vWD Patients Research Articles

Bleeding score in type 1 von Willebrand disease patients using the condensed MCMDM-1 vWD validated questionnaire.

Assessment of the severity of bleeding symptom has led to the evolution of bleeding assessment tools which are now validated. To administer the condensed molecular and clinical markers for the diagnosis and management of type 1 von Willebrand disease VWD (MCMDM-1 vWD) questionnaire to the Omani type 1 vWD patients and correlate it with the laboratory parameters. Patients and controls were personally interviewed and the condensed MCMDM-1 vWD questionnaire administered by a single investigator. Bleeding score (BS) was calculated, based on the presence or absence of the bleeding symptoms according to a standard validated questionnaire in both the patients and the controls. The median age of the patient cohort was 27 (range, 7-49) years with 60.87% of females. The median time to administer condensed MCMDM-1 BS questionnaire was 11minutes (interquartile range-IQR;7,16). Overall, bleeding from the oral cavity was the most predominant symptom (63%). The median BS was 5 (IQR;1,8) although individual scores ranged between 0 and 29. However, there was no statistically significant difference in BS between genders (males: median 4; IQR 1,6 and females: median 5, IQR 1,10) (P>.05, Kruskal-Wallis test) The Spearman's correlation value of BS was weak with FVIII:C levels and von Willebrand Ristocetin co-factor activity; very weak with von Willebrand Antigen level, and moderate with vonWillebrand Collagen Binding activity being -0.29, -0.28, -0.14 and -0.43, respectively. The BS reflects the severity of bleeding among the vWD patients. Although the BS was abnormal, it did not correlate significantly with the surrogate laboratory parameters [P>.05].

Perioperative Management of Von Willebrand Disease, a Systematic Review and Meta-Analysis

Background: von Willebrand disease (vWD) is the most common inherited bleeding disorder characterized by quantitative or qualitative defect of von Willebrand factor (vWF) production. Patients with vWD are at high risk of bleeding during surgery. We aimed to evaluate the efficacy and safety of factor VIII (FVIII)/vWF concentrates and DDAVP for bleeding prevention in vWD patients who undergo surgery or an invasive procedure.Methods:The electronic searches were performed in MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials databases. The articles published from inception to January 2016 were eligible for inclusion in this review. Studies will be included if they are observational studies or clinical trials of pediatric or adult patients who were laboratory confirmed vWD (type 1 or type 2 or type 3). We excluded case report or case series with less than 5 patients, acquired vWD and incomplete outcome reports. The primary outcome was the efficacy of hemostatic control as defined by studies. We calculated pooled incidence of the patient who achieve good or excellent hemostatic control with corresponding 95% confidence interval using single proportion meta-analysis. The secondary outcomes were adverse outcomes from FVIII/vWF and DDAVP.Results: After screening, there were 54 studies included in the meta-analysis. Forty-five studies investigated the efficacy of vWF/FVIII. Pooled analysis revealed that 98.4% (95% CI; 97.8-99.1%, I2=45.5%) of FVIII/vWF treated patients achieved excellent or good hemostasis. Subgroup analysis according to type of surgery revealed that excellent or good hemostasis was 99.4% (95%CI; 98.4-100%, I2=0%) in major surgery and 99.0% (95%CI; 98.2-99.9%, I2=0%) in minor surgery, Table 1. Adverse effects were observed in 0.2% (95% CI; 0-0.4, I2=85.1%) patients who received FVIII/vWF. The reported adverse effects from FVIII/vWF concentrates included phlebitis, shortness of breath, chest tightness, urticaria, pruritus, chill, malaise, fever, paresthesia, transient elevation of liver function test, nausea, vomiting, dizziness, headache and parvovirus B 19 seroconversion. Thromboembolism occurred in 0.17% of patients who received vWF/FIII. There were 26 studies investigating DDAVP during surgery. Hemostasis was excellent or good in 98.0% (95%CI; 97.0-99.0%, I2=26.3%. In dental procedure, 99.0% of patients receiving DDAVP (95%CI; 97.6-100%, I2=0%) achieved excellent or good hemostasis. Subgroup analysis showed that good/excellent hemostasis was found in 96.9% of pediatric patients (95%CI; 95.1-98.7%, I2=58.8%) and 96.9% (95%CI; 92.7-100%, I2= 0%) in adult patients, Table 2. Adverse effects from DDAVP were reported in 1.2% (95%CI; 0.4-2.1%, I2=95%). Hyponatremia of any level was observed in 16.1% (95%CI; 8.9-22.4%, I2=94.1%). The other reported adverse effects were headache, fatigue, rash, seizure (from hyponatremia), flushing, dizziness, peripheral edema, nausea and vomiting. No thromboembolic event was found in DDAVP treated patients.Conclusions: Both FVIII/vWF concentrates and DDAVP are effective and safe for bleeding prevention during surgery or an invasive procedure in vWD patients. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characterization of Bleeding in Hemophilia Carriers and Comparison to Women with Type 1 Von Willebrand Disease, Type 3 Von Willebrand Disease Obligate Carriers and Controls

