of children referred for VWD testing. Given the difficulty of diagnosing VWD in children, the Composite Score may be an important screening tool for diagnosis of type 1 VWD in children. Disclosures:Kerlin:Bayer Healthcare US: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; NovoNordisk A/S: Consultancy, Honoraria.

VWF Interaction With Type IV Collagen Is Mediated Through Critical VWF A1 Domain Residues

Von Willebrand factor (VWF) plays a key role in coagulation by tethering platelets to injured subendothelium via binding sites for platelet glycoprotein Ib and collagen. The binding sites for types I and III collagen in the VWF A3 domain are well characterized, and defects in this region have been implicated in von Willebrand disease (VWD). Additional collagens present in the vasculature may also be involved in interactions with VWF. A VWF A1 sequence variation, p.R1399H, has been associated with decreased binding to type VI collagen, but the clinical significance of this observation remains unclear. Type IV collagen is a common component of the basement membrane and as such may be an important ligand for VWF. While some VWD testing utilizes types I or III collagen, current clinical testing does not include collagen IV or VI.To characterize the role of the VWF A1 domain in VWF-type IV collagen interactions, we generated several A1 domain variant human and/or murine recombinant VWF (rVWF) constructs including R1399H and several type 2M VWD variants localized to the same region (S1387I, Q1402P, and an 11 amino acid deletion mutant encompassing amino acids 1392-1402). These constructs were then expressed in HEK 293T cells. To further assess the role of the A1 domain, scanning alanine mutagenesis (SAM) of residues 1387 through 1412 was conducted. VWF antigen levels (VWF:Ag), collagen binding with type III (VWF:CB3), IV (VWF:CB4), or VI (VWF:CB6) collagen were determined, and multimer distribution was assessed for all recombinant VWF variants.The role of R1399H in the context of human rVWF was characterized initially. Although VWF:Ag, VWF:CB3, and multimer distribution were normal for R1399H compared to wild-type (WT VWF), VWF:CB4 was undetectable. To examine this effect in a mouse model, the R1399H variant was expressed in the context of murine rVWF and collagen binding was determined. Similar to the human variant, murine R1399H rVWF demonstrated significantly reduced binding to murine type IV collagen, at only 7% of the binding seen with WT murine rVWF. In order to examine the behavior of R1399H under shear conditions, either WT or R1399H murine rVWF DNA was hydrodynamically injected into the tail veins of VWF -/- mice to induce expression of the proteins; blood was drawn from the vena cava 24 hours later and then examined on the VenaFlux flow apparatus. VWF expression levels and multimer distribution were similar for the R1399H- and WT-injected mice. Under static conditions, the murine plasma-derived R1399H demonstrated decreased VWF:CB4, at only 16% of the levels seen with WT VWF. No defect was seen in VWF:CB3. Furthermore, when binding to type IV collagen was assessed under flow conditions by VenaFlux, platelet adhesion was significantly decreased in mice expressing R1399H VWF as compared to mice expressing WT VWF.When examining other A1 domain variants, Q1402P and del1392-1402 demonstrated absent VWF:CB4 while S1387I demonstrated a significant reduction in VWF:CB4 compared to WT VWF. All SAM VWF A1 domain variants demonstrated normal expression, multimerization, and VWF:CB3. However, type IV collagen binding was absent for R1392A, R1395A, R1399A, and K1406A and was reduced to less than 50% of WT VWF for Q1402A, K1405A, and K1407A. These residues map to an outside face of the VWF A1 domain crystal structure, and are likely the critical residues for VWF binding to type IV collagen.Taken together, these data demonstrate that the type IV collagen binding site localizes to a specific region of the VWF A1 domain. Mutations in this region of VWF may be clinically significant due to a defect in the ability of VWF to attract platelets to exposed type IV collagen which may contribute to bleeding symptoms seen in VWD. Disclosures:No relevant conflicts of interest to declare.

