Von Willebrand Factor Assays Research Articles

Analytical and Clinical Validation of a Non-Ristocetin Based VWF Assay on 2 Automated Analyzers in a Large Reference Laboratory.

Historically, von Willebrand factor (VWF) activity assays utilized ristocetin despite limitations including poor limits of detection and high imprecision. Newer VWF activity assays such as the INNOVANCE® VWF Ac assay, however, do not rely on ristocetin to measure platelet-dependent VWF function. The purpose of this study was to evaluate the analytical and clinical performance of the Siemens Healthineers INNOVANCE VWF Ac Assay on the Siemens BCS® XP and the Sysmex® CS-2500 systems in a large reference laboratory setting. Performance indicators for the INNOVANCE VWF Ac assay were the limit of quantitation (LoQ), precision, and method comparison. Method comparison studies were performed using remnant plasma patient samples from routine coagulation tests and analyzed using both the INNOVANCE VWF Ac assay and the Siemens Healthineers ristocetin-dependent BC von Willebrand Reagent. Evaluation of the INNOVANCE VWF Ac assay on the BCS® XP and CS-2500 systems demonstrated good precision and a lower LoQ compared to the BC von Willebrand Reagent. Method comparisons support the use of the INNOVANCE VWF Ac assay on the BCS® XP and CS-2500 systems to measure platelet-dependent VWF function. The INNOVANCE VWF Ac assay was able to further assist in von Willebrand disease classification in 6/7 (86%) samples when the result was below the LoQ for the BC von Willebrand Reagent (ristocetin cofactor activity). These data are consistent with the 2021 American Society of Hematology/International Society on Thrombosis and Haemostasis/National Hemophilia Foundation/World Federation of Hemophilia von Willebrand disease guidelines that suggest using newer assays such as the INNOVANCE VWF Ac assay in place of ristocetin cofactor activity assays.

Unravelling the spectrum of von Willebrand factor variants in quantitative von Willebrand disease: results from a German cohort study

BackgroundVon Willebrand disease (VWD), the most prevalent hereditary bleeding disorder, results from deficiency of von Willebrand factor (VWF). ObjectivesThis large cohort study aims to offer a comprehensive exploration of mutation spectra and laboratory features in quantitative VWF deficiencies, shedding light on genetic underpinnings and genotype-phenotype associations. MethodsOur cohort consisted of 221 Caucasian index patients with quantitative VWD, along with 47 individuals whose plasma VWF levels fell within the lower normal boundaries (50-70 IU/dL). We conducted comprehensive VWF assays and genetic analyses, encompassing VWF gene sequencing, copy number variation investigations, and bioinformatic assessments. ResultsFollowing International Society on Thrombosis and Haemostasis-Scientific and Standardization Committee VWF guidelines, 77 index patients were characterized as having type 1 VWD (VWF antigen [VWF:Ag] < 30 IU/dL), 111 as having type 1 VWD (VWF:Ag, 30-50 IU/dL), and 33 as having type 3 VWD. Mutation detection rates were 88%, 65%, and 92%, respectively. Notably, blood group O overrepresentation was evident in type 1 with VWF:Ag of 30 to 50 IU/dL, particularly among mutation-negative patients, suggesting a potential causal role of blood group O. A total of 223 VWF variants, comprising 147 distinct variations, were identified in quantitative VWD patients, of which 57 were novel variants (39%). Additionally, approximately 70% of individuals with VWF levels within the lower normal boundaries (50-70 IU/dL) displayed VWF variants. ConclusionOur data advance our understanding of the molecular mechanisms underlying quantitative VWD, offering valuable insights for future research and clinical management. Distinct mutation patterns were observed among subgroups, particularly the contrast between type 1 VWD (VWF:Ag < 30 IU/dL) and type 1 VWD (VWF:Ag, 30-50 IU/dL), an area with limited prior investigation.

Von Willebrand factor for outcome prediction within different clinical stages of advanced chronic liver disease.