Background: Hemophilia carriers report abnormal bleeding, even when factor VIII or IX levels are normal. Information comparing bleeding events between carriers and women with other inherited bleeding disorders is lacking. Purpose: The purpose of our study was to characterize bleeding in hemophilia carriers using the International Society on Thrombosis and Hemostasis Bleeding Assessment Tool (ISTH-BAT) and to compare it with bleeding in normal controls, women with Type 1 VWD and Type 3 VWD obligate carriers (OC). Method: This was a prospective, observational, cross-sectional study performed by members of GEHEP (Global Emerging HEmostasis Panel). Study participants were recruited from GEHEP members9 clinics in North America (Kingston, Canada, Detroit and Philadelphia, USA), Europe (Malaga, Spain; Milan, Italy; Munich, Germany; Oslo, Norway) and South Africa (Johannesburg). Potential participants were identified through local patient databases and approached during clinic visits. All participants signed informed consent. Hemophilia carriers were defined by a documented FVIII or FIX mutation and/or by an appropriate family history (daughter of a man with hemophilia or mother of two sons with hemophilia or mother of one son with hemophilia with at least one other affected male relative). Demographic information was collected using a CRF and the ISTH-BAT was completed for each participant by study personnel. Existing ISTH-BAT data for women with Type 1 VWD, Type 3 VWD OC and age-matched female controls were used for comparison. Results: A total of 329 participants were included in this study; 168 hemophilia carriers, 83 women with Type 1 VWD, 32 Type 3 VWD OC and 46 female normal controls. Hemophilia carriers and normal controls were similar in age (40.1 vs 41.6, p=0.445). The mean overall ISTH-BAT bleeding score (BS) was significantly higher in carriers than in controls (5.7 vs 2.48, p Conclusion: In summary, our study showed that hemophilia carriers report significantly more bleeding by overall ISTH-BAT BS than age-matched female controls. Carriers experience both mucocutaneous bleeding as well as musculoskeletal bleeding. They score higher for mucocutaneous bleeding when compared with controls and when compared with Type 3 VWD OC. Overall Type 1 VWD patients experience more severe mucocutaneous bleeding than hemophilia carriers. However, hemophilia carriers report more musculoskeletal bleeding in the form of hemarthrosis and hematomas than all other groups. A comparison of overall ISTH-BAT BS between groups shows that bleeding in women with Type 1 VWD > hemophilia carriers > Type 3 VWD OC > controls. Additional research into the underlying pathophysiology of this abnormal bleeding is a critical next step in understanding and determining how to appropriately manage these patients. Disclosures Bidlingmaier:Novo Nordisk: Honoraria; Sobi: Honoraria; Pfizer: Honoraria; Biotest: Honoraria; Baxalta: Honoraria; Bayer: Honoraria; CSL Behring: Honoraria, Research Funding. Mingot-Castellano:Amgen: Consultancy; Pfizer: Consultancy; Novo Nordisk: Consultancy, Research Funding; Baxalta: Consultancy, Research Funding; Novartis: Consultancy; Bayer: Consultancy, Research Funding. Chitlur:Novo Nordisk: Consultancy; Baxalta: Honoraria; Bayer: Honoraria; Biogen-Idec: Honoraria; Pfizer: Honoraria. Fogarty:Bayer Healthcare: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Baxter/Baxalta: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Biogen: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity9s Board of Directors or advisory committees; CSL Behring: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Novo Nordisk: Membership on an entity9s Board of Directors or advisory committees; Pfizer: Employment, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Spark Therapeutics: Research Funding. Cuker:T2 Biosystems: Research Funding; Genzyme: Consultancy; Biogen-Idec: Consultancy, Research Funding; Amgen: Consultancy; Stago: Consultancy. Mancuso:Bayer Healthcare: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Baxalta: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Consultancy, Speakers Bureau; Sobi/Biogen Idec: Consultancy, Speakers Bureau; Pfizer: Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Kedrion: Consultancy. Holme:Baxalta, now part of Shire: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Other: Investigator Clinical Studies. Mathew:Bayer: Employment. James:CSL Behring: Research Funding; Octapharma: Research Funding; Biogen: Consultancy; Basalt: Consultancy; Bayer: Research Funding.

A Novel Microfluidic Assay to Determine Von Willebrand Factor Levels and Bleeding Phenotypes in Patients with Von Willebrand Disease and Mucocutaneous Bleeding

