Willebrand Factor Ristocetin Cofactor Activity Research Articles

Evaluation of an automated von Willebrand factor glycoprotein IbM activity assay compared with 3 alternative von Willebrand factor activity assays

BackgroundTo overcome deficiencies of the traditional von Willebrand factor (VWF) ristocetin cofactor activity assay (VWF:RCo), several automated assays for VWF platelet-binding activity have been developed. Information on the performance of these assays and their diagnostic utility remains limited. ObjectivesTo validate the VWF:glycoprotein IbM assay INNOVANCE VWF Ac and compare it with an automated VWF:RCo assay as well as with an automated assay and a manual VWF:Ab assay and to generate reference ranges and analyze reproducibility of the VWF:glycoprotein IbM assay. MethodsClinical sites enrolled healthy subjects and patients representing the intended use population; VWF activity assays were performed, and results were analyzed. The performance of the INNOVANCE VWF Ac assay was also compared between the BCS XP System and the CS-2500 and CS-5100 analyzers. ResultsThe INNOVANCE VWF Ac assay correlated well with the VWF:RCo assay and the automated HemosIL VWF:Ab assay, with Pearson coefficients of >.9 and a predicted bias of ≤5.0 IU/dL at VWF levels of 30 IU/dL and ≤5.8 IU/dL at the levels of 50 IU/dL, but correlation and bias were not as good when compared with the REAADS manual VWF:Ab assay. Reference ranges observed for healthy subjects correlated well with previously published findings. Reproducibility of the INNOVANCE VWF Ac assay on the BCS XP System and the CS analyzers was excellent, as was correlation among devices. ConclusionThe characteristics of the INNOVANCE VWF Ac assay regarding comparability with other VWF activity assays, reference ranges, and precision support the use of this assay for evaluation of patients with concern for von Willebrand disease.

Von Willebrand factor Ristocetin co-factor activity to von Willebrand factor antigen level ratio for diagnosis of acquired von Willebrand syndrome caused by aortic stenosis

BackgroundSevere aortic stenosis (AS) causes acquired von Willebrand syndrome by the excessive shear stress–dependent cleavage of high molecular weight multimers of von Willebrand factor (VWF). While the current standard diagnostic method is so-called VWF multimer analysis that is western blotting under nonreducing conditions, it remains unclear whether a ratio of VWF Ristocetin co-factor activity (VWF:RCo) to VWF antigen levels (VWF:Ag) of <0.7, which can be measured with an automated coagulation analyzer in clinical laboratories and is used for the diagnosis of hereditary von Willebrand disease. ObjectivesTo evaluated whether the VWF:RCo/VWF:Ag is useful for the diagnosis of AS-induced acquired von Willebrand syndrome. MethodsVWF:RCo and VWF:Ag were evaluated with the VWF large multimer index as a reference, which represents the percentage of a patient’s VWF high molecular weight multimer ratio to that of standard plasma in the VWF multimer analysis. ResultsWe analyzed 382 patients with AS having transaortic valve maximal pressure gradients of >30 mmHg, 27 patients with peripheral artery disease, and 46 control patients free of cardiovascular disease with osteoarthritis, diabetes, and so on. We assumed a large multimer index of <80% as loss of VWF large multimers since 59.0% of patients with severe AS had the indices of <80%, while no control patients or patients with peripheral artery disease, except for 2 patients, exhibited the indices of <80%. The VWF:RCo/VWF:Ag ratios, measured using an automated blood coagulation analyzer, were correlated with the indices (rs = 0.470, P < .001). When the ratio of <0.7 was used as a cut-off point, the sensitivity and specificity to VWF large multimer indices of <80% were 0.437 and 0.826, respectively. ConclusionVWF:RCo/VWF:Ag ratios of <0.7 may indicate loss of VWF large multimers with high specificity, but low sensitivity. VWF:RCo/VWF:Ag ratios in patients with AS having a ratio of <0.7 may be useful for monitoring the loss of VWF large multimers during their clinical courses.

Effects of Hypobaric Hypoxia on Coagulation in Healthy Subjects Exposed to 3,500 m Altitude.

