Ristocetin Cofactor Research Articles

Anticoagulation Use and Outcomes Among Patients with Atrial Fibrillation and Von Willebrand Disease

Intro: Von Willebrand disease (VWD) is a coagulopathy caused by deficiency or dysfunction of von Willebrand factor (VWF), resulting in prolonged and excessive bleeding. Patients are advised to avoid aspirin (ASA), P2Y12 inhibitors, or anticoagulation (AC) so as not to exacerbate this condition. However, typical treatment for atrial fibrillation (AF) includes anticoagulation, particularly if the risk of stroke by CHA 2DS 2-VASC score is 2+. Current recommendations suggest giving necessary antiplatelet (AP) or AC therapy over no treatment with assessment of bleeding risk throughout the course. However, this is a conditional recommendation based on low certainty in evidence, and there are no specific guidelines on treating AF in patients with VWD. This study aims to assess anticoagulation use, bleeding risk, and stroke risk in patients with VWF and AF.Methods: We conducted an IRB-approved analysis of coded data from institutional electronic medical records to select patients with diagnosis of VWD, low ristocetin cofactor level, or any abnormal VWF panel as well as patients with diagnosis of AF or atrial flutter. Three hundred and forty patients met criteria. Patients were manually screened for inclusion criteria and excluded for inaccurate diagnosis or insufficient data. Eighty-nine patients were included in the analysis. Primary endpoint was rate of major bleeding defined by ISTH criteria while on AC or AP. Categorical data were tested using the Fisher exact test at the nominal 0.05 two-sided significance level, and all person-time comparisons are made against the rate of bleeding on AC alone.Results: Most patients were female (64.0%; 57/89), and 28.1% (25/89) were deceased at the time of data collection. Date of diagnosis of AF ranged from 1980-2020. 42.7% (38/89) of patients were ever prescribed ASA, 43.8% (39/89) a P2Y12 inhibitor, 56.2% (50/89) AC, and 23.6% (21/89) had never been prescribed AP or AC. Of patients with a CHA 2DS 2-VASC of 2+, 57.5% (46/80) were ever prescribed AC. 32.0% (16/50) of patients ever prescribed AC and 25.6% (10/39) patients never prescribed AC had at least one major bleeding event (p=0.428). The rate of major bleeding on AC alone was 8.9 events per 100 person-years (32 events/359.2 years), 10.2 events per 100 person-years on AP alone (41 events/402.3 years) (p=0.572), and 1.06 events per 100 person-years (8 events/757.47 years) in patients never prescribed AC or AP (p=<0.0001). Notably, the rate of major bleeding on AC and AP together was 28.07 events per 100 person-years (23 events/81.94 years) (p=<0.0001) occurring in 7 patients, 6 of whom also had a diagnosis of acute coronary syndrome (ACS). Length of time to first major bleed is shown in Figure 1. 16.9% (15/89) of patients had thromboembolic strokes after diagnosis of AF, and 53.3% (8/15) of those strokes occurred when patients were not prescribed AC.Discussion: This retrospective observational study over 40 years characterizes AC and AP use in patients with VWD and AF. Only 57.5% of patients with CHA 2DS 2-VASC of 2+ received standard of care AC despite conditional recommendations to give necessary anticoagulation to patients with VWD. In parallel with the general population, AC use significantly increases the rate of major bleeding in patients with VWD, but there was no difference in bleeding rate between standard AC and AP monotherapy. However, major bleeding rates were notably elevated in patients prescribed concomitant AC and AP which most commonly occurred in the setting of ACS. This analysis is limited by its retrospective nature, the lack of granular details in the coded database, and incomplete data in older charts. Overall, these data do not support the use of AP monotherapy over standard AC to reduce bleeding rates for patients with VWD and AF. Additionally, AC and AP co-administration should be avoided due to high rates of major bleeding, but more studies are required to understand AP and AC strategies in patients with VWD, AF, and ACS. Although the rate of major bleeding is elevated with AC use in patients with VWD, there is no difference in lifetime prevalence of major bleeding events by AC vs no AC use. Finally, over half of thromboembolic strokes occurred when not prescribed AC. Shared decision-making around stroke and bleeding risk is advised in considering AC use for AF in patients with VWD. Prospective studies should further evaluate the risk of major bleeding and stroke in patients with VWD and AF on standard AC vs no AC. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bleeding Outcomes in People with Von Willebrand Disease Receiving Antithrombotic Therapy