The prognostic performance of von Willebrand factor (VWF) may vary across clinical stages of advanced chronic liver disease (ACLD). Therefore, we investigated the evolution of VWF and other biomarkers throughout the full ACLD spectrum and evaluated their stage-specific prognostic utility. We retrospectively included Viennese ACLD patients with available information on hepatic venous pressure gradient (HVPG), C-reactive protein (CRP)/VWF levels and outcomes. ACLD stages were defined according to D'Amico etal. We included an external validation cohort from Padua. We observed gradual increases in VWF throughout ACLD stages. In contrast, HVPG levelled off in decompensated ACLD (dACLD), whereas MELD showed only minor changes in the early stages and CRP did not increase until stage 3. VWF was associated with hepatic decompensation/liver-related death in compensated ACLD (cACLD) in a fully adjusted model, while it was not independently predictive of ACLF/liver-related death in dACLD. After backward selection, HVPG/CRP/VWF remained the main predictors of hepatic decompensation/liver-related death in cACLD. Notably, the performance of the non-invasive CRP/VWF-based model was comparable to invasive HVPG-based models (C-index:0.765 ± 0.034 vs. 0.756 ± 0.040). The discriminative ability of the CRP/VWF-based model was confirmed in an external validation cohort using another VWF assay which yielded systematically lower values. VWF is the only biomarker that gradually increases across all ACLD stages. It is of particular prognostic value in cACLD, where a CRP/VWF-based model is equivalent to an invasive HVPG-based model. Systematic differences in VWF underline the importance of interlaboratory surveys. Moreover, our findings reinforce the notion that, already in cACLD, inflammation is a key disease-driving mechanism.

Discrepant low von Willebrand factor activity results on the ACL TOP analyzer are frequent in unselected patients with myeloproliferative neoplasms and show no correlation with high-molecular-weight multimer loss or bleeding phenotype

BackgroundBleeding complications are common in patients with myeloproliferative neoplasms (MPNs), with a subset developing acquired von Willebrand disease. Despite this association, a wide spectrum of von Willebrand factor (VWF) abnormalities are described, and the performance of modern assays remains unclear. ObjectivesTo comprehensively describe the pattern of VWF laboratory abnormalities in the MPN population. MethodsWe collected samples from 74 unselected clinic patients with MPNs to evaluate VWF quantitatively and qualitatively via multiple methods, correlating findings with a retrospective analysis of clinical bleeding data. VWF assays were performed on both ACL TOP (Instrumentation Laboratory) and Acustar (Instrumentation Laboratory) analyzers using HemosIL reagents (Instrumentation Laboratory), along with multimer analysis by gel electrophoresis. ResultsFunctional VWF measurements were not concordant between assays, with a median ACL TOP VWF glycoprotein IbR to antigen ratio (VWF:GPIbR/VWF:Ag) of 0.57 (IQR, 0.43-0.71) compared to a median Acustar VWF:GPIbR/VWF:Ag of 0.91 (IQR: 0.82-1.03;P < .001). The ACL TOP showed disproportionately lower results, with 73% of patients having a ratio <0.7. Despite this, no patient experienced loss of high-molecular-weight multimers by gel electrophoresis. An inverse relationship was observed between platelet count and functional ratios on both ACL TOP (R2 = 0.20; P < .001) and Acustar (R2 = 0.18; P = .0011) analyzers. While clinically significant bleeding events were relatively common (11% patients), there was no association with VWF assay abnormalities, and generally, an alternate cause(s) was identified. ConclusionDiscrepancies in functional VWF assays are common in patients with MPN, particularly by ACL TOP VWF:GPIbR. Based on our limited series, a VWF functional to an antigenic ratio of <0.7 (“type 2 pattern”) alone is poorly predictive of bleeding risk.

In vitro field study and worldwide survey assessing how clinical haemostasis laboratories analyse recombinant and plasma-derived von Willebrand factor products.