BackgroundVon Willebrand's disease (VWD) is the most common inherited bleeding disorder. Conventional assays cannot accurately detect all abnormalities in von Willebrand factor (VWF)-platelet-collagen interactions or predict a bleeding phenotype in patients with VWD.ObjectiveWe hypothesized that a microfluidic assay (MFA) could complement current assays and better correlate with bleeding.MethodsPatients: Patients with VWD or mucocutaneous bleeding (MCB) are enrolled in an IRB-approved study. Bleeding scores (BS) are obtained via the Vincenza bleeding assessment tool. In this analysis, Type 1 VWD is defined by a VWF Antigen (VWF:Ag) level <40 IU/dL and a VWF activity (VWF:RCo)/VWF:Ag ratio >0.6, Type II VWD has a VWF:RCo/VWF:Ag ratio of <0.6, and Type III disease has VWF:Ag and VWF:RCo levels <10 IU/dL. MCB is defined as clinical mucocutaneous bleeding, a VWF:Ag level >40 IU/dL and no other laboratory evidence of a diagnosable bleeding diathesis.Standard Assays: Samples are assayed for platelet count, platelet aggregometry (agonists: collagen/ristocetin/ADP), VWF:Ag, VWF:RCo, Type III collagen binding (VWF:CBIII) and PFA-100 closure time (ADP and EPI cartridges).MFA: C ollagen (500 µg/mL) is patterned onto a glass substrate and whole blood is perfused at three distinct shear rates, low (150s-1), mid (750s-1), and high (1500s-1) for five minutes. Images of platelet adhesion are collected every 3 seconds at pre-defined points.Data Processing: Total platelet surface coverage is determined and graphed over time. Two outputs are evaluated, (1) the lag time: the time to reach 5% surface coverage and (2) the slope: slope during the linear portion of platelet adhesion.ResultsPatients: 42 samples were obtained from 35 patients with VWD/MCB. 11/35 (31%) are from type 1 VWD patients, 2/35 (6%) are from type 2 VWD patients, 1/35 (3%) are from a Type 3 VWD patients, and 22/35 (60%) are from patients with MCB.Assay Correlation: In all patients, the midshear slope correlated better with VWF:Ag (R2 = 0.52 P<0.0001) than the PFA100-ADP (R2 = 0.28 P=0.0008) and PFA100-EPI (R2 =0.32 P=0.0002) (Table 1). The midshear slope (R2 = 0.52 P=< 0.0001) also outperformed the PFA100-ADP (R2 = 0.33 P= 0.0003) and PFA100-EPI (R2 = 0.39 P= < 0.0001) in correlating with VWF:RCo levels (Table 1). In the Type 1 VWD cohort, the mid shear slope correlated with VWF:Ag (R2 = 0.54 P=0.01) (figure 1). In comparison, the PFA-100 ADP/EPI was relatively insensitive to VWF:Ag levels <40 IU/dL (figure 1). These results show that a single MFA assay correlates with VWF:Ag and VWF:RCo levels better than the PFA-100 closure time in a cohort of patients with VWD/MCB and is more sensitive to VWF:Ag levels <40 IU/dL in a cohort of individuals with Type 1 VWD.BS Correlation: No clinical assays or MFA output significantly correlated with BS in any cohort analyzed.Response to therapy: In 4 patients pre and 1hr post DDAVP, the MFA lag time remained the same or decreased (low=15.75s to 15.75s, mid=18s to 11.25s, high=30.75s to 9s) and the slope increased (low=0.0044 to 0.0057, mid=0.0044 to 0.0066, high=0.0037 to 0.0059). The increased low shear slope, 0.0044 to 0.0057, was statistically significant (p= 0.0244). This suggests that a MFA may be useful to determine response to DDAVP therapy.ConclusionsA novel collagen-based MFA outperformed the PFA100 ADP/EPI in correlating with VWF:Ag and VWF:RCo in patients with VWD/MCB. In type 1 VWD patients, it also demonstrated a significant correlation with VWF:Ag levels and a more linear relationship than the PFA100 ADP/EPI. Finally, the MFA was able to determine a response to DDAVP therapy. Thus a MFA, with a small sample input (150 µL) as compared to other shear dependent assays such as the PFA100 (1000 µL), may be useful to determine multiple clinically relevant parameters in situations where blood samples are difficult to obtain such as in neonatal settings or poor venous access.Table 1Comparison of R2 values Between Standard Assays and Shear-based Assays in All Patients with VWD/MCB.Standard AssayShear-Based AssayR2 ValueP ValueVWF:AgPFA100 (Collagen/EPI)0.320.0002PFA100 (Collagen/ADP)0.280.0008MFA Mid-shear Slope0.52<0.0001VWF:RCoPFA100 (Collagen/EPI)0.39< 0.0001PFA100 (Collagen/ADP)0.330.0003MFA Mid-shear Slope0.52< 0.0001 [Display omitted] DisclosuresNg:CSL Behring: Consultancy; Baxter: Consultancy. Manco-Johnson:Baxter, bayer, biogen, CSL Behring, NovoNordish: Honoraria.

Impact of Blood Groups on Clinical Outcomes in Patients with Von Willebrand Disease

INTRODUCTIONVon Willebrand Disease (vWD) is the most common hereditary hemorrhagic disorder with diverse clinical and genetic characteristics. Transmitted as an autosomal dominant trait (except Type III), it is caused primarily by the quantitative or qualitative deficiency of von Willebrand factor (vWF). ABO blood group types can affect the pathology, severity and hence the presentation of this clinically heterogeneous entity as the genes responsible for ABO blood groups also affects vWF levels in the plasma. We conducted a study to analyze and compare different clinico-demographic aspects among O and non-O blood group patients with Von Willebrand Disease. Our primary aim was to determine the relationship of different blood groups with venous thromboembolism (VTE) in patient with vWD. Our secondary aim was to determine the association of blood groups with different comorbid conditions such as coronary artery disease, cerebrovascular disease, peripheral vascular disease, chronic liver disease, chronic kidney disease etc. in patients with vWD.METHODSA retrospective review was carried out to include all patients diagnosed with Von Willebrand Disease from year 2002 to 2012. Patient population was obtained by using International Classification of Diseases, 9th revision (ICD9) codes. Data was collected with the help of electronic medical records. Multiple clinical variables and demographic characteristics of Von Willebrand Disease patients (n=381) were analyzed and compared between those with type O blood group (205 patients; 54%) and those with type non-O blood groups (176 patients; 46%). Univariate logistic regression was used to obtain crude odds ratios. Predictors with statistically significant p-values were included in a multivariable logistic regression model in order to yield adjusted odds ratio. Statistical significance was set at p<0.05RESULTSPatients with O blood groups were less likely to develop VTE (OR 0.29, 95% CI 0.16-0.52, p < 0.001) as compared to Non-O blood groups in which the prevalence of VTE was much higher (25% vs. 9%). Additionally, patients with O blood groups had relatively lower factor 8 activity (125.9±90.8%; OR 0.99, 95% CI 0.99-1.00, p = 0.018) when compared with Non-O blood groups (168.5±120.6%). Patients with O-blood group had significant, life threatening bleeds at an early age (40.4±20.8 years; OR 0.99, 95% CI 0.97-1.00, p = 0.008) when compared with patients with type non-O blood group (Age 47.1±21.9 years). Additionally, O-Blood groups were related to a lesser prevalence of cerebrovascular disease (OR 0.48, 95% CI 0.29-0.79, p = 0.004) and peripheral vascular disease (OR 0.47, 95% CI 0.25-0.87, p = 0.015) as compared to Non-O blood groups.DISCUSSIONOur study consolidates the fact that vWD patients with O blood group have a lesser risk of developing VTE as compared to non-O blood group patients. Additionally, strokes and peripheral vascular disease is also seen less frequently in O blood groups in patients with Von Willebrand disease. These results suggest that the genes responsible for ABO blood groups also have a significant effect on clinical outcomes in patients with vWD. There are multiple proposed mechanisms to explain this association including presence of ABO antigens on several platelet glycoproteins and glycosphingolipids as well as on the surface of vascular endothelium. More sophisticated, large-scale studies are needed to strengthen the associations we observed in our retrospective study. DisclosuresKuriakose:Kedrion: Speakers Bureau.