▪Aging brings additional challenges in the management of people with von Willebrand Disease (VWD). Plasma von Willebrand Factor (VWF) levels may increase but the impact on bleeding phenotype is unclear. With the development of age-related comorbidities, the use of antiplatelet (AP) or anticoagulant (AC) therapies may be warranted. As highlighted in the 2021 international VWD guidelines, limited evidence exists regarding the bleeding risk and safety of AP/AC use in people with VWD. 1 We sought to address this knowledge gap through a retrospective review of a large cohort of people with VWD attending a tertiary referral center.The records of all patients aged >50 years (y) attending our center registered with VWD were retrospectively reviewed. We identified all individuals treated with AP and/or AC, recording the indication for and duration of therapy. We also recorded disease subtype, baseline and most recent plasma VWF levels and bleeding on AP and/or AC. Bleeding episodes were stratified according to the World Health Organization (WHO) Bleeding Scale.From 255 eligible patients, 18 patients (7 male, 11 female) were identified who received AP and/or AC over a period of 22y. The median age at commencement was 60.2 years (range 40.0-74.5). 15 patients had Type 1 with baseline levels 30-50 IU/dL, (median VWF antigen, VWF:Ag, 57.5, range 39-87 IU/dL; median VWF ristocetin cofactor levels, VWF:RCo, 43, range 35-54 IU/dL). 3 patients had type 2 VWD (median VWF:Ag 64, range 27-90 IU/dL; median VWF:RCo of 16, range 10-66 IU/dL). The type of AP/AC used and indications for treatment are outlined in Table 1.Overall, 12 patients were treated with AP and 7 with AC therapy (19 therapies in total as one patient received first aspirin then warfarin therapy). The cumulative exposure to AP therapy was 61.9y with a median exposure time of 3.2y/patient (range 0.3-14.1y). Duration of AC therapy was shorter, with a cumulative exposure of 17.6y and a median of 1.5 y/patient (range 0.3-6.3y).Overall, 85.7% of patients on AC therapy had at least one episode of bleeding (6/7; 10 episodes total) in contrast to 58.3% of patients on AP (7/12; 12 episodes total). Of these 22 episodes, 5 (22.7%) were grade 1 bleeding. Grade 2 bleeding (iron deficiency or gastrointestinal (GI), gynecological or genitourinary bleeding) occurred in 5 patients (41.7%) treated with AP and 4 patients (57.1%) on AC (total of 14 episodes). 1 episode of grade 3 bleeding occurred in both the AP (8.3%) and AC (14.3%) group (GI bleeding requiring transfusion and abdominal hematoma respectively). The single grade 4 bleed was an intracranial hemorrhage (ICH) and occurred in a patient with type 2 VWD (VWF:RCo 10 IU/dL) on warfarin for atrial fibrillation; this required prothrombin complex concentrate, VWF concentrate, neurosurgical intervention and cessation of AC.Bleeding complications resulted in discontinuation of therapy in 2 patients (11.1%) treated; the individual with ICH and a patient with type 1 VWD on warfarin (baseline VWF:RCo 43 IU/dL) due to recurrent GI bleeding. No patients treated with AP therapy required discontinuation of use. The overall rate of major bleeding (WHO grade >/=3) in our study was 11.4 events/100 patient-years in VWD patients receiving AC therapy, in comparison to the rate of bleeding in the general population using AC of 7.2 events per 100 patient-years. 2For patients with type 1 VWD, plasma VWF levels were seen to increase during follow up (median 8.5y, VWF:Ag median +13 IU/dL, VWF:RCo +18 IU/dL), resulting in plasma VWF levels >50 IU/dL for 66.6% of patients in this cohort. Despite this, bleeding while on AP and/or AC was still experienced in 8/10 patients whose levels had normalized, necessitating cessation in one instance.In conclusion, this study provides important insights into the use of AP and/or AC in patients with VWD. Bleeding rates were higher in patients treated with AC therapy than AP resulting in cessation of therapy in 28.6% of those on AC. Bleeding events still occurred despite normalization of plasma VWF levels in patients with type 1 VWD. These data highlight the need for close follow up of patients with VWD whilst on antithrombotic therapy, particularly AC.1. Connell NT et al. ASH ISTH NHF WFH 2021 guidelines on the management of von Willebrand disease. Blood Adv 2021;5(1):301-325.2. Shoeb M, Fang M. Assessing Bleeding Risk in Patients Taking Anticoagulants. J Thromb Thrombolysis 2013;35(3):312-319. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The influence of surface roughness on the damage of von Willebrand Factor under shear flow condition.

Despite technological advances in mechanical circulatory support devices to treat end-stage heart failure, blood damage induced by non-physiological shear stress in operation often triggered clinical hemocompatibility complications. The loss of high molecular weight von Willebrand Factor (HMW-VWF) has been considered as an essential cause of gastrointestinal bleeding. In addition to the mechanics factors, interface factors may also affect blood damage, especially the surface characteristics. In this study, the effect of surface roughness on VWF damage under flow condition was investigated. A roller pump circulation experimental platform with a roughness embedded sample chamber was constructed to provide blood shearing flow condition. VWF molecular weight analysis, VWF antigen (VWF-Ag) concentration assay, and VWF ristocetin cofactor activity (VWF-Rico) assay were performed on the sheared blood samples. These variables are the main functional indicators of VWF. It was found that the surface roughness induced VWF damage is mainly caused by the loss of HMW-VWF rather than reducing the total amount of VWF. The threshold value of surface roughness for a rapid increase in the degradation of HMW-VWF under low flow rate was obtained between Ra 0.4 and 0.6 μm, which was smaller than the threshold for hemolysis. Our findings indicated that VWF is more sensitive to the interface factor of surface roughness than red blood cells, thus has a higher requirement for blood pump design. It could provide reference for the material design and processing in developing mechanical circulatory support devices.

HEMOSTASTATIC ALTERATIONS WITH DISEASE SEVERITY IN THE EARLY SYMPTOMATIC PHASE OF COVID-19

IntroductionThe main factors associated with disease severity in Covid-19 are age, sex, body weight, hypertension, and diabetes. Biomarkers of hemostatic activation have been shown to be independent predictors of disease severity in different populations.AimTo evaluate whether biomarkers of hemostatic activation were associated with clinical outcomes in patients admitted to a field hospital set up to provide initial care to patients in the early symptomatic phase of Covid-19.MethodsData and samples were obtained from June to September 2020. Laboratory evaluation included complete blood counts, PT, aPTT, fibrinogen, D-dimer, factor VIII activity, Von Willebrand Factor (VWF) (activity and antigen), C reactive protein (CRP) and P-selectin (ELISA). Patients were segregated by outcome, with clinical worsening defined as need for ICU, mechanical ventilation, pulmonary embolism, deep vein thrombosis or death.Results and discussionIn total 209 were enrolled in the study, of which 24 presented clinical deterioration (11.5%). In both groups there was more male patients. In the group of clinical worsening the mean age was 58.1 and improvement was 53.6 years old. Concerning smoking, 3.2% of patients that improved smoke. Regarding pulmonar infiltrate, it was verified in 50% in the group that worsening versus 41% in clinical improvement. No differences could be observed between patient subgroups regarding the presence of fever (63.2% vs. 62.5%), dry cough (75.1% vs. 87.5%) and dyspnea (65.9% vs. 54.2%) at admission. As main comorbidities, the groups presented chronic obstructive pulmonary disease (2.2% vs 8.3%), asthma (3.2% vs 4.2%), chronic heart failure (1.1% vs 8.3%), arterial hypertension (46% vs 41.7%) and diabetes (28.1% vs 33.3%) in comparing improved with clinical deterioration patients. In general, it was verified a significant decrease in platelet number (p = 0.0426), and an increase in the parameters of aPTT (0.0084), CRP (p = 0.0450), vWF antigen (p = 0.0022) and ristocetin cofactor (p = 0.0032).ConclusionOur results demonstrate that hemostasis activation is associated with clinical deterioration even at the early phases of Covid-19. The Ethics Research Committee of the University of Campinas approved all of the experimental procedures, and all individuals signed the informed consent form.

