ALBERT

Beschreibung: Key Points D1472H sequence variation is associated with a decreased VWF:RCo/VWF:Ag ratio in type 1 VWD subjects. D1472H sequence variation is not associated with an increase in bleeding as measured by bleeding score in type 1 VWD subjects.

Beschreibung: Abstract 381FN2 The TS Zimmerman Program for the Molecular and Clinical Biology of von Willebrand Disease (ZPMCB-VWD) is a large NIH PPG to study existing subjects with von Willebrand Disease in the United States and to contrast these with prior and ongoing studies in Canada and the UK. 569 index cases (ICs) and 1732 family members were recruited from 8 Primary Clinical Centers and 19 Secondary Clinical Center. The inclusion criteria were that the subjects had a historical diagnosis of VWD and were registered as ongoing patients in the local HTC. 247 normal controls (NCs) were studied for comparison. Data included pre-existing diagnosis, historical diagnostic VWD testing, detailed bleeding history using a modified MCMDM-1VWD QBS, and subjects had plasma and DNA drawn for studies at a central laboratory for VWD, VWF phenotyping, and full length VWF exon sequencing. ICs included 391 type 1, 105 type 2, 43 type 3, and 30 unclassified. The recent HLBI Guidelines suggested that the diagnosis of VWD be based on a VWF level of

Beschreibung: 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 (

Beschreibung: Background von Willebrand disease (VWD) is a very common inherited bleeding disorder. The current phenotypic classification of VWD variants includes disorders of both quantitative and qualitative defects in von Willebrand Factor (VWF) that determines optimal treatment of patients. Current phenotype determination is multimodal, cumbersome, performed by only a few specialized laboratories, and may delay the definitive diagnosis necessary in proper selection of therapy. We have developed an ELISA-based strip assay that is capable of rapid determination of relative qualitative and quantitative VWF functionality to correctly assign phenotypic variants of VWD. Methods 136 VWD plasma samples from the Zimmerman PPG were analyzed on a new ELISA based platform. In single, individual wells this assay measures relative values of VWF:Ag (antigen), VWF:IbCo (Ib cofactor, no ristocetin), VWF:RCo (ristocetin cofactor), VWF:F8B (binding to FVIII), VWF:CB3 (binding to collagen III), and VWF:pp (propeptide) in comparison to a 30% normal control standard in a single ELISA strip assay. The study included 22 type 1 VWD, 32 type 1C VWD, 18 type 2A VWD, 23 type 2B VWD, 20 type 2M VWD, 7 type 2N VWD, 4 type 3 VWD, and 10 hemophilia A subjects. Each sample was run in single wells for each assay and optical densities (OD) were compared to the OD of a 30% standard control plasma and a 100% VWF:Ag control. The standard ELISA plate read time was 30 minutes and full assay can be accomplished in 3 hours. Two of the coauthors were blinded as to the Zimmerman PPG VWD phenotypes of test samples. Using the ELISA strip results, phenotype assignment was determined and then compared to the unblinded Zimmerman PPG VWD diagnosis. Further statistical analysis of VWF functional profile relationships was performed using the Mann-Whitney test and ROC analysis, and can quantify the ability to identify these phenotypes. Results VWF functional profiles based on visually observed ratio relationships correctly assigned VWD phenotypic variant on first attempt in 122 of 136 subjects (89.7%). Repeat testing of the 14 incorrectly assigned subjects along with 11 random, correctly assigned subjects for a validation check, accurately re-assigned 9 of 14 previously incorrect phenotypes, suggesting initial plate to plate variability since all ELISA plates were made fresh for each run. Previously correctly assigned subjects, 11 of 11, remained correctly assigned. Comparing specific phenotypes revealed VWF:IbCo/VWF:Ag is good at separating type 1C from 2A; ROC area under the curve 0.875, with an optimal ratio threshold 0.649 (sensitivity 0.969, specificity 0.611, p