Ex vivo effects of low-dose rivaroxaban on specific coagulation assays and coagulation factor activities in patients under real life conditions

Global coagulation assays display variable effects at different concentrations of rivaroxaban. The aim of this study is to quantify the ex vivo effects of low-dose rivaroxaban on thrombophilia screening assays and coagulation factor activities based on the administration time, and to show how to mask possible interferences. Plasma samples from 40 patients receiving rivaroxaban 10 mg daily were investigated to measure activities of clotting factor II, V, VII, VIII, IX, XI, XII and XIII; protein C- and protein S-levels; lupus anticoagulants; anticardiolipin IgG and IgM; D-dimer, heparin-platelet factor 4 (HPF4) antibodies and screening tests for von Willebrand disease (VWD). Two hours after rivaroxaban administration, the activities of clotting factors were significantly decreased to different extents, except for factor XIII. Dilution of plasma samples resulted in neutralisation of these interferences. The chromogenic protein C activity assay was not affected by rivaroxaban. Depending on the timing of tablet intake in relation to blood sampling protein S activity was measured falsely high when a clotting assay was used. False-positive results for lupus anticoagulants were observed depending on the assay system used and the administration time of rivaroxaban. ELISA-based assays such as anticardiolipin IgG and IgM, D-dimer, HPF4-antibodies and the turbidimetric assays for VWD were not affected by rivaroxaban. Specific haemostasis clotting tests should be performed directly prior to rivaroxaban intake. Assay optimisation in the presence of rivaroxaban can be achieved by plasma dilution. Immunologic assays are not influenced by rivaroxaban, while chromogenic assays can be used, when they do not depend on factor Xa.

Total-Thrombus-Formation Analysis System Using a Microchip Flow Chamber Reflects Bleeding Severity of Patients with Type 1 Von Willebrand Disease.

Abstract 2237 Introduction:von Willebrand disease (VWD) has clinically heterogeneous phenotype. Routine measurements of von Willebrand factor (VWF) antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo) and FVIII activity (FVIII:C) do not always reflect clinical severity, especially in type 1 VWD. These assays evaluate VWF function under non-physiological static condition - lacking blood flow. We had reported that a new microchip flow chamber system, total-thrombus-formation analysis system (T-TAS®, Fujimori Kogyo, Tokyo) would be a clinically useful flow assay for VWD (ASH 2011). In this study, we extended this study for application to evaluation and hemostatic monitoring for type 1 VWD. Methods:Citrated or hirudin-added blood from 15 patients with type 1 VWD was utilized. Re-calcified citrated blood added corn trypsin inhibitor was injected to a microchip in T-TAS at a constant flow rate (240 s−1), which flow surface was coated by collagen and tissue factor (AR chip). Hirudin-added blood was injected to a microchip in T-TAS at higher shear rate (1,000 s−1) which surface was coated by collagen (PL chip). Flow pressure curve was visualized and time until reach to 10 kPa (T10) was evaluated. AR chip promoted thrombus formation by both platelet aggregation and fibrin generation, whilst PL chip promoted thrombus formation by platelet alone. Standard laboratory tests for VWD were also performed. Clinical severities of VWD patients were evaluated by using a quantitative bleeding score (BS, from −3 without any symptoms to +45 with all major symptoms) previously reported by Tosetto (JTH, 2006). Results:Fifteen patients with type 1 VWD showed low levels of VWF:Ag [median 14% (range 1.3–51%)], VWF:RCo [8% (1.6–32%)], and FVIII:C [31% (3.0–68%)]. T10 in AR chip or PL chip was 17.7 min (11->30) or 7.1 min (3.3->10) [normal control (n=20); 12.2 min (8.6–16.6) or 3.5 min (2.4–6.6), respectively], showing delayed thrombus formation in type 1 VWD. Correlations between VWF:Ag and VWF:RCo (r2=0.80, p<0.01) or VWF:Ag and FVIII:C (r2=0.74, p<0.01) were significant in Spearman’s correlation test. Correlation of BS [5 (0–11)] to VWF:RCo was not significant (r2=0.27, p=0.05), whilst, that of BS to T10 in PL chip of T-TAS showed more significant (r2=0.62, p<0.01) but not T10 in AR chip (r2=0.04, p=0.4). Interestingly, when focused on the patients whose VWF:RCo were less than 20% [n=10, 5.8% (1.6–13%)], correlation of VWF:RCo to BS was worse (r2=0.02). However, T10 in PL chip showed greater correlation to BS (r2=0.62, p<0.01; Figure 1). Patients who were treated with desmopressin infusion or replacement therapy showed the improvement of T-TAS parameters within normal range. Conclusion:Traditional standard assays did not always reflect the clinical phenotype of patients with VWD. In contrast, T-TAS parameters in PL chip showed good correlations to BS, indicating its usefulness for distinguishing clinical phenotype and for monitoring of treatment for patients with type 1 VWD. [Display omitted] Disclosures:Hosokawa:Fujimori Kogyo: Employment.

