Ristocetin Cofactor Research Articles

Detection of Continuous Flow Left Ventricular Assist Device -Associated Acquired Von Willebrand Factor (VWF) Abnormality by An Automated Immunoturbidimetric VWF Activity Assay

Abstract▪2273▪This icon denotes a clinically relevant abstract Background:Continuous flow left ventricular assist device (CF-LVAD) recipients have high tendency of post-surgical gastrointestinal (GI) bleeding. We previously described the loss of high molecular weight VWF multimers (HMWM), due to either an accelerated clearance or enhanced cleavage of the HMWM, in most CF-LVAD recipients (Ann Thorac Surg. 90:1263–9). However, besides the tedious plasma VWF multimer analysis, neither VWF ristocetin cofactor nor collagen binding activity assay provides sufficient sensitivity for detecting such an acquired VWF abnormality (AVWA). In this study, we examined a new automated latex particle-enhanced immunoturbidimetric VWF activity assay (VWF:Lx) for its ability of detecting CF-LVAD-AVWA. We also analyzed the VWF pro-peptide (VWF:pp) to explored the potential mechanism of CF-LVAD-AVWA. Design:As part of an on-going prospective multicenter study, pre- and post-CF-LVAD implantation (7, 30 days and 5–7 months) blood samples were collected from 15 LVAD recipients (median age 52 years; 10 male and 5 female; 2008∼2009). Plasma VWF antigen (VWF:Ag), VWF:Lx activity, VWF:pp/Ag ratios were measured; and plasma VWF multimer analyses were performed on all available plasma samples. Standard statistical analyses were employed. Result:Loss of VWF HMWM was observed in 3 patients prior to CF-LVAD implantation and virtually all patients after surgery. VWF:Ag, VWF:Lx or VWF:pp/Ag ratios of the pre- and post-implantation samples were not significantly different (P> 0.1). However, VWF:Lx/Ag ratios of the post-implantation samples were significantly decreased (P<0.05). At a cut off of 0.8, the VWF:Lx/Ag ratio has a 90% sensitivity and specificity for detecting AVWA. Conclusions:VWF:Lx/Ag ratio has excellent sensitivity and specificity in detecting CF-LVAD-AVWA, and its impact on predicting post-surgical bleeding tendency in CF-LVAD recipients is being investigated. The distinct CF-LVAD-AVWA is most likely caused by enhanced cleavage, rather than clearance, of the HMWM.Table 1:Laboratory Characteristics of VWF in Pre- and Post-CF-LVAD Implantation Patient Plasma Samples.Laboratory Results (Mean±Standard Deviation)Pre-implantation (n=15)Day 7 Post (n=13)Day 30 Post (n=13)5–7 month Post (n=4)VWF:Ag (IU/dL)318 ± 178340 ± 136263.8 ± 122.7224 ± 78VWF:Lx (U/dL)258 ± 145246 ± 87102.5 ± 64.6165 ± 44VWF:Lx/Ag Ratio0.82 ± 0.120.74 ± 0.080.75 ± 0.060.76 ± 0.12VWF:pp/Ag Ratio1.04 ± 0.361.00 ± 0.300.99 ± 0.231.02 ± 0.10Samples with HMWM Loss (Present/Total)3/1510/1312/134/4Agreement between abnormal VWF:Lx/Ag ratio (<0.8) and VWF Multimer Analysis13/1511/1312/134/4 Disclosures:No relevant conflicts of interest to declare.