Beschreibung: Abstract 3839 Menorrhagia is a common symptom of haemostatic disorders and often triggers an evaluation for a bleeding disorder. Clinical screening tools, to identify women with menorrhagia who have an underlying bleeding disorder, are very desirable. Scoring systems to quantify menstrual blood loss in women, the Pictorial Blood Loss Assessment Chart, (PBAC), and to assess the severity of bleeding symptoms in individuals diagnosed with von Willebrand disease (VWD), European Union Bleeding Score (EU-BS) (MCMDM-1VWD), were evaluated for their ability to predict the presence of low von Willebrand factor (VWF) level in a cohort of 150 women recruited from an outpatient gynecology clinic. One of us (SG or ML) completed a PBAC and an EU Bleeding questionnaire with the participant. Menorrhagia cases were defined by a PBAC ≥185 and controls by a PBAC

Beschreibung: Background Challenges in reporting subjective hemorrhagic symptoms consistently has led to the need for standardized, quantitative Bleeding Assessment Tools (BATs), some of which assign Bleeding Scores (BSs). The ISTH-BAT (International Society on Thrombosis and Hemostasis – Bleeding Assessment Tool (Rodeghiero et al JTH 2010; 8:2063)) aimed to consolidate and optimize advances made by its predecessors, which were based on the 2005 “Vicenza Bleeding Questionnaire”. It is important to note, however, that the scoring systems differ among the BATs, with each bleeding symptom scored from 0 to +3 for the original Vicenza, -1 to +4 for the MCMDM-1VWD and Condensed MCMDM-1VWD Bleeding Questionnaires and the PBQ (Pediatric Bleeding Questionnaire), and 0 to +4 for the ISTH-BAT. As a result, the normal ranges of BSs vary among questionnaires. To date, the normal range for the ISTH-BAT has not been established; the objective of this study was to determine the normal range of bleeding scores for the ISTH-BAT for both adults and pediatric patients. Patients and Methods BS data from different studies, originally generated using 4 different Vicenza-based BATs, were compiled using a bioinformatics system created to facilitate the collation and analysis using different scoring systems. Demographic and BS data, along with blood group, VWF:Ag/VWF:RCo/FVIII:C (when available) were collected from all enrolled subjects. Data were derived from multiple studies; all defined normal subjects as those without a known problem with bleeding or bruising. All BATs were expert-administered. The normal range for both adults and pediatrics was determined by: 1) removing outliers 〉 3 SD away from the mean and then, 2) selecting the mid-95th %ile. Results 1,422 normal subjects were included (adult: n=1,079, pediatric: n=343). Adult data were collected using MCMDM-1VWD (n=294), Condensed MCMDM-1VWD (n=660), and ISTH-BAT (n=125), while pediatric data were collected using PBQ (n=324) and ISTH-BAT (n=19). 48 adults were removed from the analysis because they had BSs 〉 6.3, (i.e., 〉3 SD away from the mean), leaving n=1,031 for determination of the normal range. For children, BSs 〉 3.5 were judged to be outliers and therefore 18 children were removed, leaving n=325 children for determination of the normal range. The remaining adults had a mean age of 43 yrs (range 18 – 88) with 695 females and 336 males. The remaining children had a mean age of 9 yrs (range 0.4 – 17 yrs), with 169 females and 156 males. The relationship between BSs and demographic and lab data are given in Table 1. For the ISTH-BAT, the normal range of BSs was 0 - 4 in adults (meaning that for individuals 18 yrs or older, a BS 5 or greater is positive or abnormal) and 0 - 2 in children (meaning that for individuals 〈 18 yrs, a BS 3 or greater is positive or abnormal). Conclusion The newly established normal BS ranges can now be used to objectively assess the bleeding symptoms of individuals by administration of the ISTH-BAT. They also highlight the strength of merging existing datasets to generate meaningful results. By making these data accessible to all investigators using the web-based ISTH-BAT system housed at Rockefeller University we hope to aid investigators initiating new studies and facilitate correlating bleeding symptoms with genotypic, molecular, and environmental data. Disclosures: Mauer: CSL Behring: Honoraria. James:CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Baxter: Honoraria; Bayer: Honoraria.

