ALBERT

Description: 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

Description: 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%

Description: 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.

Description: Monitoring the correction of abnormal bleeding tendencies during the treatment of patients with hemostatic disorders is essential to evaluate success of therapy. While single clotting factor assays provide valuable information, global coagulation assays are desirable to better understand the overall hemostatic condition of patients. In Hemophilia A, severity of the clotting defect is traditionally evaluated by determining FVIII activity using chromogenic or clotting assays. Evaluation of thrombin generation in plasma samples for the assessment of bleeding tendencies in hemophilic patients has been suggested. Discriminating between samples with FVIII levels below 1%, however, has been challenging using FVIII activity and thrombin generation assays. We previously reported a native whole blood thrombin generation assay (nWB-TGA) that uses recalcification of whole blood samples without the addition of tissue factor to initiate clotting. We have shown that this assay is sensitive to varying levels of FVIII in vitroand to platelet targeted FVIII gene therapy in a murine model of Hemophilia A. The objective of the present study was to determine if the nWB-TGA can be used to monitor Hemophilia A patients during FVIII therapy and if this assay allows discrimination of whole blood samples with FVIII levels below 1%. Using the nWB-TGA we evaluated thrombin generation in a severe hemophilia A patient carrying an intron 22 inversion. Numerous data points were obtained from 15 different FVIII infusions, each targeting a FVIII level of 50%. Samples collected at least 72 hours (hrs) post infusion (〉6 half-lives, calculated FVIII levels

Description: Background: Von Willebrand disease (VWD) is the most common bleeding disorder found in children and adolescents. It has a varied clinical presentation, which likely contributes to challenges and delays in the correct diagnosis and the subsequent management of the disease. Objective: To characterize diagnosis, bleeding, and treatment patterns in children (2-11 years of age) and adolescents (12-17 years of age) with VWD. Methods: This retrospective database analysis utilized data from the IQVIA PharMetrics Plus Database of medical insurance claims for patients with VWD (ICD-9 286.4) from January 1, 2006 to June 30, 2015. Patients included had ≥2 medical claims for VWD and continuous enrollment for ≥2 years, to ensure a higher likelihood of definitive VWD diagnosis, before and after their 1st VWD claim. The pre-diagnosis period included 18 months of data before diagnosis. The post-diagnosis period included 7-24 months post-diagnosis data. Data from the first 6-month post-diagnosis period were excluded due to data variability, suggestive of treatment optimization. Descriptive statistics were used to summarize patient demographic and clinical characteristics, including types of bleeding episode (BE), rates, and outcomes; treating physician specialty; and type of VWD treatment, in both the pre- and post-diagnosis periods. Results: Of 1087 patients identified, 475 were children (43% female) with a mean (SD) age at VWD diagnosis of 6.9 (2.7) years, and 612 were adolescents (74% female) with a mean (SD) age at VWD diagnosis of 14.9 (1.6) years. The top 3 treating physician specialties seen by children in the pre- and post-diagnosis periods, respectively, were hospitalists (21% and 9%), primary care physicians (16% and 7%), and hematologists (11% and 3%). Adolescents were mostly seen by hospitalists (30% and 15%), primary care physicians (25% and 16%), and obstetrician gynecologists (19% and 15%). Only 11% of children and adolescents saw a hematologist prior to diagnosis, compared with 3% and 5%, respectively, post-diagnosis. A 17% vs. a 15% decrease in bleed claims in the pre- vs. post-diagnosis period was observed among children (40% vs. 23%) and adolescents (59% vs. 44%), respectively. The most common type of BE among children in the pre- and post-diagnosis periods was epistaxis (19% and 10%), and the trend was similar for boys and girls. Heavy menstrual bleeding was the most common BE type among adolescents overall in both the pre- and post-diagnosis periods (40% and 30%; in females 54% and 40%). Epistaxis was the second most common BE among adolescents overall in both the pre- and post-diagnosis periods (11% and 7%), and in females (9% and 5%), but the highest among males (17% and 12%). Of note, 3% of children had gastro-intestinal (GI) bleeds pre-diagnosis, reducing to 1% post-diagnosis. In adolescents, 1% had GI bleeds pre-diagnosis, rising to 2% post-diagnosis. Overall, VWD related treatment claims increased between the pre- and post-diagnosis periods for both children (12% vs. 23%) and adolescents (31% vs. 50%). The most prescribed treatments for bleed management in children were aminocaproic acid (ACA), desmopressin (DDAVP) and nasal cauterization (pre-diagnosis: 5%, 4% and 4%, respectively; post-diagnosis: 11%, 13% and 3%, respectively). For adolescents, the most prescribed treatments, pre- and post-diagnosis respectively, were oral contraceptives (22% [females 29%, males 0%] and 33% [females 45%, males 0%]), DDAVP (9% [females 8%, males 12%] and 19% [females 20%, males 14%]) and ACA (4% [females 4%, males 5%] and 11% [females 12%, males 7%]). Of note, 3% and 4% of children and 2% and 4% of adolescents received plasma-derived VWF concentrates pre- and post-diagnosis respectively. Conclusions: This analysis demonstrates a decrease in BE claims following VWD diagnosis and a rise in ACA and DDAVP treatment claims in both children and adolescents, and in oral contraceptive claims among female adolescents. Nevertheless, a considerable proportion of children and adolescents continue to experience BEs 6 months post-diagnosis. This emphasizes the need for treatment optimization and improvement in the care and management of patients in these age groups. Disclosures Roberts: Shire, a Takeda company: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bioverativ: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Octapharma: Membership on an entity's Board of Directors or advisory committees; Spark: Membership on an entity's Board of Directors or advisory committees. Malec:Spark: Honoraria; Sanofi: Consultancy, Honoraria, Speakers Bureau; Bayer: Honoraria; Takeda: Honoraria; CSL: Honoraria. Halari:Charles River Associates: Employment. Hale:Baxalta US Inc., a Takeda company: Employment, Equity Ownership. Oladapo:Baxalta US Inc., a Takeda company: Employment, Equity Ownership. Sidonio:Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Grifols: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kedrion: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Shire, a Takeda company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; BioMarin: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; uniQure: Membership on an entity's Board of Directors or advisory committees.