Assessment of Von Willebrand factor antigen and activity levels in inflammatory bowel diseases

Background There is a close interaction between inflammation and coagulation. Hemostatic abnormalities are common in inflammatory bowel disease (IBD) with higher risk for a hypercoagulable state and prothrombotic conditions. In addition, a few cases of acquired coagulopathy with higher risk of bleeding have been reported. The involved pathophysiologic mechanisms are complex and incompletely understood. Objective This is a case–control study that aimed to assess the levels of Von Willebrand factor (VWF) in IBD as a marker of disease activity and its relation to higher risk of bleeding or thrombotic events. Patients and methods A total of 46 patients with IBD aged 18 years or older were enrolled in the study. After consenting, patients were divided into two groups: one group included 23 patients with active IBD and the other group included 23 patients with inactive IBD. Activity of Crohn's disease was assessed through the Harvey-Bradshaw index and activity in patients with ulcerative colitis was evaluated with the simple clinical colitis activity index. The white blood cell count, hemoglobin level, platelet count, activated partial thromboplastin time, C-reactive protein, erythrocyte sedimentation rate, albumin, fecal calprotectin, VWF antigen level (VWF:Ag), and VWF ristocetin cofactor activity (VWF:RCo) were measured. The VWF:RCo/VWF:Ag ratio was calculated. Results There were significant differences in the mean±SD of vWF antigen and vWF:RCo levels between active IBD group (189.30±62.83 and 101.73±23.42, respectively, P=0.001) and inactive IBD group 177.30±64.90 and 97.08±24.21, respectively, P=0.001). The IBD activity index was correlated with VWF antigen (r=0.78 P=0.001) and VWF:RCo levels (r=0.74 P=0.001). VWF antigen and VWF:RCo were correlated with fecal calprotectin (r=0.65, P=0.001, and r=0.67, P=0.001, respectively). The odds ratio of an elevated vWF antigen greater than 150% was 24 (95% confidence interval: 4.38–131.47) in the group with active IBD compared with the inactive IBD group. The VWF:RCo/VWF:Ag ratio of less than 0.7 which reflects the possibility of acquired Von Willebrand syndrome was detected in five patients (21.7%) with active IBD in comparison with only one patient (4.3%) in the inactive IBD group, with odds ratio of 6.1 (95% confidence interval: 0.65–57.1). Conclusion VWF antigen and activity levels can be used as markers for evaluation of IBD activity. Assessment of VWF in IBD could be significant for better hemostatic control of such patients. Activated coagulation system in IBD is well known; however, precautions for coexisting acquired functional coagulopathy should be considered.

A Comparative Evaluation of an Automated Functional Assay for Von Willebrand Factor Activity in Type 1 Von Willebrand Disease.

Backgroundvon Willebrand factor ristocetin cofactor activity (VWF:RCo) is the standard functional assay used for von Willebrand disease (VWD) diagnosis. However, it has some drawbacks including being time consuming and labor intensive and having high inter-laboratory variability. The HemosIL VWF activity assay has the advantages of both high speed and automation. The purpose of this study was to prospectively compare these two functional assays for type 1 VWD detection.MethodsPlasma samples from 108 subjects were assessed in this study. HemosIL VWF activity was measured with the HemosIL latex immunoturbidimetric commercial kits by the ACL TOP coagulation analyzer. VWF:RCo was measured by platelet aggregation method. Pearson correlation analyses were performed to estimate the correlation of HemosIL VWF activity with VWF:RCo. Receiver-operator characteristic (ROC) curve analysis was used to evaluate the performance of the two diagnostic tests.ResultsThe correlation coefficient between VWF:RCo and HemosIL VWF activity was 0.874 overall and was 0.761 and 0.811 in the cohorts of type 1 VWD and non-VWD, respectively. The sensitivity and specificity of HemosIL VWF activity assay for type 1 VWD identification were 94.7% and 80.0%, respectively, and the ROC curve of HemosIL VWF activity was larger than that of VWF:RCo (0.928 vs 0.863, p=0.0138). Finally, the positive and negative predictive values of the HemosIL VWF activity assay for type 1 VWD detection were 72.0% and 96.6%, respectively.ConclusionOur results demonstrate that the HemosIL VWF activity assay was an effective method for type 1 VWD screening and diagnosis. It carried good sensitivity and specificity and had a higher ROC curve than VWF:RCo besides showing good correlation with VWF:RCo. With its advantages of greater speed and automated performance, these results suggest that the HemosIL VWF activity assay was reliable and precise in the clinical setting.

Upregulation of pulmonary tissue factor, loss of thrombomodulin and immunothrombosis in SARS-CoV-2 infection.

BackgroundSARS-CoV-2 infection is associated with thrombotic and microvascular complications. The cause of coagulopathy in the disease is incompletely understood.MethodsA single-center cross-sectional study including 66 adult COVID-19 patients (40 moderate, 26 severe disease), and 9 controls, performed between 04/2020 and 10/2020. Markers of coagulation, endothelial cell function [angiopoietin-1,-2, P-selectin, von Willebrand Factor Antigen (WF:Ag), von Willebrand Factor Ristocetin Cofactor, ADAMTS13, thrombomodulin, soluble Endothelial cell Protein C Receptor (sEPCR), Tissue Factor Pathway Inhibitor], neutrophil activation (elastase, citrullinated histones) and fibrinolysis (tissue-type plasminogen activator, plasminogen activator inhibitor-1) were evaluated using ELISA. Tissue Factor (TF) was estimated by antithrombin-FVIIa complex (AT/FVIIa) and microparticles-TF (MP-TF). We correlated each marker and determined its association with severity. Expression of pulmonary TF, thrombomodulin and EPCR was determined by immunohistochemistry in 9 autopsies.FindingsComorbidities were frequent in both groups, with older age associated with severe disease. All patients were on prophylactic anticoagulants. Three patients (4.5%) developed pulmonary embolism. Mortality was 7.5%. Patients presented with mild alterations in the coagulogram (compensated state). Biomarkers of endothelial cell, neutrophil activation and fibrinolysis were elevated in severe vs moderate disease; AT/FVIIa and MP-TF levels were higher in severe patients. Logistic regression revealed an association of D-dimers, angiopoietin-1, vWF:Ag, thrombomodulin, white blood cells, absolute neutrophil count (ANC) and hemoglobin levels with severity, with ANC and vWF:Ag identified as independent factors. Notably, postmortem specimens demonstrated epithelial expression of TF in the lung of fatal COVID-19 cases with loss of thrombomodulin staining, implying in a shift towards a procoagulant state.InterpretationCoagulation dysregulation has multifactorial etiology in SARS-Cov-2 infection. Upregulation of pulmonary TF with loss of thrombomodulin emerge as a potential link to immunothrombosis, and therapeutic targets in the disease.FundingJohn Hopkins University School of Medicine.