Effect of the Novel Biodegradable N, O-Carboxymethylchitosan and Oligo-Chitosan on the Platelet Thrombogenicity Cascade in von Willebrand Disease

IntroductionVon Willebrand disease (vWD) is the second least common hemostatic disorder in Malaysia, and it has a low prevalence. This study examined the underlying platelet thrombogenicity cascades in the presence of different formulations of chitosan-derivatives in vWD patients. This paper aimed to determine the significant influence of chitosan biomaterial in stimulating the platelet thrombogenicity cascades that involve the von Willebrand factor, Factor 8, Thromboxane A2, P2Y12 and Glycoprotein IIb/IIIa in vWD. Materials and methodsVariable chitosan formulations of N,O-Carboxymethylchitosan (NO-CMC) and Oligo-Chitosan (O-C) were tested. Fourteen vWD subjects voluntarily participated in this study after signing informed consent forms. The patient’s demographic profiles, family history, type of vWD, clinical symptoms and laboratory profiles were recorded and analyzed. Enzyme-linked immunosorbent assay, flow cytometry and Western blot tests were used to determine the level of the chitosan-adhered-platelet-mechanisms. ResultsThe study revealed that most patients were predominantly affected by vWD type I. The O-C group of chitosan’s scaffold pores is sufficient to allow for nutrients and cells. The O-C-stimulated-mediators are capable of initiating the platelet actions and were detected to expedite the blood coagulation processes. The oligo-group of chitosans was capable of amplifying and triggering more platelet activator’s pathways via the studied mediators. The present findings suggest that the ability of each type of chitosan to coagulate blood varies depending on its chemical composition. ConclusionThe oligo group of chitosans is potentially capable of triggering platelet thrombogenicity cascades by activating platelets in vWD patients to form a platelet plug for hemostasis process.

Spinal anesthesia for emergency cesarean delivery in von willebrand disease

Von Willebrand disease(vWD) is a common hemostatic disorder encountered inobstetric anesthesia practice. This needs appropriate pre-operative evaluation to choose the best technique of anesthetic management. Evaluation needs to be individualized to consider the risk and benefits of each technique, either general or neuraxial anaesthesia. There are only a few published cases on use of spinal anesthesia in vWD patients for caesarean delivery. We present a case of vWD patient who was given spinal anesthesia for emergency caserean delivery. We were not able to determine the type of the disease, but based on clinical features it was assumed to be of type I or II. Intranasal desmopressin was given as bleeding prophylaxis. Anaesthetic considerations and peri-operative management are discussed.

Successful Ultrasound-Guided Femoral Nerve Blockade and Catheterization in a Patient with Von Willebrand Disease.

Peripheral nerve blockade (PNB) is superior to neuraxial anesthesia and/or opioid therapy for perioperative analgesia in total knee replacement (TKR). Evidence on the safety of PNB in patients with coagulopathy is lacking. We describe the first documented account of continuous femoral PNB for perioperative analgesia in a patient with Von Willebrand Disease (vWD). Given her history of opioid tolerance and after an informative discussion, a continuous femoral PNB was planned for in this 34-year-old female undergoing TKR. A Humate-P intravenous infusion was started and the patient was positioned supinely. Using sterile technique with ultrasound guidance, a Contiplex 18 Gauge Tuohy needle was advanced in plane through the fascia iliaca towards the femoral nerve. A nerve catheter was threaded through the needle and secured without complications. Postoperatively, a levobupivacaine femoral catheter infusion was maintained, and twice daily Humate-P intravenous infusions were administered for 48 hours; enoxaparin thromboprophylaxis was initiated thereafter. The patient was discharged uneventfully on postoperative day 4. Given documentation of delayed, unheralded bleeding from PNB in coagulopathic patients, we recommend individualized PNB in vWD patients. Multidisciplinary team involvement is required to guide factor supplementation and thromboprophylaxis, as is close follow-up to elicit signs of bleeding throughout the delayed postoperative period.