Von Willebrand Factor Collagen Binding Provides a Sensitive Screen for Identification of Type 2A and 2B Von Willebrand Disease

Abstract 379▪FN2▪This icon denotes a clinically relevant abstractCollagen binding is an easily performed test of von Willebrand factor (VWF) function but its role in clinical evaluation is still debated. Analysis of multimer distribution, on the other hand, is time-consuming and technically challenging. We hypothesized that VWF antigen (VWF:Ag), ristocetin cofactor activity (VWF:RCo), and collagen binding (VWF:CB) could identify the subset of von Willebrand disease (VWD) cases in which multimer analysis would be informative. Subjects from the Zimmerman Program for the Molecular and Clinical Biology of VWD were analyzed for VWF:Ag, VWF:RCo, VWF:CB (with type III human placental collagen), multimer distribution, and full VWF exon sequencing. Normal controls as well as patients with type 1, 2A, 2B, 2M, and 2N VWD were analyzed. The mean VWF:CB/VWF:Ag ratio for subjects with normal multimers was 1.10, while the mean ratio for subjects with abnormal multimers was 0.51 (p<0.001). When results were restricted to those subjects with confirmed type 2A or type 2B mutations, however, the mean ratio for subjects with abnormal multimers decreased to 0.41 (p<0.001 compared to those with normal multimers). For the 146 normal controls with multimer results available, 2 had absence of the highest molecular weight multimers, but normal collagen binding, normal bleeding scores, and no evidence of a VWF gene mutation, suggesting that the multimer results represented assay artifact. 354 type 1 subjects were examined; of those, 12 had abnormal multimer patterns. 7 had loss of the high molecular weight multimers. Of these, 5 had known type 1 VWD mutations and normal VWF:CB/VWF:Ag ratios, possibly representing sample artifacts rather than a true multimer abnormality, as no multimer issues have been previously reported for these mutations. One had no mutation found and one had a type 2A mutation. 2 had a full spectrum of multimers with relatively increased staining of the lower molecular weight bands; both with novel A1 domain mutations that are currently under investigation. 3 had larger than normal multimers observed, all with normal VWF:CB/VWF:Ag ratios. Of the 342 type 1 subjects with normal multimers, only one had a VWF:CB/VWF:Ag ratio of <0.7, likely due to very low values (VWF:CB of 2 and VWF:Ag of 4). There were 36 type 2A subjects available for analysis. 27 had loss of high molecular weight multimers. Only 3 of those had VWF:CB/VWF:Ag ratios >0.7, but none of those subjects had VWF mutations consistent with type 2A VWD. 7 subjects had a shift from high to low molecular weight multimers, 4 with VWF:CB/VWF:Ag ratios >0.7 and either known type 1 mutations or novel VWF gene mutations. 2 subjects had normal multimer distribution, one with a type1 VWD mutation and one with a novel mutation. Characterization of these novel mutations is in progress. All the 17 type 2B subjects had loss of high molecular weight multimers and abnormal collagen binding, with a VWF:CB/VWF:Ag ratio <0.7. Interestingly, however, not all had a reduced VWF:RCo/VWF:Ag ratio, suggesting VWF:CB would be required in addition to VWF:RCo if multimer distribution was omitted in initial evaluation of this type of VWD. Of 18 type 2M subjects, only one had an abnormal multimer distribution. That subject had no mutations in the VWF coding sequence and normal VWF:CB, although the VWF:RCo/VWF:Ag ratio was low at 0.53. Repeat analysis of a new sample from this subject is pending. All 7 type 2N VWD subjects had normal multimers and VWF:CB/VWF:Ag ratios >0.7. In our population, with the exception of mutations that are yet to be characterized, the combination of VWF:Ag, VWF:RCo and VWF:CB was sufficient to categorize patients as normal, type 1, type 2A, 2B or 2M in the before multimer analysis. These findings suggest that VWF:CB is a sensitive screen for detection of an abnormal multimer distribution. Collagen binding is technically much easier to perform, allowing multimer analysis to be reserved for those cases with low VWF:RCo/VWF:Ag or low VWF:CB/VWF:Ag ratios. Disclosures:No relevant conflicts of interest to declare.