Kammerer, Tobias, Anna Walzl, Thomas Müller, Philipp Groene, Giulia Roveri, Rachel Turner, Johanna Roche, Hannes Gatterer, Christoph Siebenmann, and Simon T. Schäfer. Effects of hypobaric hypoxia on coagulation in healthy subjects exposed to 3,500 m altitude. High Alt Med Biol. 24:94-103, 2023. Background: Hypoxia is discussed as a trigger for prothrombotic changes both in intensive care and high altitude medicine. This research study aimed to evaluate the effect of isolated hypobaric hypoxia (HH) on coagulation in females in a highly standardized setting. Methods: Twelve healthy female subjects were studied under HH (equivalent to 3,500 m) and normoxia (NX) during two 4-day sojourns, in a strictly controlled crossover design. Nutrition, fluid intake, hormonal status (i.e., menstrual cycle variation), and physical stress were standardized. Functional coagulation and blood lysis were measured by viscoelastometry and compared between HH and NX. In addition, plasma-based coagulation tests (PBCTs), namely prothrombin time, activated partial thromboplastin time, fibrinogen, factor VIII coagulation activity (FVIII:C), von Willebrand factor antigen (vWF:Ag), and von Willebrand factor ristocetin cofactor activity (vWF:RCo) were measured. Results: Neither for Viscoelastic Haemostatic Assays nor for PBCTs significant changes were found for HH compared with NX (all p > 0.05). Specifically, the lysis ability, as well as clotting time, clot formation, clot amplitude, and maximum clot firmness unchanged were similar between HH and NX. This also applied to all other variables. Conclusion: We demonstrate that moderate HH per se has no influence on blood coagulation in healthy females.

Platelet Dysfunction in Blood Donors Detected by Platelet Function Analyzer PFA-100™.

BackgroundPlatelet transfusions may be indicated to prevent and treat bleeding in patients with quantitative or qualitative platelet defects. Millions of platelet components are transfused worldwide. It is well known that platelet dysfunction predicts blood loss after surgery. Hence, the quality of the platelet donation and the resulting platelet concentrate are critical for the transfusion. The aim of this study is to assess platelet function in well-qualified blood donors.MethodologyBlood samples from 275 blood donors were collected in 0.129 M (3.8%) sodium citrate tubes prior to routine blood donation. Platelet function was assessed by measuring the closure time (CT) on the platelet function analyzer (PFA-100™; Siemens Health Diagnostics, Marburg, Germany) using collagen/epinephrine (CEPI) and collagen/ADP (CDP) cartridges.ResultsUsing the PFA-100™, 20.4% of donors had an abnormal platelet function test, of whom 9.4% had prolonged CT with two cartridges, 7% had only prolonged CEPI CTs consistent with aspirin-like defect, and 4% had prolonged CADP CTs only. We found no closure (>300 seconds) in 6.54% of donors, including 1.45% with the CEPI cartridge, 2.9% with the CADP cartridge, and 2.18% with CEPI and CADP cartridges. Level of von Willebrand factor ristocetin cofactor (vWF: RCo) activity was 112% (56-168%). Of the factors examined (age, sex, cigarette smoking, blood donation type, ABO, and Rhesus blood group), only blood group O was significantly linked with impaired platelet function test in qualified blood donors (p = 0.023; odds ratio = 1.981; 95% confidence interval (1.091-3.595)).ConclusionsSome qualified blood donors present abnormal platelet function results. More research is required to provide greater insight into the impact of platelet dysfunction in blood donors on the clinical efficacy of their platelet components. This study has confirmed that the influence of ABO blood group on the CT PFA-100™is not wholly dependent on vWF.

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.