A Study on the Safety of Liver Biopsy Inpatients with Von Willebrand’s Disease

Objectives: Liver biopsy remains the gold standard for diagnosis of chronic liver diseases. Outpatient percutaneous biopsy is generally safe with a mortality rate of 0.17% and hospitalization rate for bleeding of 3%. Von Willebrand disease (vWD) syndrome is the most common inherited hematological disorder with a prevalence of 1% - 3% globally. We sought to study whether vWD increases the risk of bleeding for liver biopsies. Methods: All patients (n = 120) who underwent outpatient percutaneous liver biopsies from 1997 to 2007 were analyzed. Demographics, PT/INR, platelet count, vW antigen and ristocetin induced platelet aggregation were studied. Results: No vWD patients had major bleeding that required transfusion, hospitalization or surgery but 9 (75%) had minor local bleeding and all had ecchymosis, which resolved spontaneously within 24 hours. Conclusions: Patients with vW factor deficiency can undergo percutaneous liver biopsy without major bleeding. Minor bleeding may occur at a slightly higher rate. vWD is not a contraindication to percutaneous liver biopsy.

New insights into genotype and phenotype of VWD

AbstractRecent advances in VWD research have improved our understanding of the genotype and phenotype of VWD. The VWF gene is highly polymorphic, with a large number of sequence variations reported in healthy individuals. This can lead to some difficulty when attempting to discern genotype–phenotype correlations because sequence variations may not represent disease. In type 1 VWD, mutations can be found throughout the VWF gene, but likely pathogenic sequence variations are found in only ∼2/3 of type 1 VWD patients. Sequence variations in type 2 VWD are located in the region corresponding to the defect in the VWF protein found in each type 2 variant. In type 3 VWD, sequence variations are not confined to a specific region of the VWF gene and also include large deletions that may not be picked up using conventional sequencing techniques. Use of genetic testing may be most helpful in diagnosis of type 2 VWD, in which a larger number of known, well characterized mutations are present and demonstration of one of these may help to confirm the diagnosis. Bleeding symptoms in general are more severe with decreasing VWF levels and more severe in type 2 and type 3 VWD compared with type 1 VWD. Prediction of phenotype for an individual patient, however, is still difficult, and the addition of genetic data will be most helpful in ascertaining the correct diagnosis for VWD patients.

New insights into genotype and phenotype of VWD.

Recent advances in VWD research have improved our understanding of the genotype and phenotype of VWD. The VWF gene is highly polymorphic, with a large number of sequence variations reported in healthy individuals. This can lead to some difficulty when attempting to discern genotype-phenotype correlations because sequence variations may not represent disease. In type 1 VWD, mutations can be found throughout the VWF gene, but likely pathogenic sequence variations are found in only ∼2/3 of type 1 VWD patients. Sequence variations in type 2 VWD are located in the region corresponding to the defect in the VWF protein found in each type 2 variant. In type 3 VWD, sequence variations are not confined to a specific region of the VWF gene and also include large deletions that may not be picked up using conventional sequencing techniques. Use of genetic testing may be most helpful in diagnosis of type 2 VWD, in which a larger number of known, well characterized mutations are present and demonstration of one of these may help to confirm the diagnosis. Bleeding symptoms in general are more severe with decreasing VWF levels and more severe in type 2 and type 3 VWD compared with type 1 VWD. Prediction of phenotype for an individual patient, however, is still difficult, and the addition of genetic data will be most helpful in ascertaining the correct diagnosis for VWD patients.

Von Willebrand Disease

Von Willebrand Disease

Von Willebrand Disease

1. Stacy Cooper, MD 2. Clifford Takemoto, MD 1. Charlotte Bloomberg Children’s Center, Johns Hopkins Hospital Baltimore, MD 1. 1. Nichols W, 2. Hultin MB, 3. James AH, 4. et al Von Willebrand Disease (VWD): Evidence-Based Diagnosis and Management Guidelines, The National Heart, Lung, and Blood Institute (NHLBI) Expert Panel Report (USA) . Nichols W, Hultin MB, James AH, et al. Haemophilia. 2008;14(2):171–232 [OpenUrl][1][CrossRef][2][PubMed][3][Web of Science][4] 2. 1. O’Brien SH Bleeding Scores: Are They Really Useful? O’Brien SH. Am Soc Hematol Educ Program. 2012;2012(1):152–156 [OpenUrl][5] 3. 1. Montgomery G, 2. Cox JG 1. Orkin SH, 2. Nathan DG, 3. Ginsburg D, 4. Look AT, 5. Fisher DE, 6. Lux SE, 7. Lantigua CJ Von Willebrand Disease . Montgomery G, Cox JG. In: Orkin SH, Nathan DG, Ginsburg D, Look AT, Fisher DE, Lux SE, Lantigua CJ. Nathan and Oski’s Hematology of Infancy and Childhood. 7th ed. Philadelphia, PA: Saunders; 2009:1487–1524 Von Willebrand disease (vWD) is an inherited bleeding diathesis. It is caused by quantitative or qualitative defects in von Willebrand factor (vWF), which functions to bind platelets to damaged endothelium and to stabilize factor VIII (FVIII). A range of mutations have been identified in this protein, encompassing the spectrum of single missense mutations to large deletions. Because of variation in the diagnostic criteria for vWD, debate exists regarding the prevalence; however, some studies estimate it to be as high as 1% to 2% of the population when including children and adults. There are 3 major categories of vWD, classified as partial deficiency of vWF (type 1), functional defects (type 2), and complete deficiency of vWF (type 3). Type 2 can be further subcategorized based on the 4 subtypes of specific qualitative defects of the multimers (types 2A, 2B, 2M, and 2N). The different types of vWD vary in severity. Type 1 is the most common, an autosomal dominant disorder that comprises approximately 75% of vWD patients, and is usually confined to mild bleeding. Type 2 may demonstrate either dominant or recessive inheritance, and type 3 is autosomal recessive, with … [1]: {openurl}?query=rft.jtitle%253DHaemophilia%2B%253A%2B%2Bthe%2Bofficial%2Bjournal%2Bof%2Bthe%2BWorld%2BFederation%2Bof%2BHemophilia%26rft.stitle%253DHaemophilia%26rft.aulast%253DNichols%26rft.auinit1%253DW.%2BL.%26rft.volume%253D14%26rft.issue%253D2%26rft.spage%253D171%26rft.epage%253D232%26rft.atitle%253Dvon%2BWillebrand%2Bdisease%2B%2528VWD%2529%253A%2Bevidence-based%2Bdiagnosis%2Band%2Bmanagement%2Bguidelines%252C%2Bthe%2BNational%2BHeart%252C%2BLung%252C%2Band%2BBlood%2BInstitute%2B%2528NHLBI%2529%2BExpert%2BPanel%2Breport%2B%2528USA%2529.%26rft_id%253Dinfo%253Adoi%252F10.1111%252Fj.1365-2516.2007.01643.x%26rft_id%253Dinfo%253Apmid%252F18315614%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [2]: /lookup/external-ref?access_num=10.1111/j.1365-2516.2007.01643.x&link_type=DOI [3]: /lookup/external-ref?access_num=18315614&link_type=MED&atom=%2Fpedsinreview%2F35%2F3%2F136.atom [4]: /lookup/external-ref?access_num=000253626100001&link_type=ISI [5]: {openurl}?query=rft.jtitle%253DAm%2BSoc%2BHematol%2BEduc%2BProgram%26rft.volume%253D2012%26rft.spage%253D152%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx

Critical Importance Of VWF Propeptide (VWFpp) In The Diagnosis Of Type 1 Von Willebrand Disease (VWD)

Type 1 VWD is the most common form of VWD and is characterized by quantitative deficiency of VWF. Mechanisms causing type 1 VWD include decreased VWF synthesis due to promoter polymorphisms, decreased VWF secretion with intracellular retention/degradation, and increased clearance of VWF from plasma (type 1C VWD). VWF and its propeptide (VWFpp) are released into plasma in equimolar amounts but have very different half-lives (8 – 12 hours and 2-3 hours, respectively). The assay of VWFpp can be used to assess VWF synthesis, secretion, and clearance. Reduced VWFpp level indicates reduced VWF synthesis or secretion. An increased VWFpp/VWF:Ag ratio is expected when VWF is cleared rapidly from plasma (type 1C VWD), while decreased VWF secretion/intracellular retention results in a normal ratio. We enrolled 502 type 1 VWD index cases with a pre-existing diagnosis of type 1 VWD through the Zimmerman Program for the Molecular and Clinical Biology of VWD. We confirmed 262 index cases as type 1 VWD (VWF:Ag or VWF:RCo ≤ 40 IU/dL). Of the confirmed type 1 VWD cases, 58 met the criteria for type 1C VWD with VWFpp/VWF:Ag ≥ 3 and VWF:Ag ≤ 30 IU/dL, and 12 met the criteria for “Type 1 – Severe” with VWF:Ag of 1 – 5 IU/dL and VWFpp/VWF:Ag < 3. Type 1C VWD comprised 22% of all type 1 VWD cases. The type 1C cohort included several individuals previously diagnosed as type 2A (11), type 2M (2), and “Unclassified” (3). The most significant reclassification involved 7 cases previously diagnosed as type 3 VWD – these patient had detectable VWF:Ag (2 – 6 IU/dL) and VWFpp (14 – 66 IU/dL), and elevated VWFpp/VWF:Ag (4.2 – 33.0). Although plasma VWF:Ag is very low in these patients, they might be expected to have normal platelet stores of VWF:Ag, unlike type 3 VWD patients. Type 3 VWD patients in our study had undetectable (<1 IU/dL) VWFpp and VWF:Ag. In fact, type 1C VWD comprised the majority of severe type 1 VWD cases. At very low VWF:Ag levels (2 – 10 IU/dL), 76% of index cases had a type 1C phenotype, while the remainder had normal VWFpp/VWF:Ag consistent with a decreased secretion phenotype. In the VWF:Ag of 11 – 20 IU/dL group, 38% were type 1C. At still higher VWF:Ag levels (21 – 30 IU/dL), only 7% had a type 1C phenotype and the majority had a decreased secretion phenotype. Additionally, 43% of the type 1C cohort had VWFpp levels below the normal range, indicating reduced synthesis/secretion is also involved in the mechanism causing type 1C VWD. Mutations associated with type 1C were located in the VWF D3, A1, and D4 domains while decreased secretion variants were located primarily in the D1, D2, and D3 domains. Somewhat surprisingly, no difference in bleeding score was identified between type 1C and other type 1 patients. We can conclude that a reduced VWF survival phenotype (type 1C) is very frequent in severe type 1 VWD patients with VWF:Ag < 20 IU/dL. Identification of type 1C VWD is important because the rapid clearance of VWF in these patients may preclude the use of desmopressin, which is commonly given in type 1 VWD, but may be insufficient to achieve hemostasis in type 1C patients. Disclosures:No relevant conflicts of interest to declare.