Von Willebrand factor multimeric assay: novel diagnostics capabilities

Introduction. Distinguishing between Von Willebrand disease (vWD) types often requires multimer gel analysis. The current techniques for vWF multimer structure are manual, complicated, non-standardized and time consuming. The aim of this study was to evaluate diagnostic capabilities of new automated vWF multimer screening assay.Materials and methods. Children with vWD, acquired von Willebrand Syndrome (aVWS) and 8 healthy donors as a control group were enrolled in this study. Von Willebrand factor antigen (vWF Ag); ristocetin cofactor activity (VWF:Rco); vWF collagen binding (VWF:CB); ristocetin-induced platelet aggregation (RIPA); factor VIII clotting activity (FVIII:C) and vWF factor VIII binding activity (vWF:FVIIIb) were performed to evaluate vWD. Multimer analysis was carried out using the commercial HYDRAGEL 5 von Willebrand Multimers kit on semi-automatic gel electrophoresis instrument HYDRASYS (SEBIA).Results. The samples from control group had 9—12 bands of vWF multimers with the same distribution as control plasma. Patients with type I vWD had the proportional decrease in the intensity of the bands with preservation of the normal distribution of the band. Patients with type III vWD reveal the complete absence of the multimer bands on the gel. Multimer analysis in type IIA shows the absence of high molecular weight multimer bands. In other patients the distribution of vWF multimers was normal against the changes in functional properties of vWF (types IIM, N). Most of the children with aVWS also revealed normal distribution of vWF multimers, however, in some patients, the slight decrease in large multimeric forms was observed visually on the gel.Conclusion. Multimer analysis allows to visualize the multimer distribution in various types of von Willebrand disease. The method is easy to perform and can be useful for distinguishing between the subtypes of vWD. But only the full test panel including genetic tests would allow the differentiantion of vWD types with high precision.

Coagulation function of never frozen liquid plasma stored for 40 days.

Never frozen liquid plasma (LP) has limited shelf life versus fresh frozen plasma (FFP) or plasma frozen within 24 h (PF24). Previous studies showed decreasing factor activities after Day (D)14 in thawed FFP but no differences between LP and FFP until D10. This study examined LP function through D40. FFP and PF24 were stored at -20°C until assaying. LP was assayed on D5 then stored (4°C) for testing through D40. A clinical coagulation analyzer measured Factor (F)V, FVIII, fibrinogen, prothrombin time (PT), and activated partial thromboplastin time (aPTT). Thromboelastography (TEG) and thrombogram measured functional coagulation. Ristocetin cofactor assay quantified von Willebrand factor (vWF) activity. Residual platelets were counted. FV/FVIII showed diminished activity over time in LP, while PT and aPTT both increased over time. LP vWF declined significantly by D7. Fibrinogen remained high through D40. Thrombin lagtime was delayed in LP but consistent to D40, while peak thrombin was significantly lower in LP but did not significantly decline over time. TEG R-time and angle remained constant. LP and PF24 (with residual platelets) had initially higher TEG maximum amplitudes (MA), but by D14 LP was similar to FFP. Despite significant declines in some factors in D40 LP, fibrinogen concentration and TEG MA were stable suggesting stored LP provides fibrinogen similarly to frozen plasmas even at D40. LP is easier to store and prepare for prehospital transfusion, important benefits when the alternative is crystalloid.