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 current status of remote diagnosis of von Willebrand disease in children in Russia

The diagnosis of von Willebrand disease (vWD) in children remains a challenge in many regions of our country. This encouraged the Russian Hemophilia Society to create, in 2019, a special diagnostic programme offering remote diagnosis of vWD to patients in regions. Objectives: An interim evaluation of the effectiveness of the programme for remote diagnosis of vWD in children. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of Ministry of Healthcare of the Russian Federation. Under this programme, if advised by a hematologist at the place of residence, a patient's peripheral blood was collected, frozen and sent to the Clinical Hemostasis Laboratory at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. Over the period from 11.04.2019 to 07.08.2020, we received 72 frozen peripheral blood samples from children under the age of 18 sent from 21 regions of the Russian Federation. Von Willebrand factor (vWF) ristocetin cofactor activity, vWF antigen levels as well as coagulation factor VIII activity were measured. Out of the 72 samples, only one (1.4%) was inadequate. Reduced vWF activity (30–50%) was observed in 16.7% of cases, and another 16.7% of patients had vWF activity &lt; 30%. VWF antigen levels were 30–50 % in 13.9 % of patients and &lt; 30% in 9.7% of cases. Among the patients with vWF activity &lt; 30%, 1 patient (8.3%) had type 1 vWD, 10 patients (83.3%) – type 2 vWD, 1 child (8.3%) – type 3 vWD. Six children (8.3%) demonstrated reduced factor VIII activity while maintaining normal vWF activity and antigen levels. The coagulation factor VIII activity/vWF antigen ratio was decreased in 8 children (11.1%). The vWD remote diagnosis programme has allowed us to detect abnormalities in 30 patients (41.7%), 16.7% of whom demonstrated laboratory features characteristic of vWD. Eighteen children (25%) require further, more comprehensive laboratory testing. This programme has great potential, especially in underpopulated regions where the development of local diagnostic capabilities may not be economically viable.

Von Willebrand factor variant D1472H has no effect in mice with humanized VWF-platelet interactions

AbstractThe von Willebrand factor ristocetin cofactor activity assay (VWF:RCo) is used for diagnosis of von Willebrand disease (VWD) because of its ability to evaluate VWF binding to platelets. VWF sequence variant p.D1472H is associated with lower VWF:RCo levels in the absence of associated bleeding symptoms, indicating the VWF:RCo may not be accurate for characterizing VWF function in individuals with this variant. Thus, this study aimed to determine the implications of the variant on VWF functioning in vivo. Mice were engineered with humanized wild-type (WT*) VWF A1/A2 and VWF with the p.D1472H (1472H) variant along with humanized platelet GPIbα and bred to homozygosity. VWF antigen and VWF binding to GPIbα were measured using enzyme-linked immunosorbent assay. Gel electrophoresis was used for VWF multimer analysis. Tail bleeding assays were performed at a 3-mm defined length. Normal VWF multimers were preserved in both WT* and 1472H mice. VWF expression was normal in the WT* and 1472H mice, and VWF binding to GPIbα did not statistically differ between the groups. Additionally, tail bleeding times were similar for WT* and 1472H mice. These results show the p.D1472H variant does not impair hemostasis in mice, and support the conclusion that p.D1472H is a normal variant in humans.

Whole blood ristocetin-induced platelet impedance aggregometry does not reflect clinical severity in patients with type 1 von Willebrand disease.

The haemorrhagic phenotype in patients with von Willebrand disease (VWD) is heterogeneous, and assays of von Willebrand factor ristocetin cofactor activity (VWF:RCo) do not always reflect clinical severity, especially in those individuals classed as type 1 VWD. Recent studies have shown that whole blood ristocetin-induced platelet agglutination (WB-RIPA) using an easy-to-use analyzer, Multiplate® platelet impedance technique, could be informative as a diagnostic test in VWD, although inconsistencies were evident in patients with the type 1 disorder, possibly associated with clinical symptoms. To investigate the relationship between WB-RIPA, bleeding scores (BS) and VWF-related measurements in type 1 VWD. WB-RIPA assay using the Multiplate® was performed using whole blood from 55 patients with type 1 VWD. BS was determined using a standardized questionnaire. WB-RIPA values were significantly lower in type 1 VWD than in healthy controls (P<0.0001). Weak correlations were apparent between WB-RIPA and VWF:RCo or VWF antigen (VWF:Ag; r=0.22 or 0.28, respectively). There were significant differences in VWF:RCo (P=0.036) and VWF:Ag (P=0.0013) between patients with BS ≥4 (defined as abnormal bleeding tendency) and BS <4 (defined as no abnormal bleeding tendency), respectively. However, no significant difference was observed in WB-RIPA between the BS ≥4 group and BS <4 group. Overall, VWD patients with a WB-RIPA level >70U did not seem to have an abnormal bleeding tendency, but low levels of WB-RIPA did not correlate with BS. WB-RIPA did not reflect clinical severity in type 1 VWD patients.