Platelet Function Defects in Adolescents with Menorrhagia

Abstract▪2284▪This icon denotes a clinically relevant abstract Background:Platelet function defects (PFDs) are reported to occur frequently in adult women with menorrhagia. There is limited information and lack of uniform testing for platelet dysfunction in the adolescent population, with the few available studies reporting varying incidence from 2 to 44%, depending on the testing methodology employed. We sought to look at our experience in detecting and managing platelet dysfunction in the adolescent menorrhagia population seen in the Texas Children's Hematology Center (TCHC) and Gynecology sub-specialty clinics at Texas Children's Hospital, Houston, Texas. Methods:Adolescents with menorrhagia aged </= 18 years seen at the TCHC undergo a comprehensive bleeding disorders work-up by both hematology and gynecology providers. Menorrhagia was defined as menses lasting longer than 7 days, pictorial blood assessment chart score of > 100 points, using more than 8–10 pads and/or tampons per day, and/or passing clots greater than 1 inch in diameter. Whole blood platelet aggregometry with low/high dose ADP, arachidonic acid, collagen, ristocetin and thrombin agonists are performed as a second tier test in these patients after ruling out thrombocytopenia, coagulation factor deficiencies, and von Willebrand disease. We retrospectively reviewed the medical records of adolescents with menorrhagia seen at the TCHC between June 2009 and November 2010, as approved by the Institutional Review Board. Patient demographics, laboratory results including results of whole blood platelet aggregometry, platelet count, von Willebrand antigen and ristocetin cofactor activity levels were analyzed; menorrhagia therapy details, patient outcome, and follow-up information were obtained. Results:During the study period, 114 female patients with menorrhagia were evaluated for a bleeding disorder at the TCHC. The median age was 14 years with a range of 9–19 years. Of these, 68 patients (59%) had platelet aggregation studies performed. Patients were instructed to avoid non-steroidal anti-inflammatory medications for at least 1 week prior to testing. All patients tested had a platelet count of 150,000 or above. Twenty-eight percent (n=19) of the patients had decreased aggregation and/or secretion results for 1 or more agonist/s. Of these, 6 had defects in aggregation only, 6 had defects in secretion only and 7 had both aggregation and secretion defects. Eleven patients (16%) had 2 or more aggregation and/or secretion defects. Of these, 3 had decreased secretion only, 1 had decreased aggregation only, and 7 patients had combined aggregation and secretion defects.Five of 19 patients with defects in the platelet aggregation studies also had von Willebrand ristocetin cofactor activity < 50%. Of these, 2 had abnormal platelet aggregation to ristocetin only, and 3 had decreased aggregation to ristocetin and ADP. Seventy-nine percent (n=15/19) of patients with platelet dysfunction were managed with hormonal therapy, 42% (n=8/19) received anti-fibrinolytic agents and 16% (n=3/19) received intra-nasal DDAVP for menorrhagia. Of these, 84% (n=16) had improved outcome, with a median follow-up of 15.6 months with a range of 1–66 months.Table 1Platelet aggregation results in adolescent menorrhagia patientsCategoryNumber(%)Adolescents with menorrhagia evaluated114Patients tested with whole blood aggregometry6859Patients with abnormal platelet aggregation19281 Defect Only8122 or More Defects1116Aggregation only34Secretion only11Both710 Conclusion:We conclude that platelet aggregation abnormalities are present in up to one-third of adolescents with menorrhagia. This percentage may be higher as only 59% of our patients were tested for PFDs. Prospective studies to evaluate for PFDs in adolescents with menorrhagia are needed to identify their true prevalence in this population, which will enable accurate diagnosis of PFDs and appropriate management of menorrhagia. Disclosures:Dietrich:CSL Behring: Consultancy, Research Funding; Duramed: Consultancy, Research Funding. Shah:CSL Behring: Research Funding.