Several well-established clinical laboratory methods are available to measure von Willebrand factor (VWF) in plasma samples, but few data are available on their use for analysing recombinant VWF (rVWF). To evaluate how clinical diagnostic laboratories analyse rVWF and plasma-derived VWF (pdVWF) spiked in vitro into VWF-deficient plasma using quantitative protein and functional assays of VWF. Human VWF-deficient plasma samples were spiked with rVWF (vonicog alfa; Takeda) or pdVWF/factor VIII (pdVWF/FVIII; antihemophilic factor/VWF complex [human], CSL Behring), each at final concentrations of 1.0, 0.6, 0.2, 0.1 IU/mL VWF:ristocetin cofactor activity (VWF:RCo) according to labelled VWF activity. The ISTH SSC secondary coagulation standard was used as a control. Participating laboratories received three sets of these blinded aliquots. Mean results per assay were compared with the expected potency based on the labelled VWF:RCo activity. Among 39 laboratories, the most commonly established assay was VWF:RCo; 22 laboratories reported data from 2214 tests. Despite a trend to lower values, VWF:RCo activities for rVWF were in agreement with target concentrations (71%-109%), whereas VWF:platelet glycoprotein Ib (VWF:GpIb) and VWF collagen-binding activity (VWF:CB) assays gave high recoveries (up to 132% and 127%, respectively). In contrast, pdVWF/FVIII was substantially underestimated by VWF:GpIb and VWF:CB assays (56%-86% recoveries), whereas the VWF:RCo assay gave recoveries of 47%-112% for pdVWF/FVIII. The results of VWF assays used in clinical laboratories differ between rVWF and pdVWF, particularly for VWF:GpIb and VWF:CB assays. These differences may arise from the higher multimeric structure of rVWF compared to pdVWF.

External Quality Assessment Data for Investigation of von Willebrand Disease: Focus on Relative Utility of Contemporary Functional von Willebrand Factor Assays. The United Kingdom National External Quality Assessment Scheme (UK NEQAS) Experience.

Von Willebrand disease (VWD) is one of the most common hereditary bleeding disorders. Effective management of patients and their families depends on accurate diagnosis and subtype classification, and quality assurance including participation in proficiency testing programs is essential to ensure the accuracy of the panel of assays required to achieve this diagnosis. We report here findings from recent external quality assessment (EQA) exercises, as well as from a questionnaire about diagnostic practices employed by centers in the United Kingdom National Quality Assessment Scheme (UK NEQAS) performing von Willebrand factor (VWF) assays. Plasma samples from donors with VWD, "normal" donors, the International Society for Thrombosis and Haemostasis Scientific Subcommittee (ISTH SSC) plasma standard, and whole blood samples were sent to participants in the UK NEQAS BC program for VWF investigation. Calibration of lot#5 of the ISTH SSC plasma standard was shown to give improved comparability between the recovered value from an EQA exercise and the assigned potency for VWF activity assays. Diagnostic accuracy and precision amongst UK NEQAS participants was good, with an average 99% of centers reporting the correct interpretation for normal, type 1 and type 2 VWD samples, 100% diagnostic accuracy for centers performing FVIII binding assays, and good agreement amongst centers performing multimeric analysis. Genetic analysis of the VWF gene by specialist centers demonstrated errors in the genotyping process in one center, but also demonstrated failings in the interpretation of results in other centers. Despite evidence of good laboratory accuracy and precision in their assays, a questionnaire identified marked variation in diagnostic criteria employed, underlining the importance of guidelines to support the diagnosis of VWD.

Trancatheter Aortic Valve Implantation in the Treatment of Heyde's Syndrome: A Case Report

Calcified aortic stenosis is the most common valvulopathy of the elderly. Its association with angiodysplasia and digestive bleeding is known as Heyde syndrome. This clinical case highlights the importance of the diagnosis of this syndrome whose complications can be fatal. The cessation of gastrointestinal bleeding is often remarkable after aortic valve replacement. Transcatheter aortic valve implantation (TAVI) can represents a good alternative to surgical valve replacement, especially in patients with high surgical risk. We report a case of 62-year-old patient, admitted for management of global cardiac decompensation on a tight aortic stenosis, confirmed by transthoracic echocardiography as well as a few episodes of syncope. He had a history of repeated lower digestive hemorrhages that required several transfusions. The evolution was marked by the appearance of heavy rectal bleeding on cecum angiodysplasia lesions confirmed by colonoscopy. The association of digestive bleeding and aortic stenosis raised the diagnosis of Heyde syndrome. An assay of von Willebrand factor (vWF) antigen, vWF binding collagen activity and their ratio confirmed a qualitative deficiency of vWF type 2A. The proposed treatment was TAVI because of the high surgical risk and the patient's refusal of the surgery. The evolution at 1 month post TAVI was marked by the improvement of hemostatic parameters related to the vWF and the disappearance of cardiac symptomatology. The cessation of gastrointestinal bleeding in Heyde's syndrome is conditioned by percutaneous aortic valve replacement in the high-risk surgical subject with a good clinical and biological evolution.