Beschreibung: While von Willebrand disease (VWD) is the most common inherited bleeding disorder, most patients have quantitative defects in von Willebrand factor (VWF). The qualitative variants, collectively termed type 2 VWD, are less common, but also in general more severe than type 1 VWD. However, despite a common laboratory phenotype of decreased VWF:RCo/VWF:Ag ratio for types 2A, 2B, and 2M VWD, the clinical phenotype is highly variable. We examined index cases and affected family members enrolled in the Zimmerman Program with a phenotypic diagnosis of type 2 VWD. All subjects had factor VIII (FVIII), VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and multimer distribution analyzed in a central laboratory. For calculation of mean VWF:RCo values, a level of 5 was assigned to subjects with VWF:RCo below the laboratory lower limit of detection of 10 IU/dL. A platelet binding assay was also performed using a gain of function GPIb containing 2 mutations that enable spontaneous binding to VWF in the absence of ristocetin (VWF:GPIbM). Full length VWF gene sequencing was performed for all index cases. Targeted sequencing was performed for family members to ascertain the presence or absence of sequence variations found in the index case. Bleeding symptoms were quantified using the ISTH bleeding assessment tool and reported as bleeding scores (BS). Mean FVIII, VWF:Ag, VWF:RCo, and BS are listed in the table below for each type 2 variant. For type 2A VWD, 113 subjects have been enrolled to date. All had an abnormal multimer distribution with loss of high molecular weight multimers. 6 type 2A subjects had a VWF:RCo/VWF:Ag ratio of ≤0.7. The lowest VWF:RCo levels were seen in the type 2A cohort with 60%