Detection of Non Inhibitory Binding Antibodies to Von Willebrand Factor Affecting the Clearance of VWF:Ag in Von Willebrand Disease

Abstract▪2275▪This icon denotes a clinically relevant abstractVon Willebrand Disease (VWD) is an inherited rare bleeding disorder caused by a deficiency of von Willebrand factor (VWF). VWF, the largest multimeric plasma glycoprotein, facilitates platelet aggregation and stabilizes FVIII in the circulation. Inhibitory antibodies to VWF have been reported in 10% – 15% of type 3 VWD patients repetitively treated with plasma-derived VWF/FVIII concentrates. The clinical impact of non-inhibitory antibodies to VWF has not been previously reported. To investigate the immunogenicity of a novel recombinant human VWF (rhVWF), both total binding anti-VWF antibodies and inhibitory anti-VWF antibodies (VWF:Ristocetin Cofactor Activity [RCo], VWF:Collagen Binding Activity [CB], VWF:FVIII Binding [FVIIIB]) were assessed in a Phase 1 multicenter clinical study. Total binding anti-VWF antibodies were determined by an enzyme-linked immunosorbent assay (ELISA) employing polyclonal anti-human IgG antibodies. Plasma samples were analyzed in two steps employing a screening assay determining the titer of the binding antibodies and in a second step, confirming the specificity of the antibody in a competition assay. Samples were considered positive, when a sample was at least two titer steps lower than the antibody titer detected in the screening assay necessitating a screening titer of at least 1:80. Neutralizing antibodies to the key functional activities of VWF, such as VWF:RCo, VWF:CB and VWF:FVIIIB, were measured by assays based on the Bethesda assay established for quantitative analysis of FVIII inhibitors (Nijmegen modification of the Bethesda assay). To exclude false positive results, the detection limit for anti-VWF inhibitors was set to 1 BU/mL for all 3 assays. Three of 39 subjects screened had a pre-existing high titer non-neutralizing binding antibody (all 1/1280) to VWF. One of these 3 subjects was excluded from the study due to the presence of an inhibitory antibody to VWF:CB (1.3 BU/mL) at screening. Two of the 3 subjects were enrolled and treated with rVWF and/or pdVWF concentrate as part of the safety, tolerability and PK assessments. The high titer binding anti-VWF antibodies were associated with a significant decreased VWF:Ag activity post infusion of either pdVWF or rVWF and consequential decreased activity of VWF:RCo, VWF:CB and FVIII:C. For example, one of the subject had a reduced VWF:Ag incremental recovery of 1.1 IU/dL/(U VWF:RCo/kg) (mean of cohort 1.6 IU/dL/[U VWF:RCo/kg]) and a reduced VWF:Ag t1/2 2.4 hours (mean of cohort t1/2 25.3 hours) post infusion of rhVWF dosed at 50 IU VWF:RCo/kg. Relevant data of all PK assessments and antibody titers will be presented. None of the study subjects developed an inhibitory antibody to either VWF or FVIII and there was no impact on the subsequent treatment of bleeding episodes with commercial pdFVIII/VWF concentrates noted by the treating physician. The clinical relevance of non-neutralizing antibodies to VWF requires additional investigation. Disclosures:Suiter:Baxter Innovations GmbH: Employment. Horling:Baxter Innovations GmbH: Employment. Reipert:Baxter Innovations GmbH: Employment. Turecek:Baxter BioScience: Employment. Varadi:Baxter Innovations GmbH: Employment. Chapman:Baxter Innovations GmbH: Employment. Engl:Baxter Innovations GmbH: Employment. Wong:Baxter Healthcare Corp: Employment. Ewenstein:Baxter Healthcare Corp: Employment.

Transient Decrease in Factor VIII Following Delivery: Clearance or Consumption?