Evaluating Performance of Contemporary and Historical von Willebrand Factor (VWF) Assays in the Laboratory Identification of von Willebrand Disease (VWD): The Australasian Experience.

von Willebrand disease (VWD) is a common bleeding disorder that arises from deficiency and/or defects of von Willebrand factor (VWF). Appropriate diagnosis of VWD, including differential identification of qualitative (types 2A, 2B, 2M, 2N VWD) versus quantitative (types 1 and 3 VWD) defects remains problematic but has important management implications, given differential therapy. Complete assessment for VWD in a patient with a bleeding history requires comprehensive test panels, including VWF activity and antigen. We describe the Australasian experience, using data from the Royal College of Pathologists of Australasia (RCPA) Quality Assurance Program (QAP) related to VWF testing in their VWD test module. The RCPAQAP has been providing samples for VWF testing since 1998, representing 25 years of proficiency testing related to VWD diagnosis. A total of 109 samples have been dispatched to participants over these years, with current assessment involving dispatches of two samples (=4 samples) per year. Samples have represented all types of VWD, as well as normal or other samples, including acquired von Willebrand syndrome and plasma VWF concentrates as used in VWD therapy. Different VWF assays and activity/antigen ratios show different utility in VWD and type identification. In the past 9 years of data capture, a total of 166 errors were identified from a total of 1,839 interpretations, representing a base error rate of 9.0%. Identification errors were highest for type 2 VWD samples (15.3%), intermediate for type 1 VWD samples (7.5%), and lowest for normal samples (2.4%). Errors can be linked to assay limitations, including assay variability and low-level VWF detection limits, as well as laboratory issues (including test result misinterpretation, which accounts for approximately 40% of all errors for type 2 VWD). For test-associated errors, VWF:RCo and VWF:GPIbM were associated with the highest variability and error rate, which was up to 10x higher than that using VWF:CB. As a test group, chemiluminescence-based procedures were associated with lowest inter-laboratory variability, best low-level VWF detection (down to <1 U/dL), and least errors overall. These findings inform on reasons behind high rates of errors associated with VWD diagnosis, with some assays and methodologies performing substantially better than others.

Advances in laboratory assessment of thrombosis and hemostasis.

Technologies in laboratory diagnostics are changing fast with progress in understanding and therapy of diseases. Unfortunately, new analyzers are often needed to be installed in a clinical laboratory to implement such techniques. The demand for new hardware is a bottleneck in improving the diagnostic services for many facilities with limited resources. In this regard, hemostasis laboratories take a slightly different position. Because many in vitro diagnostic tests target the functional aspects of hemostasis, further meaningful information can be obtained from the same analyzers as in current use. Automated coagulometers are good candidates for such further utilization. Clot waveform analysis is a leading example. Behind the simple values reported as clotting time, clotting curves exist that represent the process of fibrin clot formation. Clot waveform analysis examines the clotting curves and derives new parameters other than clotting times. The clot waveform parameters are now in active use in assessing the hemostatic potential of hemorrhagic patients. Clinical application of coagulometers can also be widened by modifying the reagent formulation. For example, the chromogenic factor VIII assay with bovine source reagent compositions has recently been introduced for hemophilia A patients on emicizumab prophylaxis. Also, new immunoturbidimetric functional assays for von Willebrand factor have been developed recently. Thus, new clinically relevant information can be mined from the automated coagulometers that are based on old technology.

In vitro study of red blood cell and VWF damage in mechanical circulatory support devices based on blood-shearing platform.