Contribution of Select ISTH-BAT Subscores to the Total Score in Type 1 VWD

Background:von Willebrand Disease (VWD) is the most common inherited bleeding disorder with partial quantitative deficiency in von Willebrand Factor (VWF); Type 1 comprises 80% of VWD. Clinical manifestations include mucocutaneous bleeding, prolonged bleeding after surgery or injury and excessive menorrhagia in females which have also been reported in normal subjects. A diagnosis of Type 1 VWD is based upon reduced plasma VWF, abnormal mucocutaneous bleeding symptoms and usually a family history of bleeding and VWD. Evaluating abnormal bleeding symptoms involves a detailed bleeding history, which can be quite subjective and time consuming. To address these challenges, the Vicenza-based Bleeding Assessment Tool (BAT) was developed as a validated score and has evolved into the current International Society on Thrombosis and Haemostasis-Bleeding Assessment Tool (ISTH-BAT). A large multi-center NIH supported study (The Zimmerman Program for the Molecular and Clinical Biology of VWD), began in 2005 to correlate clinical laboratory testing, bleeding severity determined by the ISTH-BAT and genetic variability in VWD. In this current study, we investigated which bleeding symptoms were most sensitive in applying the ISTH-BAT to the diagnosis of Type I VWD.Methods:Subjects were enrolled in the Zimmerman Program . Each symptom in the validated ISTH-BAT questionnaire was scored from 0-4 (no symptoms to symptom intervention) and included epistaxis, bruising, oral bleeding, hematuria, gastrointestinal (GI) bleeding, head bleeding, hemarthrosis, hematoma and prolonged bleeding with minor wounds, tooth extraction, surgery, menstruation and child birth. Other scored bleeding symptoms included bleeding at circumcision, excessive umbilical stump bleeding, venipuncture bleeding, vacuum extraction delivery bleeding, ovulatory bleeding and cephalohematoma. The scores of each symptom were analyzed in both healthy controls and subjects diagnosed with Type 1 VWD. Only subjects with VWF antigen (VWF:Ag) and/or VWF ristocetin cofactor activity (VWF:RCo) <30 IU/dL were considered in the comparative analysis. VWF levels were performed by a central laboratory, including VWF:Ag, VWF:RCo, VWF propeptide (VWFpp) and VWF collagen binding with type III collagen (VWF:CB). Statistical analysis included Mann-Whitney test, Chi-square or a Fisher's exact test, Pearson correlations and Spearman correlations. Results were considered significant for a two-sided p-value <0.05. A regression analysis was performed to examine which subscores contributed the most to the total score of the ISTH-BAT.Results:Analysis included 383 subjects (Female=262, Male=121) enrolled in the Zimmerman Program . Of these subjects, 244 were healthy controls (mean age of 38.93 [range 10-73 years of age]) and 139 had Type 1 VWD (mean age of 19.46 [range 1-76 years of age]). Within the Type 1 subjects, there were more females >12 years of age (no gender difference < 12 years of age) and greater number of Caucasians and younger subjects. Type 1 VWD subjects scored significantly higher than the healthy control group in both ISTH-BAT total scores (p<0.0001) as well as all subscores (p<0.001). In comparing bleeding symptoms from healthy controls with Type 1 VWD subjects: epistaxis 4% vs 46%, bruising 16% vs 64%, minor wound bleeding 1% vs 42%, oral bleeding 3% vs 35%, tooth extraction bleeding 3% vs 18%, GI bleeding 3% vs 11%, hematuria 0% vs 11%, surgical bleeding 4% vs 23%, menstrual bleeding 60% vs 85%, bleeding after delivery 2% vs 12%, other bleeding 0% vs 11% (47% were circumcision bleeding), head bleeding 0% vs 4%, hemarthrosis 0% vs 11%, and hematoma 0% vs 7%. Regression analysis revealed that epistaxis, bruising, excessive bleeding with minor wounds, oral bleeding, and menstrual bleeding subscores contributed the most to the ISTH-BAT total score (p<0.016).Conclusion:The ISTH-BAT currently is strongly predictive of bleeding symptoms in the diagnosis of Type 1 VWD. Though all ISTH-BAT subscores correlated with a diagnosis of Type 1 VWD, some subscores contributed more significantly to the total score than others and were more prevalent than in normal subjects. Accordingly, the sensitivity and specificity of ISTH-BAT subscores are critical to the assessment of bleeding in the diagnosis of Type 1 VWD. DisclosuresFlood:Shire: Consultancy; CSL Behring: Consultancy. Gill:Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. James:CSL Behring: Research Funding; Shire: Research Funding; Bayer: Research Funding.

Severe Hemophilia A in a Patient with Sexual Differentiation Disorder: A Case Report

Hemophilia A is an inherited X-linked disease where the VIII Factor (FVIII) production is altered, causing hemorrhagic diathesis. It can be classified into mild, moderate or severe, depending on the levels of circulating FVIII. It is suffered by males with an incidence of 1 in 5000 live births. Women are carriers, and they transmit the mutation in 90% of the cases, whereas 10% are de novo mutations. Females are usually asymptomatic, showing normal or intermediate FVIII factor, although it is estimated that 1 in 100.000 carriers will show severe bleeding manifestations due to FVIII severe deficit. For this to occur, some of the following physiopathologic mechanisms should be present: 1) heterozygous compound mutations affecting both alleles of the FVIII; 2) Homozygous mutations; 3) Extreme lionization of all the normal X chromosomes; 4) Numerical or structural alterations of the X chromosomes, as in Turner´s syndrome; 5) Genetical mutation in a patient with XY genotype and female phenotype.The objective of this report is to describe the case of a girl with severe Hempohilia A and XY cariotype.Case report:A 2-year-old female patient presented to consultation, with no remarkable previous perinatal history nor family history of hematologic diseases, referred due to spontaneous hematomas and bleeding events of difficult medical management with a 4 ISTH score. Coagulation studies were performed, showing a FVIII level of less than 1%, with the rest of the blood parameters being normal. Coagulation studies of the mother showed FVIII levels of 30%, Von Willebrand antigen of 59% and ristocetin cofactor of 43%. The genetic analysis of both the child and mother informed a type I inversion of the 22 intron. A karyotype analysis of the patient is done. She is phenotypically a female, but she has a 46XY genotype. At present she is being studied to rule out if the sexual differentiation disorder is due to the lack of SRY gen (Swyer´s syndrome) or to insensitivity to androgens (Morris´syndrome).From the moment of the diagnosis, the patient started prophylaxis with recombinant FVIII factor, not showing complications.DiscussionThe severe manifestations of the X linked diseases are infrequent in females. Approximately 30% of the carriers of hemophilia have an activity of the FVIII of less than 40%, with the consequent risk of bleeding. The most common cause for this is the selective inactivation of the normal X chromosome, that usually occurs in a balanced and randomized fashion. The diagnosis of hemophilia is made based on the FVIII activity dosage, and is confirmed with the molecular study of the mutations of the gene. In asymptomatic carriers, the status of homozygous or heterozygous mutation must be determined.Similarly, it is important to know what is the patient´s karyotype, because although its frequency is low, the disease can be the result of a sexual differentiation disorder, as it is the case presented here, or a chromosomopathy. In both cases is important to follow up the patient interdisciplinary with endocrinology, to treat the hormonal disorders related to each situation.ConclusionIt is of paramount importance to obtain a complete clinical and molecular diagnosis, to perform a correct treatment and follow up, and also offer an adequate genetic counseling. DisclosuresNo relevant conflicts of interest to declare.