Abstract 2286 Background:Von Willebrand factor (VWF) and factor VIII (FVIII) levels increase during pregnancy and return to baseline by one month postpartum (PP). Understanding the normal levels during this period has implications for the management of women with bleeding disorders. As part of a larger study of von Willebrand disease (VWD) postpartum, we obtained VWF ristocetin cofactor (VWF:RCo), VWF antigen (VWF:Ag) and FVIII levels on 26 women without a known bleeding disorder to establish normal ranges during the PP period. Methods:Subjects were enrolled during the last trimester of pregnancy from obstetric clinics and physician practices affiliated with 4 university medical centers. VWF:RCo, VWF:Ag, and FVIII were obtained at enrollment, on admission to the hospital for childbirth, and at 4 hrs, 12 hrs, 24 hrs, 48 hrs, 72 hrs, and at 7, 14, 21, 28, and 42 days PP (6 weeks PP). Specimens were processed within an hour of venipuncture and centrally analyzed at the Duke Clinical Coagulation Laboratory. Means, standard deviations and 95% confidence intervals (CI) were established for each assay at each time point. Results:In the third trimester, mean VWF levels (VWF:RCo = 134 IU/dL [95% CI 113, 154]; VWF:Ag = 182 IU/dL [156, 209]) were 60–80% higher than the baseline levels at 6 weeks PP (VWF:RCo = 85 IU/dL [65, 105]; VWF:Ag = 103 IU/dL [87, 119]). On admission for childbirth, mean VWF levels were another 10–20% higher than the third trimester values (VWF:RCo = 147 IU/dL [108, 186]; VWF:Ag = 213 IU/dL [171, 255]). VWF levels peaked at 12 hrs PP (VWF:RCo = 214 IU/dL [171, 256]; VWF:Ag = 248 IU/dL [204, 293]) which was 140–160% above baseline (6 weeks PP). VWF levels gradually declined to 10% below baseline (VWF:RCo = 74 IU/dL [56, 93]; VWF:Ag = 97 IU/dL [80, 114]) at 3 weeks PP before returning to baseline at 6 weeks PP. This is in contrast to FVIII levels. In the third trimester, mean FVIII levels (FVIII = 127 IU/dL [111, 143]) were 40–50% higher than baseline (FVIII = 86 IU/dL [75, 97]), but on admission for childbirth, FVIII levels were 5% lower (FVIII = 121IU/dL [94, 148]) than the third trimester values. By 24 hrs PP, FVIII levels had dropped another 15% (FVIII = 103 IU/dL [91, 114]) before rising 30% to 134 IU/dL (101, 167). They gradually declined to 10% below baseline at 3 weeks PP (FVIII = 80 IU/dL [69, 91]; see figure) before returning to baseline at 6 weeks PP. These patterns were consistent despite age, race/ethnicity, parity or mode of delivery, although in women who underwent cesarean delivery, the initial nadir in FVIII levels occurred sooner than in women with vaginal delivery (4–12 hrs PP as opposed to 24 hours) and the subsequent peak in levels occurred sooner than in women with vaginal delivery (2 days as opposed to 3 days PP). Conclusions:Unlike VWF levels which increase after delivery, FVIII levels drop 15% after delivery before rising again over the next 1–2 days, and then decline gradually. The explanation for this drop in FVIII is not, clear, but may be due to altered clearance or consumption of FVIII at delivery. [Display omitted] Disclosures:James:CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kouides:CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding. Konkle:CSL Behring: Membership on an entity's Board of Directors or advisory committees. Philipp:CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Evaluation of Comprehensive Hemostatic Function of Patients with Von Willebrand Disease (VWD) Under Flow Using a New Microchip Flow Chamber System