Critical von Willebrand factor A1 domain residues influence type VI collagen binding

von Willebrand factor (VWF) binds to subendothelial collagen at sites of vascular injury. Laboratory testing for von Willebrand disease (VWD), however, does not always include collagen binding assays (VWF:CB) and standard VWF:CB assays use type I and/or type III collagen rather than type VI collagen. We report here on several mutations that exclusively alter binding to type VI collagen. Healthy controls and index cases from the Zimmerman Program for the Molecular and Clinical Biology of VWD were analyzed for VWF antigen (VWF:Ag), VWF ristocetin cofactor activity and VWF:CB with types I, III and VI collagen. VWF gene sequencing was performed for all subjects. Two healthy controls and one type 1 VWD subject were heterozygous for an A1 domain sequence variation, R1399H, and displayed a selective decreased binding to type VI collagen but not types I and III. Expression of recombinant 1399H VWF resulted in absent binding to type VI collagen. Two other VWF A1 domain mutations, S1387I and Q1402P, displayed diminished binding to type VI collagen. An 11 amino acid deletion in the A1 domain also abrogated binding to type VI collagen. VWF:CB may be useful in diagnosis of VWD, as a decreased VWF:CB/VWF:Ag ratio may reflect specific loss of collagen binding ability. Mutations that exclusively affect type VI collagen binding may be associated with bleeding, yet missed by current VWF testing.

Can von Willebrand Disease Be Investigated on Combined Hormonal Contraceptives?

Study ObjectiveTo review the medical literature and determine whether testing for von Willebrand disease can be performed in adolescents using combined hormonal contraceptives (CHC). DesignLiterature review where Embase and Medline were searched using the key words “von Willebrand factor,” “von Willebrand disease,” “contraceptive agents,” and “menorrhagia.” Articles were included in the review if they were controlled trials comparing a current form of CHC versus a control group and testing for von Willebrand factor was performed and reported. Main Outcome MeasuresImpact of combined hormonal contraceptives on von Willebrand factor antigen. Secondary outcomes included effects on Factor VIII and von Willebrand factor activity known as the Ristocetin cofactor. ResultsSeven articles met inclusion criteria. All seven assessed VWF Ag with CHC use; six of the seven demonstrated no change and one, Gevers Leuven, demonstrated a significant decrease after CHC use. Three studies measured Factor VIII and showed no significant change with use. One study by Kadir assessed the Ristocetin cofactor and also failed to demonstrate change on CHC. ConclusionFrom the literature it appears that adolescents, assessed for menorrhagia and already on combined hormonal contraceptives, can be tested for von Willebrand disease if this diagnosis is suspected by the physician. By allowing adolescents to remain on combined hormonal contraceptives during testing, one avoids the risk of recurrent and severe menorrhagia which could result in admission and transfusion.