A New Frontier Has Emerged That Enables the Reliable Diagnosis and Subtyping of Von Willebrand Disease (vWD) Clinical Patients in 48 Hours

Remarkably, classical von Willebrand disease (vWD) laboratory work-ups have been shown to require repeat laboratory testing 1 to 20 times to confirm diagnosis [1]. Furthermore, most diagnostic laboratory algorithms terminate with molecular confirmation [2, 3]. Here we present the method validation of a diagnostic laboratory approach that allows for the simultaneous functional and genetic diagnosis and subtyping von Willebrand disease (vWD) and pseudo- or platelet-type vWD within 48 hours. The clinical sensitivity of this test is approximately 85% for subtypes 2A, 2B, 2N, 2M and 3 vWD [4].The method validation is compliant with current Clinical Laboratory Improvement Act (CLIA) and applicable current Good Laboratory Practices (cGLP, 21 CFH Part 58) guidelines and therefore is suitable for clinical utilization and clinical trial utilization. The method validation protocol included the creation of reference intervals, reportable ranges, intra-run precision, inter-run precision, accuracy, lower limit of quantification (LLQ), linearity, analytical and clinical sensitivity assessment, analytical and clinical specificity assessment, interfering substances, stability and longitudinal quality control performance [Table 1.].Historically, no one test has been able to diagnose vWD. Classical laboratory work-ups include aPTT, factor VIII (8) activity, Ristocetin cofactor activity, vWF antigen, ratio calculations, multimeric composition and others. Each of these tests is influenced by numerous factors, such as inflammation, stress, infection, hormone replacement therapy, age, acute phase response, menstrual cycle, pregnancy exercise, ABO blood type, lupus anticoagulant and other factors, making traditional diagnosis a challenging moving target [3, 5, 6].The methodology used in this study employed simultaneous classical vWD laboratory testing and rapid next generation sequencing (NGS) optimized for 48-hour result availability [Table 2]. Furthermore, normal healthy and vWD 2N subtype samples can clearly be distinguished in this testing approach [Table 3].vWD occurs with bleeding symptoms at rate of 1 in 1,000 in the general population [6]. In 10% of those cases the bleeding is severe [3]. This prevalence makes vWD the most common congenital bleeding disorder known [6]. Acquire vWD (1-5% of cases) can be assessed by contrasting functional von Willebrand testing with genetic testing in a specialized coagulation, platelet and genetics laboratory. Common symptoms of vWD include menorrhagia, prolonged bleeding or bruising from minor trauma, recurrent nose bloods, excessive bleeding at childbirth and excessive bleeding associated with surgery.In conclusion, the standard clinical approach of diagnosing vWD can take months to years and require one or more referrals to specialized clinicians to establish diagnosis and subtyping. The data presented in this study proposes that an alternative method of rapidly characterizing suspected vWD patients simultaneously with both functional and genetic tests can revolutionize speed of diagnosis and treatment.vWD Subtypes: (each can have severe clinical presentations)Type 1 = 60%Type 2 = 30%Type 2A = 15%Type 2B = 5%Type 2M < 5%Type 2N <5% (clinically similar to mild hemophilia A)Type 3 = 5-10%Pseudo-vWD or Platelet-type vWD = rare (defect in GP1BA gene)Acquired vWD = 1-5%

Platelet Function Tests and Inflammatory Markers for the Differentiation of Primary Thrombocytosis and Secondary Thrombocytosis

BACKGROUNDPrimary thrombocytosis (PT) that results from clonal bone marrow abnormality such as myeloproliferative neoplasm (MPN) can induce abnormal platelet function and acquired von Willebrand disease. Secondary thrombocytosis (ST) that may accompany inflammations, infections, malignancies, or stress is associated with elevated markers of the acute phase reactants, including C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). The aim of this study is to evaluate role of platelet function tests by light transmission platelet aggregometry (LTA), plasma von Willebrand factor antigen (vWF:Ag), ristocetin cofactor activity (vWF:RCo) and inflammatory markers (ESR, CRP) for the differentiation of PT and ST.METHODSThis was a prospective study in patients age 18 years or older with platelet counts higher than 450 x 109/L who admitted or attended the out-patient clinics in Chiang Mai University Hospital during November 2016 to June 2017. They were classified to PT from MPN according to WHO 2016 Classification and ST. Primary outcomes were sensitivity (Se) and specificity (Sp) of platelet function tests by LTA for the differentiation of PT and ST. Secondary outcomes were Se and Sp of ESR, CRP, vWF, and vWF:RCo for the differentiation of PT and ST as well as correlation between these markers and thrombohemorrhagic complications in patients with PT and ST. Categorical variables were compared using the Chi-squared test or Fisher exact test as appropriate. Continuous variables were compared by t-test or Wilcoxon rank-sum test (Mann-Whitney test). Level of significance was defined as p-value < 0.05.RESULTSThere were 49 patients enrolled in the study, mean age was 50.26 ± 18.26 years. Twenty-four patients (49%) had PT including 13 chronic myeloid leukemia (54%), 9 essential thrombocythemia (38%), 1 primary myelofibrosis (4%), and 1 polycythemia vera (4%). Twenty-five patients (51%) had ST including 10 malignancies (40%), 8 chronic infections (32%), 4 post-splenectomy (16%), and 3 autoimmune diseases (12%). Mean platelet count was higher in PT compared with ST (1083.79 ± 469.46 x 109/L and 716.28 ± 200.01 x 109/L respectively, p = 0.006). There was no difference in age and sex between groups. Abnormal LTA induced by collagen, arachidonic acid (AA), and epinephrine (Epi) were significantly presented in PT compared with ST (29%, 25%, and 50% respectively in PT; and 0%, 0%, 3% respectively in ST; p = 0.004, 0.010, and 0.004 respectively). The Se and Sp of collagen vs. AA vs. Epi-induced abnormal platelet aggregation for the differentiation of PT from ST were Se of 29.2% vs. 25% vs. 50%, and Sp of 100% vs. 100% vs. 88%, respectively. Mean ESR and CRP were significantly higher in ST (ESR 51.08 ± 22.38 mm/hr and CRP 56 ± 61.74 mg/L) compared with PT (ESR 16.30 ± 18.17 mm/hr and CRP 17.39 ± 37.96 mg/L; p < 0.001 and p = 0.006, respectively). The Se and Sp of ESR > 48 mm/hr in the differentiation of ST from PT were 76% and 91.7%, respectively while Se and Sp of CRP > 14.8 mg/L were 60% and 83.3%, respectively. No significant difference in proportion of patients with low vWF:Ag between groups (8.3% in PT and 0% in ST; p = 0.141) whereas PT group had significant higher proportion of patients with low vWF:RCo (45.8% in PT and 12.0% in ST; p = 0.009). The Se and Sp of abnormal vWF:RCo to differentiation of PT from ST were 45.8% and 88%, respectively. Thrombohemorrhagic complications at the time of diagnosis was 21% in PT while no complication in ST (p = 0.006). Risk factors of thrombohemorrhagic complications were platelet counts more than 1,569.8 x 109/L (p = 0.03) and Epi-induced abnormal platelet aggregometry (p = 0.02).CONCLUSIONAbnormal platelet function determined by LTA with collagen, AA, epinephrine had high specificity to differentiate between PT and ST. vWF:RCo, ESR and CRP levels might be helpful for the differentiation of PT and ST. PT has higher incidence of thrombohemorrhagic complication compare to ST and is correlated with high platelet counts and Epi-induced abnormal platelet aggregometry. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Recombinant Von Willebrand Factor Administration: Dosing Considerations and Rapid Stabilization of Endogenous Plasma FVIII Levels in Patients with Severe Von Willebrand Disease