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.

Role of red blood cells in the anemia-associated bleeding under high shear conditions.

Red blood cells (RBCs) contribute to hemostasis under blood-flow, and anemia might contribute to a hemorrhagic diathesis. The majority of current laboratory techniques to assess hemostasis do not consider the effects of RBCs. An assay to determine the role of RBCs in hemostasis could be beneficial for clinical management. To investigate the influence of RBCs in hemostasis. Hemostasis was investigated using a novel microchip flow-chamber system (T-TAS® ) in an anemic patient with von Willebrand disease. Subsequently, the effects of RBCs in total thrombus analysis system (T-TAS) were examined using reconstituted whole blood at various hematocrit levels. In vivo: When the patient was anemic and demonstrated persisted hemorrhagic symptoms despite the maintained adequate von Willebrand factor ristocetin cofactor activity levels, thrombus formation determined by T-TAS was delayed. However, transfusions of RBCs resolved bleeding symptom and, accordingly, the thrombus formation in T-TAS improved. In vitro: Thrombus formation determined by T-TAS at 1000 s-1 was dose-dependent on hematocrit (the time to reach 10 kPa (T10 ): 10.0 ± 0, 9.5 ± 1.4, 6.7 ± 2.4, 2.8 ± 1.6 min at hematocrits of 0%, 12.5%, 25% and 50%, respectively). Markedly defective thrombus formation (T10 >10 min) was confirmed at a hematocrit <25% at 2000 s-1 . Red blood cells play an essential role in hemostasis under high shear, and RBC transfusions could be effective for refractory bleeding in patients with anemia. T-TAS measurements appear to reflect the hemostatic consequences of diminished red cell numbers under blood-flow, and could provide a valuable means for monitoring patients.

Laboratory Diagnosis of von Willebrand Disease

Von Willebrand disease (VWD) is considered the most common congenital bleeding abnormality in the world and an accurate diagnosis is often very challenging. Clinical laboratories usually provide screening assays along with more complex assays to aid clinicians with the diagnosis. These assays measure different properties and activities of von Willebrand factor. They may be adversely affected by numerous preanalytical variables and often have variable performance characteristics that may contribute to an inadequate interpretation. This review describes methods used in the laboratory for diagnosing VWD and basic mechanisms of each of these assays. A thorough understanding of the assays and associated preanalytical variables in VWD testing is essential for accurate diagnosis. ABBREVIATIONS:ADAMTS13 - a disintegrin and metalloproteinase with a thrombospondin type 1 motif, DDAVP - 1-desamino-8-D-arginine vasopressin, EC - endothelial cell, VWD - von Willebrand disease, VWF - von Willebrand factor, VWF:Ag - von Willebrand factor antigen, VWF:CBA - von Willebrand factor collagen binding assay, VWF:RCo - von Willebrand factor ristocetin cofactor activity

Detection and analysis of plasma VWF levels in healthy Chinese by automatic standardized assay