Laboratory Diagnosis and Management ofvon Willebrand Disease in South Africa

Patients with Von Willebrand disease (VWD) in South Africa are cared for in 17 Hemophilia Treatment Centers. The exact prevalence of the disease is uncertain, but 539 patients are annotated in registries. VWD patients are mostly diagnosed in the five largest academic centers, and the classification of the subtypes is performed by one of these, the VWD testing facility. An algorithm is used for the diagnosis of VWD. The distribution of subtypes diagnosed by the VWD reference center is 38%, 58%, and 4% for type 1, 2, and 3, respectively, and ~15% of plasma samples received are rejected due to poor storage and transport conditions. A novel single nucleotide polymorphism has been found in an African patient with type 2B VWD. From the type 1 VWD patients who were diagnosed by the VWD testing facility, 45% seem to have an increased VWF clearance phenotype with a propeptide-to-antigen ratio of 1.9 ± 0.3. VWD patients are treated with desmopressin, factor (F)VIII/VWF concentrate (Haemosolvate FVIII; National Bioproducts Institute, Durban, South Africa), and tranexamic acid. Haemosolvate FVIII contains a VWF antigen concentration of 167 ± 27 IU/mL, a ristocetin cofactor activity of 100 ± 29 IU/mL, a collagen binding activity of 99 ± 29 IU/mL, normal VWF multimers, and a FVIII concentration of 50 IU/mL. Not all patients with VWD are currently classified, and many VWD patients in South Africa are probably undiagnosed.

Diagnosis and Management ofvon Willebrand Disease in Australia

Von Willebrand disease (VWD) is the most common inherited bleeding disorder and arises from deficiencies and/or defects in the plasma protein Von Willebrand factor (VWF). VWD is classified into six different types, with type 1 identifying a (partial) quantitative deficiency of VWF, type 3 defining a (virtual) total deficiency of VWF, and type 2 identifying four separate types (2A, 2B, 2M, and 2N) characterized by qualitative defects. The classification is based on phenotypic assays including factor VIII coagulant, VWF antigen, and VWF activity, primarily by ristocetin cofactor and collagen binding, as supplemented by additional testing. In Australia, >30 pathology-based laboratories perform VWD testing, and tests and test panels reflect a wide variety of practice. In our own referral laboratory, diagnosis is a staged process reflecting a combination of clinical and laboratory findings with a large panel of tests. We also use data from desmopressin trials to assist in VWD type assignment. The current report presents an overview of the VWD diagnostic process as applied within Australia, includes summary data from the Australian Bleeding Disorders Registry, and provides specific details of the diagnostic and management practice undertaken in our reference laboratory, which also maintains a local bleeding disorders database. This database currently contains 4070 entries, including 1832 suspected or confirmed cases of VWD. Excluding 311 as yet unclassified cases, 1254 cases (82.4%) would define (potential) quantitative deficiencies of VWF ("low VWF" or type 1 VWD), 241 (15.8%) qualitative defects (type 2 VWD), and 23 (1.5%) type 3 VWD. Most of the quantitative defects reflect only mild loss of VWF, and <15% of total cases would be identified to have VWF levels <35 U/dL. Most cases of type 2 remain unclassified (34.9%) because available data are limited. Type 2A and 2M VWD represent the most common qualitative defects, representing 22.8% and 22.2% of defined type 2 VWD cases. Type 2B and 2N reflect 8.3% and 12.9%, respectively, of type 2 VWD cases.

Cost-Utility Analysis of von Willebrand Disease Screening in Adolescents with Menorrhagia

To construct a decision analysis model to evaluate the cost utility of von Willebrand disease (VWD) testing in adolescents with menorrhagia. A 20-year Markov decision analytic model was constructed to evaluate the cost utility of two strategies: testing or not testing for VWD. The model includes probabilities of remaining well, suffering an acute menorrhagia bleeding event, surgical complications, oral contraceptive pill complications, or dying. Probabilities, costs, and utilities were estimated from published literature. The prevalence of type 1 VWD in adolescent females with menorrhagia was estimated at 13%. The cost of testing adolescents with menorrhagia for VWD was $1790, versus $1251 for not testing for VWD. The effectiveness of not testing in quality-adjusted life-years (QALYs) gained (14.237 QALYs) was similar to the VWD testing strategy (14.246 QALYs). Compared with not testing for VWD, screening for VWD had an incremental cost-effectiveness ratio of $62,791 per QALY, a value typically considered economically reasonable. In adolescents with menorrhagia, testing for VWD before the initiation of oral contraceptives is cost-effective.