Recombinant Von Willebrand Factor Administration: Dosing Considerations and Rapid Stabilization of Endogenous Plasma FVIII Levels in Patients with Severe Von Willebrand Disease

Monitoring of von Willebrand factor inhibitors in patients with type 3 von Willebrand disease using a quantitative assay.

Antibodies inhibiting von Willebrand factor (VWF) develop in a subset of patients with type 3 von Willebrand disease (VWD3) and may be detected by their inhibition of ristocetin cofactor activity (VWF:RCo). Some also inhibit factor VIII activity (VIII:C). To describe monitoring of ten VWD3 patients for VWF inhibitors using a quantitative assay. VWF inhibitor was measured by comparing VWF:RCo activity of a mix of patient and pooled normal plasma (PNP) with a mix of buffer and PNP, using agglutination of fixed normal platelets in microtiter plates or lyophilized platelets in an aggregometer. VIII:C inhibitor was measured by Bethesda assay. Preanalytical heat treatment of patient plasma was used during treatment episodes. Four of 10 patients monitored developed VWF inhibitors, two detected during bleeding episodes refractory to treatment and two on routine screening. Data from the first five patients were used to establish an arbitrary unit, VWU, defined as the amount of inhibitor per millilitre of patient plasma inactivating 25% of the activity of 1mL of PNP. In three of four patients, both VWF:RCo and VIIII:C were inhibited at some time points, although VIII:C inhibition sometimes disappeared. In one patient, no VIII:C inhibition was seen. Two patients remained inhibitor positive more than 15 years after inhibitor detection, one became negative following immune tolerance induction, and one was deceased. VWF inhibitors can be quantitatively monitored in VWD3 patients. Preanalytical heat treatment may be required for their detection post infusion.

Increased VWF and Decreased ADAMTS-13 in COVID-19: Creating a Milieu for (Micro)Thrombosis.

von Willebrand factor (VWF) is a large adhesive multimeric protein involved in hemostasis. The larger the size (or number of VWF multimers), the greater the functionality of the protein. A deficiency or defect of VWF can lead to von Willebrand disease (VWD) and cause bleeding. Conversely, an increase in VWF may create an environment that promotes thrombosis. ADAMS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), sometimes called VWF-cleaving protease, is primarily responsible for controlling the size of VWF. The most severe deficiency (<10% of normal levels) of ADAMTS-13 arises in thrombotic thrombocytopenic purpura, a condition characterized by the presence of ultralarge VWF and clinically resulting in enhanced risk of thrombosis. However, ADAMTS-13 deficiency may result from other pathological processes. Of relevance is the recent finding that COVID-19 (coronavirus disease 2019) is associated with both increased levels and activity of VWF as well as generally decreased (or occasionally normal) activity levels of ADAMTS-13. Thus, in COVID-19 there is an alteration in the VWF/ADAMTS-13 axis, most often described by increased VWF/ADAMTS-13 ratio (or reduced ADAMTS-13/VWF ratio). COVID-19 is also associated with high prothrombotic risk. Thus, the imbalance of VWF and ADAMTS-13 in COVID-19 may be providing a milieu that promotes (micro)thrombosis, in a clinical picture resembling a secondary thrombotic microangiopathy in some patients. This review therefore assesses the literature on VWF, ADAMTS-13, and COVID-19. Whenever reported in COVID-19, VWF has always been identified as raised (compared with normal reference ranges or control populations). Reports have included VWF level (i.e., VWF antigen) and in some cases one or more VWF "activity" (e.g., collagen binding; platelet glycoprotein Ib [GPIb] binding, using ristocetin cofactor or more modern versions including VWF:GPIbR [recombinant] and VWF:GPIbM [mutant]). Whenever reported, ADAMTS-13 has been reported as "normal" or reduced; however, it should be recognized that "normal" levels may still identify a relative reduction in individual cases. Some reports also discuss the raised VWF/ADAMTS-13 (or reduced ADAMTS-13/VWF) ratio, but very few provide actual numerical data.