Abstract▪2283▪This icon denotes a clinically relevant abstract Introduction:von Willebrand factor (VWF) acts as bridging molecule between platelets and vessel wall or as a career for plasma factor VIII (FVIII). Earlier reports using flow chamber had revealed that VWF acts as an “initiator” through its interactionwith glycoprotein (GP) Ib, whereas fibrinogen, via its binding to GP IIb-IIIa, acts as a “stabilizer” against high shear. VWD is categorized as partial or complete quantitative defect (type 1 or 3) or qualitative defect (type 2) of VWF based on laboratory tests, such as VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and FVIII activity (FVIII:C). However, the diagnosis of VWD remains difficult because the clinical and laboratory phenotype has wide-spectrum. An assay reflecting clinical phenotypes of VWD and effect of the treatment would be helpful for clinicians. Methods: We evaluated a thrombus formation with a new applicative microchip flow chamber instrument, total-thrombus-formation analysis system (T-TAS®). Citrated or hirudin-added blood from healthy volunteers (n=5) or 3 patients with VWD [symptomatic type 1 (S-1), asymptomatic type 1 (AS-1), and symptomatic type 3 (S-3)] were utilized. Normal blood was mixed with inhibitor for GP Ib (OS-1), GP IIb-IIIa (abciximab) and monoclonal antibody against for VWF (mAb-VWF). Patients’ blood was infused with FVIII/VWF concentrates in (ex) vivo. Re-calcified citrated blood (450 μl) added corn trypsin inhibitor (30 μg/ml) was infused to AR chip of T-TAS at constant flow rate (240–600 s−1), which surface was coated by collagen and tissue factor. Hirudin-added blood (350 μl) was infused to PL chip of T-TAS at higher shear (1,000–2,000 s−1) which surface was coated by collagen. Flow pressure curve was plotted and time to 10 kPa (T10) was evaluated. Furthermore, flow images were recorded with a micro camera. AR chip promoted white thrombus formation (WTF) which reflected both platelet aggregation and fibrin generation, whilst PL chip did only platelet thrombus formation (PTF). Rotational thromboerastometry (ROTEM) were simultaneously investigated. Results: Both abciximab (0.5–2.0 μg/ml) and OS-1 (100–400 nM) inhibited WTF in AR chip dose-dependently. OS-1 (200 nM) inhibited WTF partially at 240 s−1 (T10 15.4±1.6 min, control 8.9±0.4 min), but completely at 600 s−1 (T10 >30 min, control 7.5±0.2 min). In PL chip, both agents inhibited PTF at much lower dose than those in AR chip. ROTEM parameters showed little change in both agents. Using mAb-VWF, similar results to OS-1 were obtained. These data showed that GP Ib-VWF interaction was shear-dependent, consistent with earlier reports. In in (ex) vivo assays, S-1 and AS-1 had comparable VWF levels (VWF:Ag/VWF:RCo 20%/6.4% and 5.8%/3.2%, respectively) in spite of clear difference of clinical phenotype. ROTEM parameters of S-1 were rather better than those of AS-1 likely reflecting FVIII:C (60% and 7.2%). Interestingly, however, PTF in PL chip (at 1,000 s−1) showed significant delay of T10 in S-1 (9.0 min, control 4.1±0.5 min) compared to AS-1 (5.2 min) and after in vivo infusion of FVIII/VWF concentrates, T10 in S-1 was improved (5.0 min). In S-3, ROTEM parameters declined reflecting low FVIII:C (1.2%), and prolonged T10 (>30 min) in AR chip likely reflected complete defect of VWF:Ag (<1.0%) and VWF:RCo (<1.6%). Ex vivo infusion of FVIII concentrates improved ROTEM parameters but not T-TAS parameters, whilst infusion of FVIII/VWF concentrates improved both parameters. Interestingly, scanned images with micro camera in OS-1, mAb-VWF, S-1 and S-3 was quite similar, whilst the image of AS-1 was similar to normal control. S-3 showed the normal images after infusions of FVIII/VWF concentrates (Figure). Conclusion: T-TAS has potentials for diagnosis of VWD reflecting clinical phenotypes and for monitoring the drug effects. Further, AR chip of T-TAS might have a possibility of being a potent tool for categorizing wide-range bleeding disorders “at a glance”. [Display omitted] Disclosures:Hosokawa:Fujimori Kogyo: Employment. Nogami:Bayer Award 2009: Research Funding.