目的采用全自动标准化方法检测正常人血管性血友病因子（VWF）各指标并分析其影响因素。方法采用全自动、标准化检测仪及配套试剂测定70名正常人VWF抗原（VWF∶Ag）、VWF瑞斯托霉素辅因子活性（VWF∶Rco）、VWF胶原结合试验（VWF∶CB）、凝血因子FⅧ活性（FⅧ∶C），比较年龄、ABO血型、性别及地域因素对正常人VWF各指标的影响。对英国国家生物标准与检定所（NIBSC）确认的8个标准血浆样本（2名正常人、6例2型VWD）进行VWF各项指标检测。结果①70名正常人血浆VWF∶Ag为（95.4±44.9）%，VWF∶Rco为（105.9±35.4）%，VWF∶CB为（89.8±28.4）%；VWF∶Rco/VWF∶Ag比值为1.18±0.25，VWF∶CB/VWF∶Ag比值为1.03±0.29。②年龄≥30岁组与<30岁组比较，血浆VWF各指标差异均无统计学意义（P>0.05）。③O型血组VWF∶Rco、VWF∶CB低于非O型血组（t=2.074，P=0.042；t=3.949，P=0.001），VWF∶Ag、VWF∶Rco/VWF∶Ag、VWF∶CB/VWF∶Ag比值差异无统计学意义（P>0.05）。④男、女性组VWF各指标差异无统计学意义（P>0.05）。⑤北方（淮河以北地区）组VWF∶Ag、VWF∶CB高于南方（苏州地区）组（t=4.525，P=0.001；t=3.214，P=0.002），VWF∶Rco/VWF∶Ag、VWF∶CB/VWF∶Ag比值低于南方组（t=6.373，P=0.001；t=2.902，P=0.005），VWF∶Rco差异无统计学意义（t=1.598，P>0.115）。⑥8个标准血浆样本各项VWF指标检测结果符合已知诊断。结论通过使用全自动标准化检测设备首次获得较完整的正常人血浆VWF水平检测数据；ABO血型和地域因素对VWF水平有显著影响，而年龄和性别的影响不显著。

Assessing the clinical severity of type 1 von Willebrand disease patients with a microchip flow‐chamber system

The clinical phenotype of von Willebrand disease (VWD) is heterogeneous, and von Willebrand factor ristocetin cofactor activity (VWF:RCo) does not always reflect clinical severity, especially in VWD type 1. We have reported the potential of a microchip flow-chamber system (Total-Thrombus Formation Analysis System [T-TAS®]) for assessing physiologic hemostasis in VWD. Aim To evaluate the relationship between T-TAS, bleeding score (BS) and laboratory test results in type 1 VWD patients. Microchips coated with collagen (platelet chip [PL-chip]) or collagen/thromboplastin (AR-chip) were used to assess platelet thrombus formation (PTF) at high shear rates or fibrin-rich PTF at low shear rates, respectively, in whole blood from 50 patients. The times needed for the flow pressure to increase by 10 kPa and 30 kPa (T10 and T30 ) from baseline were calculated from flow pressure curves. BS was determined by the use of a standardized questionnaire. PL-T10 values correlated with BS (R(2) ~ 0.45) better than VWF:RCo (R(2) ~ 0.36), irrespective of the flow rate, whereas AR-T10 showed only a weak correlation with BS (R(2) ~ 0.18). Patients with PL-T10 > 10 min or AR-T10 > 30 min had lower VWF levels and higher BS than those with PL-T10 ≤ 10 min or AR-T10 ≤ 30 min, and the greatest differences were observed with PL-T10. Clinical severity appeared to correlate best with PL-T10 > 8 min. BS was significantly higher in patients with VWF:RCo of < 10 IU dL(-1) than in those with VWF:RCo of 10 IU dL(-1) to < 25 IU dL(-1) and 25-40 IU dL(-1). In patients with VWF:RCo of < 10 IU dL(-1) , BS was significantly higher in those with PL-T10 > 8 min than in those with PL-T10 ≤ 8 min. T-TAS could be a useful technique for discriminating and predicting BS in VWD type 1 patients.

Increased plasma serotonin metabolite 5-hydroxyindole acetic acid concentrations are associated with impaired systolic and late diastolic forward flows during cardiac cycle and elevated resistive index at popliteal artery and renal insufficiency in type 2 diabetic patients with microalbuminuria.