Description: 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

Description: Key Points A novel ELISA-based VWF multiplex activity assay assigns VWD phenotype among a cohort of type 1 and 2 VWD with an overall accuracy of 〉88%. This assay shows correlation with traditional quantitative clinical VWF assays and may provide a rapid diagnostic method for variant VWD.

Description: Introduction:Antiphospholipid syndrome (APS) is characterized by arterial and/or venous thrombosis in the presence of antiphospholipid antibodies. Recently, derangement in the complement pathway has been implicated in APS pathophysiology. Refractory cases of APS with recurrent and potentially fatal thrombosis have prompted use of the monoclonal antibody eculizumab, which inhibits generation of the terminal complement complex. We present the successful use of eculizumab in controlling and preventing recurrent thrombosis in a refractory case of APS. Case Description:An 18-year-old female received a diagnosis of antiphospholipid syndrome after developing extensive unprovoked deep vein thrombosis of axillary, inferior vena caval and brachiocephalic veins. Thrombophilia evaluation revealed triple positivity for lupus anti-coagulant (LA), beta-2 glycoprotein I (GP) IgG 84.9 SGU Units, IgM 76.5 SMU Units, IgA 66.7 SAU Units and strongly positive anti-cardiolipin (aCL) antibodies (each 〉〉40U/mL) with persistent positive titers after 12 weeks of initial evaluation. She was refractory with trials of multiple anticoagulants alone and with antiplatelet and adjunctive therapies. Anticoagulants used were enoxaparin, fondaparinux, apixaban, rivaroxaban, and warfarin; antiplatelet agents used were aspirin and clopidogrel; and adjunctive therapies included hydroxychloroquine, immunosuppression with steroids and rituximab, and plasmapheresis. Despite these interventions, she continued to develop recurrent thrombosis of subclavian, femoral, common femoral, iliac, popliteal and saphenous veins. She additionally developed a potentially life-threatening hepatic infarction and pulmonary embolism, and 6 weeks of plasma exchange failed to decrease antiphospholipid antibody titers. Following this event, eculizumab (600mg weekly x 4 weeks, followed by 900mg once on week 5, followed by 900mg every 2 weeks) was initiated in combination with fondaparinux, aspirin, clopidogrel, and hydroxychloroquine. She has remained on this regimen with additional anticoagulation and antiplatelet therapy without recurrence of thrombosis over the subsequent year. Discussion:Recent evidence has shown complement activation playing a role in pathophysiology of antiphospholipid syndrome. This is thought to occur through C3 and C5a causing platelet and endothelial activation, leading to microvascular thrombosis. Histological evidence of such processes is supported by the demonstration of anti-beta-2 GP1 IgG-C5b-9 immune complexes in microvascular organ thrombi in individuals with APS leading to thrombotic microangiopathy and multiorgan failure. Such evidence of complement induced thrombosis in antiphospholipid syndrome led us to use eculizumab following failure of available therapies including anticoagulation, antiplatelet therapy, immune modulation and plasmapheresis in a potentially life-threatening event. Absence of recurrent thrombotic events over approximately one year with eculizumab combined with anticoagulation and antiplatelet therapy highlights the potential role of complement inhibition in preventing thrombosis in APS. While the role of complement in thrombosis continues to be elucidated, we have observed no decline in antiphospholipid antibody levels in a span of one year in our patient. Importantly, there has been no