Impaired ristocetin–induced Platelet Aggregation in Whole Blood Assessed with a Multiplate© Analyser Suggests a New Mechanism of Antiplatelet Effect of Aspirin and Clopidogrel,

Abstract 3362The five channel computerized Whole Blood Aggregation instrument (Multiple Platelet Function Analyzer or Multiplate®), assesses platelet aggregation based on a modified whole blood impedance aggregation method. It permits platelet aggregation to be measured after adding commonly used agonists as arachidonic acid (ASPItest), ADP (ADPtest), collagen (COLtest), ristocetin (RISTOtest) and TRAP (TRAPtest), by detecting changes in electrical resistance in whole blood. Instrument handling is easy. Results are available within 9 minutes.Our objective was to evaluate the effect of aspirin (irreversible inhibitor of COX-1) and/or clopidogrel (irreversible inhibitor of the platelet P2Y12 receptor) on whole blood platelet aggregations induced by the 5 agonists using the Multiplate® in patients treated by aspirin and/or clopidogrel.Patients and controls. Two hundred and twenty two consecutive patients were recruited: 83 treated daily by 75 or 100 mg aspirin (group A); 42 treated daily by 75 mg clopidogrel (group C); 70 treated daily by 75 or 100 mg aspirin plus 75mg clopidogrel (group AC) and 27 who were daily on 100 mg aspirin before coronary intervention were tested 12 h after dual loading dose of aspirin between 75 et 500 mg and 75 to 900 mg clopidogrel according to cardiologists' recommendations: group loading aspirin-clopidogrel (LAC). Among group AC, 23 consecutive patients requiring intracranial stent placement of supra-aortic vessel were tested first at preoperative, without antiplatelet therapy, then 1 month after initiation of daily continuous dual antiplatelet therapy by 100 mg aspirin + 75mg clopidogrel. Ninety six volunteers without pathology or drugs influencing platelet functions constitute the normal control group (N).Blood samples. All patient and controls gave informed consent prior to blood sampling. Blood samples were collected by venipuncture or obtained from the arterial sheath directly into vacutainer Becton Dickinson tube containing 0.129M sodium citrate.Results. Patients under medication showing lower aggregation values than the arbitrary cutoff (fifth percentile of the aggregation in the normal control group was selected for each agonist) were classified as abnormal and having biological sensitivity to the agonist tested. Aggregation values above the cutoff with ASPItest or ADPtest for patients on antiplatelet agents were considered as a persistent platelet aggregation and as a biological resistance. According to the literature, resistance to aspirin was found in 8.6% of patients under aspirin alone or in combination and in 25.1% of patients under clopidogrel alone or in combination. Our main result shows an inhibition in platelet aggregation using ristocetin as agonist for 73.9% of patients taking aspirin alone, for 27.8% on clopidogrel and in 94% of patients receiving combination of the 2 drugs. This inhibition appears after aspirin + clopidogrel intake as we could observe it among patients candidates for intracranial stent placement tested before and after one month of treatment by dual antiplatelet therapy.This effect is not related to von Willebrand Factor (vWF) deficiency since the measurement of ristocetin cofactor activity, and vWF antigen carried out among 14 patients exhibiting an inhibition in whole blood platelet aggregation using RISTOtest were normal and unchanged before and after antiplatelet treatment. VWF is essential platelet-to-platelet interactions which is promoted by the binding of VWF with platelet-receptor glycoprotein IbIX (GPIbIX).Our results suggest: 1) aspirin inhibits the interaction of vWF to GP IbIX. This inhibition appears increased by the association of clopidogrel to aspirin. 2) a new mechanism of inhibition of the platelet function GPIbIX-vWF dependant conjointly to inhibition of cyclooxygenase by aspirin and P2Y12 receptor by clopidogrel.Table I:Biological sensibility according to the five tests (%) in the 4 groups testedGroup (n)ASPItestADPtestCOLtestRISTOtestTRAPtestA (83)84.312.038.373.98.4C (42)38.176.219.227.816.7AC (70)90.074.348.288.222.9LAC (27)100.074.163.0100.029.6 Disclosures:No relevant conflicts of interest to declare.