Blood damage induced by mechanical circulatory support devices (MCSDs) remains a significant challenge to optimal clinical care. Although researchers have been conducting in vitro studies, the major determinant of blood damage is still unclear. An optimized capillary tube blood-shearing platform with custom designed parts was constructed to investigate the influence of two flow-dependent parameters (shear stress and exposure time) on the shear-induced damage of red blood cells and von Willebrand factor (VWF). Blood samples under different high shear stress and instantaneous exposure time were obtained by changing the flow rate and the length of capillary tube. Plasma free hemoglobin assay and immunoblotting of VWF were then performed on the sheared blood samples. The quantitative correlation between the hemolysis index and the two flow-dependent parameters was found following the power law mathematical model under the flow condition with high shear stress and instantaneous exposure time. The degradation of high molecular weight VWF was not obvious under high shear stress factor. However, the degradation of high molecular weight VWF was found as the result of the accumulation over exposure time under non-physiological shear stress, which was consistent with the different mechanism of VWF damage comparing to red blood cell damage. Compared to peak shear stress, exposure time has a greater effect on both red blood cell and VWF damage. To improve the hemocompatibility of MCSDs, it is more important to avoid regions of slow blood flow with non-physiological shear stress under laminar flow conditions.

Von Willebrand factor assays in patients with acquired immune thrombotic thrombocytopenia purpura treated with caplacizumab.

Acquired immune thrombotic thrombocytopenic purpura (iTTP) is a rare disease with a poor prognosis if undiagnosed. It is caused by autoantibody production to the von Willebrand factor (VWF) cleaving protease, A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13). Caplacizumab, an immunoglobulin directed to the platelet glycoprotein Ibα receptor of VWF, has been reported to induce quicker resolution of iTTP compared to placebo. The laboratory measurement of VWF activity was significantly reduced in clinical trials of caplacizumab. Several VWF assays are available in the UK and this study investigated whether differences in VWF parameters were present in 11 patients diagnosed with iTTP and treated with daily caplacizumab. Chromogenic factor VIII activity, VWF antigen, collagen binding activity, VWF multimers and six VWF activity assays were measured prior to caplacizumab therapy and on several occasions during treatment. VWF antigen and collagen binding activity levels were normal or borderline normal in all patients. Ultra-large molecular weight multimers were present in all patients following treatment. VWF activity assays were normal or reduced during treatment, but this was reagent and patient dependant. In the unusual scenario of a caplacizumab-treated patient requiring measurement of VWF activity, it is important that laboratories understand how their local reagents perform as results cannot be predicted.

Incorporation of Evidence-based Guidelines on Bleeding Risk Assessment Prior to Surgery into Practice: Real-time Experience.

ObjectivesDespite guidelines recommending no need for coagulation testing before surgeries when a history of bleeding is negative, surgeons still overuse it in this part of the world. We aim to measure unbiased estimates of hemostatic outcomes in ear, nose, and throat (ENT) surgeries and assess the surgeons’ behavior of preoperative coagulation testing.MethodsWe enrolled all patients who underwent ENT surgeries from July 2017 to January 2018. The primary outcome was postoperative bleeding. Surgeons were asked about their decision on history alone or doing coagulation testing and their reason.ResultsWe recruited 730 patients; 372 were interviewed for a challenging bleeding history alone (group 1), and 358 had preoperative coagulation testing (group 2). Coagulation testing was repeated twice or more in 55.0% of patients, and more than half had coagulation factor and Von Willebrand factor assays. Most surgeons performed coagulation testing because of habitual practice.ConclusionsAlmost half of the local surgeons consider coagulation testing as standard to evaluate bleeding risk before surgical procedures. This resulted in unnecessary delays in surgeries, parent/patient anxiety, and additional total cost. We recommend awareness campaigns for surgeons and the involvement of surgical societies to adhere to guidelines of detailed hemostatic history.