Although lower extremity arterial disease is frequently accompanied by diabetes mellitus, the association of circulating biomarkers with flow components during the cardiac cycle in lower-leg arteries has yet to be fully elucidated. We enrolled 165 type 2 diabetic patients with normal ankle-brachial index (ABI 1.0-1.4), comprising 106 normoalbuminuric and 59 microalbuminuric patients, and 40 age-matched nondiabetic subjects consecutively admitted to our hospital. Serum high sensitivity C-reactive protein (hsCRP) level and plasma von Willebrand factor ristocetin cofactor activity (VWF) and vasoconstrictor serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA) concentrations were measured. An automatic device was used to measure ABI and brachial-ankle pulse wave velocity (baPWV). Flow components during the cardiac cycle, total flow volume, and resistive index at popliteal artery were evaluated using gated magnetic resonance imaging. Although estimated glomerular filtration rate (eGFR), early diastolic flow reversal, heart rate, and ABI were similar between the groups, diabetic patients had higher log hsCRP (p<0.001), VWF (p<0.001), 5-HIAA (p=0.002), resistive index (p<0.001) and baPWV (p<0.001) and lower systolic (p=0.026) and late diastolic (p<0.001) forward flows and total flow volume (p<0.001) than nondiabetic subjects. Multivariate analyses demonstrated that 5-HIAA in microalbuminuric patients showed higher associations with systolic and late diastolic forward flows during the cardiac cycle, total flow volume and resistive index at popliteal artery, and eGFR compared to normoalbuminuric patients. In microalbuminuric patients, 5-HIAA was a significant independent determinant among these factors. Thus, increased plasma 5-HIAA levels are involved in the pathogenesis of impaired blood flow in lower extremities and renal insufficiency in diabetic patients with microalbuminuria.

Evaluation of an heterogeneous group of patients with von Willebrand disease using an assay alternative to ristocetin induced platelet agglutination

Diagnosis of von Willebrand disease (VWD) type 2 usually relies on the discrepancy between the von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) and VWF antigen (VWF:Ag). Type 2B patients can be discriminated from other qualitative VWD variants by using ristocetin-induced platelet agglutination (RIPA) test. The major limitation of RIPA isthe requirement of fresh blood sample. In this study, we evaluated the VWF gain-of-function mutant GPIb binding (VWF:GPIbM) and VWF:RCo assays to investigate whether the VWF:GPIbM/VWF:RCo ratio was able to identify the type 2B variant among an heterogeneous VWD population, previously characterized following the ISTH-SSC guidelines. Seventy-six VWD patients and 31 healthy subjects were evaluated by using VWF:Ag, VWF:RCo, and VWF:GPIbM assays. The mean (minimum-maximum values) VWF:GPIbM/VWF:RCo ratio was higher in type 2B patients (2.53, 0.84-6.11) than in healthy controls (1.05, 0.87-1.34), type 1 (0.85, 0.51-1.15), 2A (1.20, 0.36-2.82), and 2M (1.07, 0.91-1.38) (P<0.0001). Type 2B variants were divided into four groups (A, B, C, and D) according to their different multimeric patterns. The mean value of the VWF:GPIbM/VWF:RCo ratio in the four groups showed an increasing trend from group A (1.08) to D (3.69), proportional to the loss of high molecular weight multimers. Among 32 type 2B patients, previously diagnosed with RIPA, 8 (mainly with a type I New York/Malmö phenotype) were not confirmed using the VWF:GPIbM/VWF:RCo ratio. Whenever the RIPA test is not feasible, the VWF:GPIbM/VWF:RCo ratio might help to identify severe type 2B VWD patients.

Optimization of the automated, CS-2000i™ method for measuring low levels of von Willebrand factor ristocetin cofactor activity (VWF:RCo).

Measurements of von Willebrand factor ristocetin cofactor activity (VWF:RCo) are necessary for the diagnosis of von Willebrand disease (VWD). However, the conventional manual glass-plate method is technically demanding, and exhibits high intra-/inter-laboratory variation. An automated technique has been recently described utilizing the CS-2000i™ with specific VWF:RCo reagents, but measurements at lower levels (<10 IU/dL) lack reliability. We have optimized this automated system for measuring low levels of VWF:RCo. Using a sample volume (72 μL) and initial stirring speed (400 rpm), the lowest level of VWF:RCo detected with a CV < 20 % was 2.5 IU/dL. Greater variability was evident at this stirring speed with smaller sample volumes. Decreasing the stirring speed (200 rpm) with a sample volume (72 μL), however, significantly improved the CV, and enabled measurements at the lowest levels (≤2.5 IU/dL). The optimized assay demonstrated a high correlation (R(2) = 0.841) with the glass-plate method at <10 IU/dL VWF:RCo, suggesting that the different VWF:RCo reagents did not affect the results. In addition, changes in VWF:RCo post-DDAVP infusions were parallel in both assays, indicating that the automated method is suitable for the clinical monitoring of treatment in VWD. The optimized automated measurement of VWF:RCo offers a promising method for evaluating VWF at low levels of hemostatic activity.