Genetic testing in von Willebrand disease: reply to rebuttal

See also Mannucci PM. Genetic testing in von Willebrand disease: a rebuttal. This issue, p 860.

Cost-Utility Analysis of Von Willebrand Disease Screening in Adolescents with Menorrhagia.

Abstract 2476Poster Board II-453 Introduction:It is unknown whether it is cost effective to screen adolescents with menorrhagia for von Willebrand disease (VWD) prior to starting oral contraceptive pills (OCPs). Because OCPs can mask a diagnosis of VWD, a woman is at risk for potential future traumatic or surgical bleeding, once OCPs are stopped. We, therefore, designed a decision analysis model to evaluate the cost utility of VWD testing in adolescents with menorrhagia. Methods:A 20-year Markov decision analytic model was constructed to evaluate the cost-utility of two strategies: testing or not testing for VWD. The hypothetical patient is a 15-year-old female presenting with menorrhagia and a decision regarding VWD testing is made. The model includes probabilities of remaining well, suffering an acute menorrhagia bleeding event, surgical complications, OCP complications or dying. Probabilities, costs and utilities were estimated from published literature. The prevalence of type 1 VWD in adolescent females with menorrhagia was estimated to be 13%. We assumed all patients were treated with OCPs regardless of whether hemostatic testing was performed. All patients were assumed to have mild type I VWD, the most common subtype, and all were responsive to intravenous DDAVP. Probabilities of surgical complications in patients with and without VWD were based on adenotonsillectomy hemorrhage rates in children. The probability of acute menorrhagia bleeding necessitating hospitalization or red blood cell transfusion was estimated to be 1% in an adolescent with VWD and 0% in an adolescent with normal hemostatic testing. The sole complication from OCPs incorporated into this decision analytic model was acute deep vein thrombosis. Results:The cost of testing adolescents with menorrhagia for VWD was $1,638, vs. $1,251 for not testing for VWD. The effectiveness of not testing in quality-adjusted life-year (QALY) gained (14.237 QALY) was similar to the VWD testing strategy (14.246 QALY). Compared to not testing for VWD, screening for VWD had an incremental cost-effectiveness ratio (ICER) per QALY of $45,061/QALY, a value typically considered economically reasonable. In one-way sensitivity analysis, the outcome and ICER were most sensitive to the probabilities of an acute bleeding event, surgical complication, and the prevalence of VWD. Monte Carlo probabilistic sensitivity analysis, substituting input distributions rather than a single point estimate for the probabilities, costs and utilities, was also performed. Under this assumption, testing for VWD is a cost-effective strategy approximately 60% of the time when the cost-effectiveness threshold is $100,000/QALY. Conclusions:It is cost effective to perform VWD testing in adolescents presenting with menorrhagia prior to initiating OCPs. This is related to avoiding cost from postoperative and other bleeding when the diagnosis is masked by OCPs. Disclosures:No relevant conflicts of interest to declare.

A Novel VWF A3 Domain Mutation with Absent Binding to Type I and Type III Collagen.