Assessment of the Diagnostic Value of Plasma Level of Von Willebrand Factor and ADAMTS13 in Patients with Cerebral Infarction

Background: Von Willebrand Factor (VWF) is a large multimeric plasma gly-coprotein that plays an important role in primary haemostasis. VWF together with ADAMTS13 plays a key role in arterial thrombosis. VWF is integral for platelet adhesion to collagen fibres and atherosclerotic plaues and platelets aggregation under high shear conditions. Upon vascular wall damage, plasma VWF binds to collagen in the exposed endothelial matrix and platelet glycoprotein triggers platelet aggregation and thrombous formation. In the early stages of acute ischaemic stroke, VWF increased significantly and the ADAMTS13 level decreased. The increase of plasma VWF and FVIII in a patient with ischaemic stroke indicated an increased risk of complications and severe neurological deficit. Objective: to evaluate the validity of VWF and ADFAMTS13 as diagnostic markers of cerebral infarction by comparing the two markers’ validity in diagnosis. Methods: this case control study enrolled 38 cerebral infarction patients 20 acute infarction and 18 recurrent infarction. Patients were compared with 65 age and sex with control patients. We performed the VWF assay using the minividas immunofluorescence technique and the ADAMTS13 assay using the ELISA technique. Results: VWF level of acute infarction patients (280.97 ± 109.83 IU/dl) while its level in recurrent infarction (264.93 ± 172.8 IU/dl) were highly significantly increased compared to control (75.8 ± 16.24 IU/dl). ADAMTS13 level of acute infarction patients was (506.11 ± 225.24 ng/dl) and (1386.03 ± 667.64 ng/dl) in recurrent infarction patients with highly significant differences between the acute and control group and non-significant different between recurrent and control. ADAMTS13 is an excellent diagnostic test in acute cerebral infarction but a weak diagnostic test in recurrent cerebral infarction but it is a good diagnostic test in differentiation between acute and recurrent cerebral infarction patients. VWF is an excellent diagnostic test in both acute and recurrent cases. In acute infarction, VWF is positively correlated with kidney function while ADAMTS13 level did not correlate with different laboratory parameters in recurrent infarction. A positive correlation is present between ADAMTS13 and PT & PTT concentration. No correlation exists between VWF & ADAMTS13 in the 3 groups. Conclusion: ADAMTS13 is closely related to the occurrence, development and progression of acute cerebral infarction, protecting the brain from ischaemic reperfusion injury. It is an excellent diagnostic test in acute cerebral infarction but a weak diagnostic test in recurrent cerebral infarction while VWF is an excellent diagnostic test in acute & recurrent cerebral infarction. ADAMTS13 is expected to become a new therapeutic agent in acute cerebral infarction patients.

Evaluating errors in the laboratory identification of von Willebrand disease using contemporary von Willebrand factor assays

von Willebrand disease (VWD) arises from deficiency and/or defects of von Willebrand factor (VWF). Assessment requires test panels, including VWF activity and antigen. Appropriate diagnosis including differential identification of qualitative versus quantitative defects remains problematic but has important management implications. Data using a large set (n=27) of varied plasma samples comprising both quantitative VWF deficiency ('Type 1 and 3') vs qualitative defects ('Type 2') tested in a cross-laboratory setting have been evaluated to assess contemporary VWF assays for utility to differentially identify sample types. Different VWF assays and activity/antigen ratios showed different utility in VWD and type identification. Identification errors were linked to assay limitations, including variability, and laboratory issues (e.g., test result misinterpretation). Quantitative deficient (type 1) samples were misinterpreted as qualitative defects (type 2) on 35/467 occasions (7.5% error rate); 11.4% of these errors were due to laboratories misinterpreting their own data, which was instead consistent with quantitative deficiencies. Conversely, qualitative defects were misinterpreted as quantitative deficiencies at a higher error rate (14.3%), but this was more often due to laboratories misinterpreting their data (40% of errors). For test-associated errors, VWF:RCo and VWF:GPIbM were associated with the highest variability and error rate, which was many-fold higher than that using VWF:CB. Chemiluminescence ('CLIA') procedures were associated with lowest inter-laboratory variability and errors overall. These findings in part explain the high rate of errors associated with VWD diagnosis. VWF:GPIbM showed a surprisingly high rate of test associated errors, whilst CLIA procedures performed best overall.