The Management of Bleeding Risk in Von Willebrand Disease: Should Blood Group O Make a Difference?

▪Type 1 von Willebrand disease (VWD) is a common inherited bleeding disorder characterized by a quantitative deficiency of von Willebrand factor (VWF) and excessive mucocutaneous bleeding. It is well known that von Willebrand factor (VWF) levels are significantly lower in persons with ABO blood group O and that there is a significantly higher prevalence of blood group O in patients with a diagnosis of type 1 VWD. This raises a question important to the clinical care of patients with low VWF; is there a difference in bleeding risk in patients with a deficiency of VWF on the basis of blood group O versus those with a deficiency of VWF due to a sequence variation in the VWF gene. To explore this question, we examined index cases enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWD with a quantitative deficiency of VWF including those with a phenotypic diagnosis of low VWF [von Willebrand factor antigen (VWF:Ag) or von Willebrand factor ristocetin cofactor activity (VWF:RCo)≤ 50 U/dL and >40 U/dL], type 1 VWD (VWF:Ag or VWF:RCo≤40 U/dL) and type 1C VWD (VWF:Ag ≤30 U/dL and VWF propeptide/Ag ratio >3 indicating increased clearance of VWF). VWF:Ag, VWF:RCo, VWF propeptide and multimer distribution were analyzed in the clinical hemostasis laboratory at the BloodCenter of Wisconsin. Full length VWF gene sequencing including intron-exon boundaries was performed for all index cases. Bleeding symptoms were scored utilizing the ISTH, MCMDM1 and PBQ bleeding assessment tools. Since there was a high correlation among bleeding scores with these three methods, the ISTH score was utilized for this analysis.Complete data were available for 91 subjects with low VWF, 161 subjects with type 1 VWD, 53 subjects with type 1C VWD and 74 normal control subjects. Median ages were not different among the diagnostic groups. As expected based on previous reports, the groups of VWD patients with lower VWF:Ag values (Mean±1SD) had an increased percentage of subjects with an identified sequence variation; low VWF group, VWF:Ag 50.4±8.4, 38% with sequence variation; Type 1 VWD, VWF:Ag 30.8±11.6, 70% with sequence variation; and Type 1C VWD, VWF:Ag 9.8±5.9, 83% with sequence variation. The low VWF group and the type 1 VWD groups both had a significantly increased frequency of subjects with blood group O (70 – 80%) vs. nonO (19-30%) in comparison to those with type 1C in whom the frequency of blood group O (57% ) vs. nonO (43%) was not different than that in the normal control population (43% O vs. 57% nonO). This suggests that the type 1 and low VWF groups have a significant proportion of subjects with low VWF on the basis of blood group O alone. Since those subjects with a diagnosis of low VWF or Type 1 VWD who have no identifiable sequence variation are more likely to be affected by blood group O alone, we examined the ISTH bleeding scores in subjects with similar VWF levels and blood group O with and without an identifiable VWF gene sequence variation. We found that there was no difference in the ISTH bleeding scores in blood group O subjects regardless of the presence of a VWF gene sequence variation; low VWF, no sequence change (N=45), median bleeding score 6.0 vs. 5.0 with sequence change (N=27); type 1 VWD, no sequence change (N=39), median bleeding score 5.0 vs. 5.0 with sequence change (N=73); type 1C VWD, no sequence change (N=6), median bleeding score 7.0 vs. 7.0 with sequence change (N=24). There were no differences in median bleeding scores in blood group O vs. nonO groups, and none in the nonO groups with or without sequence variation in any of the VWD phenotypic diagnostic categories. There were no significant differences in bleeding scores by gender in any of the diagnostic categories.In conclusion, we found no differences in ISTH bleeding scores in the groups of subjects most likely to have low VWF on the basis of blood group O alone, i.e., those with blood group O and no identifiable VWF gene sequence variation compared to those with VWF gene sequence variations. Thus, management of persons with VWF deficiency should be based on baseline VWF levels and not modified in those with blood group O. DisclosuresNo relevant conflicts of interest to declare.