Beschreibung: The diagnosis of variant von Willebrand Disease (VWD) currently involves evaluating multiple aspects of von Willebrand Factor (VWF) functions using separate, individual assays. This process can be time consuming and may delay definitive diagnosis of variant VWD. To address this issue, we have previously described a novel, rapid ELISA-based VWF functional screening assay that allows for variant VWD phenotype assignment and can provide same-day results. When considering application of this assay as a screening test, it is important to investigate its correlation with conventional clinical assays of VWF function. Therefore, the objective of our study was to investigate the relationship of our qualitative and semi-quantitative assay with traditional quantitative assays of VWF functions. 161 plasma samples from the Zimmerman PPG were analyzed on a novel ELISA-based platform, which measures relative values of VWF:Ag (antigen), VWF:IbCo (Ib cofactor, no ristocetin), VWF:RCo (ristocetin cofactor), VWF:FVIIIB (binding to FVIII), VWF:CBIII (binding to collagen III), and VWFpp (propeptide). Samples were compared to a 30% standard control plasma by a ratio of each unique VWF functional activity over the VWF:Ag. Data were compared to available quantitative data from the BloodCenter of Wisconsin clinical laboratory on the same patient plasma samples. Plasma samples included 21 type 1, 30 type 1C, 23 type 2A, 23 type 2B, 20 type 2M, 17 type 2N, 5 type 2N carriers (heterozygote), and 22 potential hemophilia A (HA) subjects. Data was analyzed by linear regression and power function. In all VWD samples analyzed by power function, VWF:Ag by our assay correlated with the clinical assay with an R2 of 0.794. For VWFpp from 111 VWD subjects, the power function revealed our assay correlated with the clinical assay with an R2 of 0.630, and sub-analysis of 51 type 1 and 1C VWD subjects showed an R2 of 0.673. VWF:CBIII analysis of all type 2A, 2B, and 2M subjects through linear regression revealed an R2 of 0.675. VWF:IbCo analysis was separated into type 2B and non-type 2B VWD subjects due to the significant VWF:IbCo enhancement seen in our assay because of augmented binding of type 2B VWF to glycoprotein Ib. While correlation of our VWF:RCo with the clinical VWF:RCo was poor, the VWF:IbCo was able to discriminate type 2B from non-type 2B. VWF:FVIIIB analysis showed complete discrimination of type 2N and potential HA subjects. In addition, there was discrimination of type 2N VWD subjects from type 2N carriers; however these data were not fit into the statistical model because there were too few subjects in each category. In summary, our study shows that each individual component of the VWF functional screening assay shows correlation with traditional quantitative assays of VWF functions. The best correlation was seen with the VWF:Ag, and there was also discrimination of type 2B, type 2N, type 2N carriers, and HA subjects demonstrated in our analysis. These data indicate the potential use of our assay as a laboratory screening test to expedite diagnosis of variant VWD. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Introduction: In recent years, the role of copy number variation (CNV) in the pathogenesis of type 1 von Willebrand disease (VWD) has been extensively explored. Large cohort studies including the MCMDM-1VWD and French studies have identified a total of eight partial VWF gene deletions, involving single and multiple exons, as well as one duplication. Furthermore, breakpoints have been identified within the MCMDM-1VWD cohort study for deletions involving exons 3, 32-34 and 33-34. Aim: To characterize and determine heterozygous deletion breakpoints of a deletion within the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD) cohort, previously identified through array CGH analysis and to determine the pathogenesis of the deletion through in vitro analysis. Methods: Identification of deletion breakpoints utilized a deletion-specific multiplex PCR approach, whereby two forward primers and one reverse primer were designed to determine the heterozygous exon deletion, based on previous optimization within the MCMDM-1VWD cohort. VWF antigen (VWF:Ag), VWF propeptide (VWFpp), and multimer distribution were performed in a central laboratory. Recombinant exon 33-34 deletion mutant (rVWFdel33-34) was created using site directed mutagenesis. Recombinant vectors were transiently transfected into HEK293T cells and Renilla used as a transfection control. Conditioned media and cellular lysates were collected 48hr post-transfection and analysed by VWF:Ag ELISA, comparing to wild-type VWF (rVWFwt) expression. Results: Three families enrolled in the ZPMCB-VWD displayed a deletion of VWF exon 33-34. The deletion was determined to be 3.4kb in size and led to an in-frame loss of exons 33-34, with breakpoints being identical to those identified in the MCMDM-1VWD study (c.5620+872_5842+2440delinsGCAGCATAAGCATAAAGC). VWFpp/VWF:Ag ratios were elevated in all affected family members with the exon 33-34 deletion (mean 4.3) as opposed to unaffected family members (mean 1.3). These results are consistent with findings in the MCMDM-1VWD family (mean VWFpp/VWF:Ag of 4.8), suggesting increased clearance of VWF. In silico analysis of the breakpoint region suggested no significant repetitive elements and microhomology mediated end-joining was hypothesised as the potential mechanism leading to the deletion. Expression of recombinant mutant rVWFdel33-34 revealed that secretion reduced by 52% in the homozygous state (p

Beschreibung: Background: Diagnosis of von Willebrand disease (VWD) is challenging in clinical practice due to variability in laboratory testing and clinical bleeding history. We investigated the prospective diagnosis of VWD in academic hematology clinics across the U.S. and Kingston, ON and report on the final cohort here. Methods: Subjects were enrolled as new consults to their hematologist for evaluation of a bleeding disorder from 11 centers. Laboratory results including VWF antigen (VWF:Ag) and VWF platelet binding activity were determined both locally (VWF ristocetin cofactor activity [VWF:RCo] or VWF:GPIbM) and centrally (VWF:GPIbM) to determine the comparative effectiveness in VWD diagnosis. Some centers defined type 1 VWD with levels