recurrent thrombosis during this time. Our case suggests that eculizumab may have a role as a therapeutic option in refractory thrombosis in APS. Chronology of Events:Eculizumab initiated on 9/5/2019. No evidence of venous or arterial thrombosis since 10/2019 to present. Figure Disclosures Tarantino: Pfizer:Other;Genentech:Membership on an entity's Board of Directors or advisory committees;Octapharma:Membership on an entity's Board of Directors or advisory committees;Dova:Membership on an entity's Board of Directors or advisory committees;CDC:Membership on an entity's Board of Directors or advisory committees;Grifols:Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Amgen:Membership on an entity's Board of Directors or advisory committees;Biomarin:Membership on an entity's Board of Directors or advisory committees;NovoNordisk:Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau;Takeda:Research Funding;HRSA:Membership on an entity's Board of Directors or advisory committees;Spark:Membership on an entity's Board of Directors or advisory committees;Sobi:Membership on an entity's Board of Directors or advisory committees.McCrae:Rigel:Consultancy;Novartis:Honoraria;Momenta Pharmaceuticals:Consultancy;Dova:Consultancy.Chaturvedi:Sanofi:Honoraria, Membership on an entity's Board of Directors or advisory committees;Alexion:Honoraria, Membership on an entity's Board of Directors or advisory committees.Roberts:Octapharma:Consultancy, Speakers Bureau;uniQure:Consultancy;Sanofi:Consultancy, Speakers Bureau;Novo Nordisk:Consultancy, Speakers Bureau;Pfizer:Consultancy;Takeda:Consultancy, Research Funding, Speakers Bureau. OffLabel Disclosure: eculizumab for treatment of refractory thrombosis in antiphospholipid syndrome

Description: Background: Von Willebrand Disease (VWD) is the most common inherited bleeding disorder, thought to occur in ~0.1% of the population. VWD results from a quantitative (Type 1 or 3) or qualitative (Type 2) defect in von Willebrand Factor (VWF), a multifunctional plasma protein involved in primary and secondary hemostasis. Diagnosis of VWD can be difficult due to pre-analytical variables, a wide coefficient of variation in testing, and incomplete penetrance. Treatment of VWD is aimed at replacement of the defective or missing protein through plasma derived or recombinant VWF, release of endogenous VWF through desmopressin (DDAVP) or clot stabilization with anti-fibrinolytic therapy. Though individuals with mild VWD and bleeding symptoms are common, less is known regarding individuals with VWD and a clinically severe bleeding phenotype. Aims: To characterize the bleeding phenotype and treatment regimens in patients with clinically severe VWD in the United States. Study Design and Methods: ATHN 9 is sponsored by the American Thrombosis and Hemostasis Network (ATHN) and is being conducted at ATHN-affiliated sites across the US. Participants were identified by the site investigators with the projected goal to enroll 130 individuals. Inclusion criteria were patients with severe VWD defined as type 3 VWD, or VWF:RCo, VWF:GPIbM or VWF:Ag≤ 30% or patients with "clinically severe VWD" defined by VWF:RCo, VWF:GPIbM or VWF:Ag ≤ 40% a with severe bleeding phenotype (need for recurrent use of factor concentrates) and prior enrollment in the ATHN dataset national surveillance data collection project. Patients with platelet-type or acquired VWD were excluded. Laboratory assessment including a standardized diagnostic battery, VWF genetic analysis, and inhibitor testing, was performed by a central laboratory. Bleeding was assessed using the International Society for Thrombosis and Haemostasis (ISTH) Bleeding Assessment Tool (BAT) (normal adult 0-4, normal score