Abstract 541Von Willebrand factor (VWF) plays a key role in coagulation by tethering platelets to injured subendothelium through binding sites for collagen and platelet GPIb. Laboratory testing for von Willebrand disease (VWD) utilizes various assays of VWF protein and function, but collagen binding (VWF:CB) has not always been performed. Samples obtained from a large, multi-center US study of von Willebrand disease (VWD), the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD), were evaluated for VWF:CB. Whole blood was collected for DNA sequencing and plasma samples were collected for VWF testing. VWF antigen (VWF:Ag), ristocetin cofactor activity (VWF:RCo), and VWF:CB were performed in the BloodCenter of Wisconsin hemostasis laboratory. A normal range for the VWF:CB was established using 233 healthy controls enrolled in the ZPMCB-VWD. The mean VWF:Ag was 105 IU/dL and the mean VWF:CB was 123 U/dL, with a mean VWF:CB/VWF:Ag ratio of 1.19. This yields a normal range of 0.86-1.51 (mean ± 2 SD). No sequence variations in the VWF A3 domain affecting VWF:CB were observed in the healthy controls. Collagen binding was then examined in subjects enrolled with a diagnosis of VWD. The index case in one family was noted to have a low VWF:CB/VWF:Ag ratio of 0.54. The VWF:Ag was 41 IU/dL, VWF:RCo was 44 IU/dL, VWF:CB was 22 U/dL, and multimer distribution was normal. Decreased VWF:CB/VWF:Ag was found in the father and sister of the index case, while the mother had normal VWF:CB/VWF:Ag. The index case initially presented with epistaxis and easy bruising. Her bleeding score was 8, as determined by the revised European Union questionnaire. DNA sequencing of exons 28-32 was performed and revealed a heterozygous mutation (5356C>G) predicting replacement of wild-type histidine 1786 by aspartic acid in all affected family members. This amino acid is located in a critical face of the A3 loop. The 1786D mutation was cloned into a full-length VWF expression vector and expressed, as was a previously reported collagen-binding mutation, 1731T. Collagen binding assays were performed using type I human placental collagen at 5 μg/mL or type III human placental collagen at 1 μg/mL to capture either plasma or recombinant VWF (rVWF). Both mutations had normal expression, at 97% and 87% of the expression seen with wild-type VWF, respectively. Multimer distribution was also normal, including presence of high molecular weight multimers. Plasma from family members heterozygous for the H1786D mutation showed a mean VWF:CB/VWF:Ag ratio of 0.23 ± 0.05 (mean ± SD) for type I collagen and 0.50 ± 0.11 for type III collagen. When the rVWF constructs were examined, the novel 1786D mutation showed essentially no binding to either type I or type III collagen, at <1% of wild-type rVWF. In contrast, the previously reported collagen binding mutation 1731T showed VWF:CB/VWF:Ag ratios of 45% for type I and 50% for type III collagen when compared to wild-type rVWF. As the proband had a minimally decreased VWF:Ag, we then examined collagen binding in ZPMCB-VWD subjects with VWF:Ag between 30 and 50 IU/dL. The mean VWF:CB/VWF:Ag ratio in this group was 1.02, with a range of 0.62 to 1.28. These results, taken together with the data in the healthy controls, suggest that decreased VWF:CB/VWF:Ag ratios may identify a specific defect in VWF function. In particular, the H1786D mutation appears to result in a complete loss of VWF's ability to bind either type I or type III collagen. The subjects with this mutation were all heterozygous, which may account for reduction, rather than absence, of in vitro VWF:CB. The VWF:CB may be useful in diagnosis of VWD, and a decreased VWF:CB/VWF:Ag ratio in the face of normal VWF multimer distribution may reflect specific loss of collagen binding ability leading to bleeding symptoms. Disclosures:Montgomery:GTI Diagnostics: Consultancy; Baxter: Consultancy; AstraZeneca: Consultancy; Bayer: Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees.

Genetic testing for von Willebrand disease: the case against.

Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, Westmead, NSW, AustraliaTo cite this article: Favaloro EJ. Genetic testing for von Willebrand disease: the case against. J Thromb Haemost 2010; 8: 6–12.See also Peake IR, Goodeve AC. Genetic testing for von Willebrand disease: the case for. This issue, pp 13–6.