Comparison of von Willebrand factor platelet‐binding activity assays: ELISA overreads type 2B with loss of HMW multimers

BackgroundA number of new assays with different measuring principles are available to measure von Willebrand factor (VWF) glycoprotein Ib (GPIb)‐binding activity, but little is known about how these assays might behave differently for subtypes of von Willebrand disease (VWD). ObjectivesThe Comparison of Assays to Measure VWF Activity (COMPASS‐VWF) study was designed to compare all available VWF GPIb‐binding activity assays for VWF. We specifically searched for particular assay behavior differences. Patients/MethodsTo sort out random differences from systematic assay behavior deviations, all assays were performed in different laboratories on the same samples in a blinded fashion. Samples from 53 normal controls and 42 well‐characterized VWD patients were reanalyzed in this study to dissect assay‐specific discrepancies. ResultsNo assay behavior differences were found for 53 normal controls. For VWD patients, we found the following systematic assay behavior patterns: (a) All ELISA assays for VWF:GPIbR as well as VWF:GPIbM are insensitive to detect the low VWF activity of VWD type 2B patients with loss of high molecular weight multimers; (b) VWF:Ab assay reports higher activity for the p.V1665E mutation than all other assays; and (c) all ristocetin‐based assays (including VWF:RCo using fixed platelets) but the AcuStar assay report discrepantly low VWF activity for the p.P1467S polymorphism. No systematic assay‐specific difference was observed for either the particle agglutination VWF:GPIbM assay or the AcuStar assay using magnetic beads. ConclusionsDifferent assay principles may lead to discrepant results for certain VWD types or mutations. Therefore, a more extensive study for a large number of patients is needed to better characterize the incidence and relevance of such assay‐specific differences.

Evaluation of a fully automated von Willebrand factor assay panel for the diagnosis of von Willebrand disease.

von Willebrand disease (VWD) diagnosis starts with first level tests: factor VIII coagulant activity, VWF antigen (VWF:Ag) and platelet-dependent VWF activity (VWF:RCo, VWF:Ab, VWF:GPIbR or VWF:GPIbM). The VWF collagen binding (VWF:CB) assay measures the binding capacity of von Willebrand factor (VWF) to collagen. To assess, in previously diagnosed VWD patients, the performance of a fully automated chemiluminescent test panel including VWF:Ag, VWF:GPIbR and VWF:CB assays. The patients, historically evaluated using in-house VWF:Ag and VWF:CB assays and an automated latex enhanced immunoassay VWF:GPIbR method, were re-evaluated using the VWF test panel HemosIL AcuStar. The VWF:GPIbR/VWF:Ag and VWF:CB/VWF:Ag obtained by means of AcuStar showed an overall good concordance with the corresponding data obtained at the time of the historical diagnosis. When discrepancies occurred, these were generally due to the lower VWF:CB/VWF:Ag obtained with AcuStar as compared with that obtained with the historical methods and this affected particularly the diagnosis of VWD type 2M. Together, the AcuStar VWF:GPIbR/VWF:Ag and VWF:CB/VWF:Ag were able to distinguish type 1 from types 2A, 2B and 2M, whereas no distinction was possible between type 2A and 2B. The AcuStar panel offers a good performance in the differential diagnosis between VWD type 1 and 2A/2B patients. A high rate of coincidence with historical diagnosis was obtained for VWD types 3, 2A/2B and 1. Even though in some cases more tests (eg, RIPA/multimeric analysis) are needed to complete an accurate VWD classification, the AcuStar panel is considered a sensitive, rapid and reliable tool to diagnose VWD patients.

Von Willebrand Disease Minimize Menorrhagia (VWDMin) Trial

Von Willebrand Disease Minimize Menorrhagia (VWDMin) Trial

The Mystery of “Magic Blood” - Familial Macrothrombocytopenia Associated with a Novel Variant in GP1BA Gene