Assessment Of The Validation and Performance Of The Cone and Plate(let) Analyzer (CPA) In The Diagnosis Of Von Willebrand's Disease

BackgroundVon Willebrand disease (VWD) is the most common significant bleeding disorder in the western world. VWD presents with a wide spectrum of clinical manifestations, ranging from totally asymptomatic individuals to severe, occasionally life-threatening bleeding episodes and hence early and accurate diagnosis is crucial for starting treatment.Diagnosis of VWD requires specialized laboratories with skilled personnel and the testing process itself is time and labor consuming, and is costly. In recent years several methods for VWD diagnosis were developed including several flow bedside instruments which test whole blood coagulation following shear stress activation of clotting. One such tool is the cone and plate(let) analyzer (CPA). In the CPA test whole blood is activated by a rotating conical disc on a polystyrene surface at high shear rate. The test result is expressed as the % of the plastic surface covered with adherent platelets (SC- surface coverage %) and the average size of the platelet aggregates (average size- AS μm2).The aim of the current study was to test the properties of the CPA as a diagnostic tool for VWD in a non-selected group of patients, to compare its diagnostic abilities with current methods of diagnosis, and to test for presence of (patient and physician) bias in the diagnostic process. MethodsPatients referred for evaluation of bleeding symptoms, post-surgical bleeding, abnormal coagulation tests or with a known familial bleeding disorder were compared to a group of healthy controls and warfarin treated patients. Diagnosis of VWD and all other bleeding disorders was achieved by standard clinical and laboratory methods and in the final analysis VWD patients were compared to all other non VWD groups. The CPA was tested in all patients and controls, first on an inception cohort (268 subjects) and results from that sample were then applied to a validation cohort (221 subjects). The optimal SC and AS values were determined by ROC analysis in each sample and both samples overall. Sensitivity, specificity and likelihood ratios (likelihood of having (LR+) or not having (LR-) VWD given a specific cut-off value) were computed for each cut-off value. ROC method was also applied to test whether age, gender, severity of VWD, presence of bleeding symptoms, genetic/familial diagnosis of VWD and comorbidity of patients had any effect on CPA's testing accuracy. ResultsDuring a 10 year period 489 consecutive patients referred to Sheba medical Center were studied. Following a complete workup we diagnosed (in both cohorts) 149 patients with VWD (type 1-72, type 2-42, type 3+acquired VWD- 35), that were compared to 217 healthy controls, 35 VKA treated patients, and 88 patients with other bleeding disorders (hemophilia A/B – 39, Factor II, VII, XI deficiencies – 49). The surface coverage was found as the best CPA diagnostic parameter for diagnosis of VWD, with an optimal cut-off value of 8.3% in the inception cohort and 6.9 % (but with better sensitivity) in the validation cohort (table and figure). In all of the study cohorts SC optimal cut-off maintained its high specificity with an improved sensitivity and a cut-off value of 7.1%. Age, sex, severity of VWD, or additional genetic/familial diagnosis of VWD had no effect on CPA's testing accuracy. However, patients tested soon after a bleeding event or during hospitalization (originated mainly from the validation cohort) were older, had higher levels VW activity and higher levels of SC and therefore different SC cut-off value with different test characteristics (table).CohortSC %Sensitivity (%)Specificity (%)LR+LR-Inception8.397888.30.03Validation6.9749617.30.27Both7.1849622.10.16 ConclusionIn this relatively large and representative cohort of healthy volunteers and patients with various bleeding disorders the CPA test was found sensitive and highly specific in the diagnosis of VWD. The test was robust to patient's age, sex, and VWD severity but a mild spectrum bias cannot be ruled out (as found in the validation cohort) deriving mainly from patient's comorbidity and recent bleeding. This highly specific test may prove its utility in preliminary or urgent diagnosis of VWD before a complete clinical and laboratory workup is done. [Display omitted] Disclosures:Savion:Mattis medical: Equity Ownership.

P.P2063S: a neutral VWF variant masquerading as a mutation

Dear Editor, Kasatkar and colleagues recently reported that the previously described von Willebrand factor (VWF) variant c.6187C>T (p.P2063S) appears to be a common cause of type 3 von Willebrand disease (VWD3) in the Indian population [1]. In support of their conclusion, p.P2063S was the only VWF variant observed in 11 Indian VWD3 index cases (IC), tracked with disease phenotype and was predicted to be deleterious via in silico protein prediction analysis. In order to ensure accurate molecular characterisation of VWF variants, it is essential to consider all available information. Regarding p.P2063S, there are several lines of evidence that contradict the conclusion drawn. p.P2063S is also observed in healthy controls with reported population frequencies between 0.6–2.5% in African-Americans / Caucasians [2–4] and (as reported by Kasatkar and colleagues [1]) 3% in Indians. Given a frequency of 3% in the general population, Hardy-Weinberg equilibrium predicts homozygosity for this variant in approximately 1/1100 individuals. This would suggest a high population frequency of VWD3 if this variant were causative. It is also notable that p.P2063S has not been reported as causal in previous studies of VWD3 in Indians [5–7]. The majority of missense mutations reported in VWD3 patients occur in the VWF propeptide and C-terminus where they are reported to affect protein folding and multimerisation resulting in intracellular retention of the protein [2,7,8]. Kasatkar and colleagues argue that diagnosis of VWD3 relies on defective release or synthesis of VWF [1], however given that p.P2063S has no effect on VWF expression in vitro [2] it seems unlikely that this variant would affect VWF in vivo. This is supported by observations of p.P2063S in VWD patients inherited together with other VWF variants that more readily explain their VWD phenotype (Table 1), most notably in a Turkish VWD3 IC [8]. Table 1 Co-inheritance of p.P2063S and actual VWD causing variants In previous Indian VWD3 studies [5–7], molecular analysis failed to fully account for the genetic cause in 8/54 IC (15%). It is therefore likely that the actual disease causing mutation has yet to be identified by Kasatkar and colleagues, who imply that DNA scanning methods were used to identify mutations in their patient cohort [1]. DNA scanning methods are not 100% sensitive to all sequence variations so may fail to detect the ‘true’ mutation, for example chemical cleavage mismatch analysis identified a homozygous p.P2063S variant in a Turkish VWD3 IC but failed to identify the actual p.Q1734* causative mutation (Table 1) [8]. Alternatively, the causative mutation may have been overlooked if, for example, it were a synonymous variant that disrupts normal splicing or protein folding, or a deep intronic variant causing disruption of splicing outside the VWF regions analysed [9]. In summary, review of the available information supports the conclusion that p.P2063S is a common neutral VWF polymorphic variant. Further studies (for example using mRNA as the template investigated) may identify the underlying cause of VWD in this interesting patient group.