Decreased von Willebrand factor ristocetin cofactor activity and increased ADAMTS13 antigen increase postoperative drainage after coronary artery bypass grafting

Routine coagulation tests and bleed-scores fail to identify patients at risk of excessive postoperative drainage following coronary artery bypass grafting (CABG). We sought to investigate whether lower von Willebrand factor (VWF) and higher ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) are associated with a high postoperative drainage after CABG. In the prospective cohort study, VWF antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCO), VWF collagen-binding (VWF:CB), ADAMTS13 antigen (ADAMTS13:Ag) and ADAMTS13 activity were measured on the day of elective on-pump CABG in 232 consecutive patients without a prior history of haemorrhagic diathesis, including von Willebrand disease (95% discontinued aspirin preoperatively). Postoperative drainage and blood product use were recorded. A comparison of extreme drainage quartiles (n=56) showed that individuals with the highest drainage volumes have mean VWF:RCO lower by 19% (P<0.0001), median VWF:Ag lower by 19% (P<0.0001), ADAMTS13:Ag higher by 8% (P=0.0002), ADAMTS13 activity higher by 9% (P=0.01) and fibrinogen lower by 14% (P=0.03) than those with the lowest drainage. Linear regression analysis showed that preoperative VWF:RCO (b=-4.83, P=0.002) and fibrinogen (b=-61.52, P=0.04) are the only independent predictors of postoperative drainage. Multivariate logistic regression demonstrated that preoperative VWF:RCO in the lowest quartile and ADAMTS13:Ag levels in the highest quartile increased the risk of high (≥1000 ml) drainage (odds ratio [95% confidence interval]=4.88 [1.83-13.02], P=0.001 and 3.77 [1.49-9.52], P=0.005; respectively). Patients undergoing elective CABG with lower preoperative VWF:RCO are at risk of having larger postoperative drainage, which suggests a novel contributor to increased perioperative bleeding in cardiac surgery.

An approach to outreach patients with von Willebrand disease in Egypt by targeting women with heavy menstrual bleeding and/or bleeding symptoms

von Willebrand disease (VWD) is frequently ignored as a cause of menorrhagia. We investigated Egyptian women complaining of heavy menstrual bleeding (HMB) and/or other bleeding symptoms to detect potential VWD cases. Seventy-five female patients complaining of HMB and/or bleeding symptoms and 38 age-matched healthy female controls went through a family history questionnaire, a physical examination and were evaluated for bleeding score, pictorial blood assessment chart (PBAC), complete blood count, serum ferritin, blood group, prothrombin time, activated partial thromboplastin time, factor VIII (FVIII) activity, von Willebrand factor (VWF) ristocetin cofactor (RCo) activity, antigen (Ag), and RCo/Ag ratio. Sixty-eight of 75 patients presented with HMB, out of which 46 had no organic pathology and 7 presented other bleeding symptoms. Six patients were diagnosed with VWD, three with HMB, two with other bleeding symptoms and one with family history of VWD. Two related VWD patients were diagnosed in the control group. There were significant differences in bleeding and PBAC scores, ferritin level, FVIII activity, VWF:RCo and VWF:Ag between VWD patients and controls. This study indicated a high prevalence of VWD among patients with HMB without organic pathology (6.5%) and demonstrated the sensitivity of diagnostic parameters of VWD patients in an outreach campaign. The inexpensive bleeding and PBAC scoring systems are valuable to exclude cases without objective bleeding symptoms. Raising gynaecologists awareness about hereditary bleeding disorders is important to ensure a proper diagnosis and possible referral of these patients. Management of these patients with comprehensive medical care services under a multidisciplinary team would be ideal.