Introduction: Inherited macrothrombocytopenias comprise a heterogeneous group of rare inherited disorders characterized by decreased platelet count with enlarged platelet size. Glycoprotein Ib (GP Ib) is a platelet surface membrane glycoprotein that is encoded by GP1BA gene, and functions as a receptor for von Willebrand factor (VWF). Mutations in the GP1BA gene are seen in Bernard-Soulier syndrome (BSS). Here we describe a family with an isolated giant platelet disorder and a novel variant in the GP1BA gene following an autosomal dominant mode of inheritance. In our kindred this variant was not associated with clinical history or specific laboratory evidence of BSS. Case Presentation: 34-year-old female reported first being diagnosed with macrothrombocytopenia at the age of 13 on routine blood work. The patient and 11 of her relatives spanning 5 generations have reported asymptomatic macrothrombocytopenia (Figure 1), described as "Magic Blood" by her family. Her platelet count fell below 20,000/microL during one of her pregnancies. She was treated as idiopathic thrombocytopenic purpura (ITP) and was given corticosteroids to increase platelet count without improvement. On presentation, patient's complete blood count was significant for low platelet at 55,000/microL and macrothrombocytes were observed on blood smear. Methods/Results: VWF assays, including Factor VIII activity, VWF Ag, and Ristocetin Cofactor, were within normal limits. VWF multimer analysis revealed a normal pattern and distribution of bands. Patient had a normal platelet aggregation in response to ADP, Collagen, Epinephrine, Ristocetin and Arachidonate. Flow Cytometry detected normal GP Ib and GP IIb/ IIIa expression. Whole exome sequencing and copy number analysis of 29 genes associated with thrombocytopenia revealed a c.97T&gt;G substitution in the GP1BA gene predicted to result in the amino acid substitution p.Cys33Gly (Figure 2). To our knowledge, this variant has not been reported in the literature or public databases. To confirm this novel variant is the cause of the familiar macrothrombocytopenia, two relatives with macrothrombocytopenia, a maternal uncle and a first cousin, also underwent genetic testing and were found to have the same variant. Discussion: Variants in GP1BA are associated with both autosomal dominant and recessive forms of BSS and with autosomal dominant platelet-type VWD. Our kindred is surprisingly asymptomatic given the location and specific amino acid substitution generated by the variant. Amino acid residue p.Cys33 resides in an extracellular N-terminal domain that is critical for VWF binding and proper assembly of the GP1B-IX complex. A heterozygous substitution involving the same amino acid residue defined as p.Cys33Arg was observed in two patients with macrothrombocytopenia with no reported bleeding complications (MC, unpublished data). Substitution of a nearby cysteine residue defined as p.Cys20Gly has been reported in a case of monoallelic chronic macrothrombocytopenia without bleeding diathesis similar to our patient. Amino acid residues p.Cys20 and p.Cyc33 are highly conserved among divergent species and substitution of these amino acid residues appears to not be tolerated. Substitution of other cysteine amino acid residues in the extracellular domain of the GP1BA protein (p.Cys81 and p.Cys225) has also been reported in patients with biallelic BSS, suggesting that perturbation of cysteine amino acid residues is likely to affect protein structure and function. Conclusion:We think the p.Cys33Gly substitution found in our patient and her relatives is likely to be a primary cause of monoallelic GP1BA-associated macrothrombocytopenia. It is important to distinguish inherited macrothrombocytopenia from ITP in order to avoid unnecessary and potentially toxic treatment. Disclosures No relevant conflicts of interest to declare.

The Discordance between Offsite to Onsite Testing for Von Willebrand Disease in Post-Menarchal Females: A Multi-Center Study

The Discordance between Offsite to Onsite Testing for Von Willebrand Disease in Post-Menarchal Females: A Multi-Center Study

Automated assays for von Willebrand factor activity.

von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) by platelet aggregometry has been considered the gold standard for evaluating the ability of VWF to bind platelets for over 40 years. Many automated systems no longer require platelets and rather rely on agglutination of latex particles. Automated methods of measuring VWF activity have improved performance characteristics and are performed on the same coagulation instruments used for routine testing via immunoturbidimetric methodology. Alternatively, a newer chemiluminescence assay system for measuring VWF activity demonstrates excellent performance characteristics. As these methods are becoming widely used, it is important to assess their performance in diagnosing and monitoring different types of von Willebrand disease. We review the automated methodologies and the published performance of these VWF assays. Advantages and limitations of these automated methods are discussed.