Cellular and molecular basis of von Willebrand disease: studies on blood outgrowth endothelial cells

AbstractVon Willebrand disease (VWD) is a heterogeneous bleeding disorder caused by decrease or dysfunction of von Willebrand factor (VWF). A wide range of mutations in the VWF gene have been characterized; however, their cellular consequences are still poorly understood. Here we have used a recently developed approach to study the molecular and cellular basis of VWD. We isolated blood outgrowth endothelial cells (BOECs) from peripheral blood of 4 type 1 VWD and 4 type 2 VWD patients and 9 healthy controls. We confirmed the endothelial lineage of BOECs, then measured VWF messenger RNA (mRNA) and protein levels (before and after stimulation) and VWF multimers. Decreased mRNA levels were predictive of plasma VWF levels in type 1 VWD, confirming a defect in VWF synthesis. However, BOECs from this group of patients also showed defects in processing, storage, and/or secretion of VWF. Levels of VWF mRNA and protein were normal in BOECs from 3 type 2 VWD patients, supporting the dysfunctional VWF model. However, 1 type 2M patient showed decreased VWF synthesis and storage, indicating a complex cellular defect. These results demonstrate for the first time that isolation of endothelial cells from VWD patients provides novel insight into cellular mechanisms of the disease.

PM.33 The Obstetric Management of Haemophilia Carriers and Patients with Von Willebrand’s Disease in Leeds

Background Haemophilia and von Willebrand’s disease (vWD) are inherited bleeding disorders that present significant obstetric challenges, including risks of neonatal intracranial haemorrhage and postpartum haemorrhage (PPH). The ideal mode of...

Phenotypic and molecular characterisation of type 3 von Willebrand disease in a cohort of Indian patients

Severe type 3 VWD (VWD3) is characterised by complete absence or presence of trace amounts of non-functional von Willebrand factor (VWF). The study was designed to evaluate the VWF mutations in VWD3 patients and characterise the breakpoints of two identified homozygous novel large deletions. Patients were diagnosed by conventional tests and VWF multimer analysis. Mutation screening was performed in 19 VWD3 patients by direct sequencing of VWF including flanking intronic sequence and multiplex ligation-dependent probe amplification (MLPA) analysis. Breakpoint characterisation of two identified novel large deletions was done using walking primers and long spanning PCR. A total of 21 different mutations including 15 (71.4%) novel ones were identified in 17 (89.5%) patients. Of these mutations, five (23.8%) were nonsense (p.R1659*, p.R1779*, p.R1853*, p.Q2470*, p.Q2520*), one was a putative splice site (p.M814I) and seven (33.3%) were deletions (p.L254fs*48, p.C849fs*60, p.L1871fs*6, p.E2720fs*24) including three novel large deletions of exon 14-15, 80,830bp (-41510_657+7928A*del) and 2,231bp [1534-2072T_c.1692G*del(p.512fs*terminus)] respectively. A patient carried gene conversion comprising of pseudogene harbouring mutations. The missense mutations (p.G19R, p.K355R, p.D437Y, p.C633R, p.M771V, p.G2044D, p.C2491R) appear to play a major role and were identified in seven (36.8%) patients. In conclusion, a high frequency of novel mutations suggests the high propensity of VWF for new mutations. Missense and deletion mutations found to be a common cause of VWD3 in cohort of Indian VWD3 patients. Breakpoints characterisation of two large deletions reveals the double strand break and non-homologous recombination as deletions mechanism.

Perioperative Management of Type 2N Von Willebrand’s Disease with Recombinant Factor VIII in a Patient Undergoing Knee-Replacement Surgery

Type 2N Von Willebrand's disease (2N VWD) is a rare, recessively inherited bleeding disorder, comprising 1-2% of all VWD patients, usually manifesting as a mild bleeding diathesis. Treatment includes desmopressin (DDAVP) or intermediate purity plasma-derived FVIII concentrates containing residual VWF. We present a case of a 75-year-old gentleman, incidentally diagnosed with type 2N VWD in 2002 on routine blood testing before surgery with an ISTH bleeding score of 1-2, who was treated with recombinant FVIII preoperatively.