Relationship between Von Willebrand Factor and Gastrointestinal Bleeding Compared EVAHEART and HeartMate II

Purpose Gastrointestinal (GI) bleeding is crucial issue in long-term management of continuous-flow (CF) left ventricular assist device (LVAD) patients, and it has been reported that lack of high-molecular-weight (HMW) von Willebrand factor (vWF) multimers is one of the most critical cause of GI bleeding. Aim of this study was to investigate a relationship between the prevalence of GI bleeding in LVAD and the effects of vWF activities including the comparison between EVAHEART and HeartMate II. Methods We performed EVAHEART implantation for 36 patients and HeartMate II for 6 patients with advanced heart failure (INTERMACS profile I - IV, eligible for heart transplantation, mean age 43 years old) between March 2011 and December 2017. Laboratory data of factor VIII-antigen (VIII-Ag), vWF ristocetin cofactor (RCo), and the presence of HMW-vWF multimers were measured post-operatively over time. The primary outcome was comparison of VIII-Ag and vWF RCo between two groups. The analysis was performed using Wilcoxon signed-rank test and a two-factor repeated-measures analysis of variance. Results Thirty patients (71%) survived with a mean follow-up period of 1157 (range 14-2255) days, and 25 of them underwent heart transplantation. There was no significant difference of survival rate (75% vs 67%, p=0.67), the loss of HMW-vWF multimers (3% vs 0%, p=0.68), and the incidence of GI bleeding (8% vs 0%, p=0.46). The EVAHEART group showed higher vWF RCo than the HeartMate II (p Conclusion This result suggested that EVAHEART maintained vWF RCo, and there was a possibility that EVAHEART provided low incidence of GI bleeding by means of maintenance the HMW-vWF multimers.

Acquired von Willebrand syndrome in monoclonal gammopathy – A scoping review on hemostatic management

BackgroundAcquired von Willebrand syndrome (AVWS) has been associated with monoclonal gammopathy of undetermined significance (MGUS), with limited data on its management. MethodsWe conducted a systematic literature search in Medline (Ovid), Embase, and Scopus up to September 11, 2019, for studies reporting on the management of AVWS associated with MGUS (AVWS‐MGUS). Data on patient characteristics, laboratory parameters at presentation, and clinical and laboratory outcomes were extracted. ObjectivesTo describe the clinical presentation and outcomes of different therapeutic approaches. ResultsSeventy‐five studies were included in the final review, for a total of 137 patients. Most patients had von Willebrand factor ristocetin cofactor activity <30 IU/dL (86.6%) and factor VIII levels <50 IU/dL (91.8%). Bleeding severity ranged from no bleeding (16.1%) to minor bleeding (46.4%) and major bleeding (37.5%). The overall clinical success rates for 1‐deamino‐8‐D‐arginine vasopressin (DDAVP), factor replacement therapy, and intravenous immunoglobulin (IVIG) were 43.8%, 33.3%, and 85.4%, respectively. The laboratory response rates for DDAVP, factor replacement therapy, and IVIG were 39.0%, 62.9%, and 88.6%, respectively. Several other treatments were also reported in small numbers, out of which myeloma‐directed therapies, plasma exchange, recombinant factor VIIa, and antifibrinolytics appeared most successful, while immunosuppressive agents were largely ineffective. ConclusionIVIG appears to be an effective treatment for AVWS‐MGUS bleeding, conferring a high clinical success rate with measurable laboratory outcomes; albeit temporary. DDAVP and factor replacement therapy may be partially successful in controlling minor bleeds, but not major bleeds. Other less commonly used agents may be effective in certain cases, although data are limited.

The diagnosis and management of von Willebrand disease

Von Willebrand disease (VWD) is a congenital bleeding disorder caused by quantitative and qualitative abnormalities in von Willebrand factor (VWF). VWD was first reported in 1926 by Dr. Erik von Willebrand, a Swedish physician, who reported a bleeding disorder in a family. VWD manifests as bleeding from the skin and mucous membranes (abnormal primary hemostasis). The symptoms are less severe than those of hemophilia and may be undiagnosed in many cases. Evidence-based guidelines are required for selection and patient management. Since ristocetin cofactor activity (vWF:RCo) and VWF antigen levels (vWF:Ag) are not routinely measured in clinical laboratories, the actual diagnosis is often determined by a mild prolongation of the activated partial thromboplastic time (APTT) associated with a relative decrease in factor VIII activity. Particularly, it is desirable to provide standard treatment measures on how to introduce the recently proposed bleeding score (BS; clinically scored for various bleeding symptoms of VWD) and how to deal with gynecological symptoms, such as excessive menstruation and irregular vaginal bleeding. In terms of treatment options, recombinant vWF concentrates were introduced in 2020, which has led to the expansion in the range of treatment options for patients.

Altered von Willebrand Factor and ADAMTS13 Levels in Children With Cirrhosis and Extrahepatic Portal Hypertension.

This study was concerned with whether vWF (von Willebrand factor) and a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13 (ADAMTS13) has altered in patients with cirrhosis and extrahepatic portal hypertension (EPH). We aimed to investigate changes to vWF and ADAMTS13 in children with cirrhosis and EPH. This study was conducted between January and October 2019 with both cirrhosis and EPH patients and with healthy volunteers. The von Willebrand factor antigen (vWF:Ag), von Willebrand Ristocetin cofactor (vWF:RCo), and ADAMTS13 antigen and activity were studied. Twenty-eight children with cirrhosis, 16 children with EPH, and 20 healthy controls were included in the study. vWF:Ag and vWF:RCo levels were higher in patients with cirrhosis than in healthy controls (171.65±101.67 vs. 85.86±30.58, P<0.01 and 121.62±55.83 vs. 61.52±27.03, P<0.01, respectively). vWF:Ag and vWF:RCo levels were higher in patients with EPH than in healthy controls (133.93±80.13 vs. 85.86±30.58, P<0.01 and 103.18±58.55 vs. 61.52±27.03, P=0.02, respectively). The ADAMTS13 antigen and activity levels were lower in patients with cirrhosis than in healthy controls (0.58±0.23 vs. 0.97±0.15, P<0.01 and 49.91±22.43 vs. 86.51±22.07, P=0.02, respectively). The ADAMTS13 antigen and activity levels were lower in patients with EPH than in healthy controls (0.69±0.11 vs. 0.97±0.15, P=0.03; and 68.50±13.29 vs. 86.51±22.07, P=0.02, respectively). The increase in vWF and the decrease in ADAMTS13 were more pronounced in cirrhotic patients with autoimmune hepatitis (AIH) than in non-AIH patients. While levels of vWF:Ag and vWF:RCo increased in children with cirrhosis and EPH, levels of the ADAMTS13 antigen and ADAMTS13 activity decreased. These alterations were more pronounced in patients with AIH-derived cirrhosis.