Comparative Evaluation of Five Different Assays for Ristocetin Cofactor Activity Determination,

Abstract 3317 Introduction:Despite significant advances in the comprehension of the molecular pathogenesis of Von Willebrand Disease (VWD), the diagnosis of this condition remains one of the greatest challenges of daily hematology practice, especially in settings of low prevalence such as primary care services. Ristocetin cofactor activity (VWF:RCo) is one of the pillars of the diagnosis of VWD and is also essential for its management. However, reproducibility of tests available to measure VWF:RCo is still a major issue, as evidenced by coefficient of variations (CV) as high as 30%, 45% and 27% in the ECAT, NEQAS and PALQ external quality assessment program. Classical methods to measure VWF:RCo include light-transmission platelet agregometry (LPA) and visual agglutination with formaldehyde fixed human platelet (VA), but alternative methods continue to evolve. An automated method (BC) has been recently shown superior to LPA and VA. Other methods for which limited comparative precision studies are lacking include a new LIA-based automated assay (LIA) and flow citometry (FC), using fluorescence-marked platelets and ristocetin. Methods:Here we evaluate five different VWF:RCo assays, including classical LPA and VA, two different commercially available automated coagulometer-based methods (BC and LIA), and FC. Reference ranges for the each method were established in twenty healthy adults. Plasma samples from thirty-three patients with VWD (type 1: n=26; type 2A or 2M: n=4; type 3: n=3) followed at our comprehensive care center (IHTC-Campinas, Brazil) were assayed for the comparative studies. Diagnosis of VWD was based on historical levels of VWF:Ag (assayed by ELISA) and VWF:RCo (assayed by both LTA and VA) obtained from medical records, and from bleeding history (evaluated by VWD bleeding score). Imprecision studies (intra-assay variability), were performed in different samples: commercial lyophilized normal and abnormal plasma, as well as plasma from two additional VWD patients with borderline results. All analyses were performed in the same samples, in an external quality assessment program-participating university laboratory. Results:Despite using similar conditions than those described in an original report, and repeated experiments, the flow cytometry-based assay (FC) was not reproducible in our hands, yielding extremely high CV that prevented its use in a clinical laboratory. Mean and reference ranges for VWF: RCo assays levels in healthy adults were: VA: 0.84 IU/ml (0.62–1.07); LTA: 1.13 IU/ml (0.92–1.35); BC: 0.93 IU/ml (0.75–1.12). The automated BC method yielded the lower intra-assay variability, in the range of 1.13% to 5.46% in different samples, compared to values as high as 29.4% for LTA. Correlation coefficient (r) ranged from 0.93 between BC reagent and LTA to 0.85 between VA and BC reagent. Mean VWF: RCo levels in patients ranged from 22.99 ± 17.80 with BC to 22.60 ± 18.50 with LTA and 20.11± 14.50 with VA. In twenty-six type I VWD patients, mean VWF:RCo/VW:Ag ratio ranged from was 0.7 (range 0.4–1.5) with the VA method to 0.8 (range 0.8–1.3) with the BC method. In four type 2 VWD patients, mean VWF:RCo/VW:Ag ratio was 0.2 (range 0.3–0.4) using VA method to 0.4 (range 0.4–0.5) with the BC method. Concordance, evaluated using the Bland-Altman plot, was higher between LTA and BC method. Conclusion:In our study, LTA and VA performance was consistent with historical data, presenting very high CV, but good correlation and concordance with new automated methods. Of note, VA, a simple, inexpensive and equipment-independent method, presented better CV than LTA and should be regarded as a good alternative the diagnosis of VWD in areas with limited resources. Unfortunately, flow cytometry-based VWF:RCo methods apparently suffer from the same problems of LTA and VA regarding standardization. New automated assay present enhanced precision compared to VA and LTA, with the added benefits of automation in the setting of a Hemostasis laboratory. Ongoing improvements in protocols of these new assays, leading to even higher precision, could translate into more confident limits of normal and pathological VWF:RCo levels, enabling critical changes in the diagnosis, management and even in the definition of VWD. Disclosures:No relevant conflicts of interest to declare.

No evidence for an association of AB0 blood group and manifestation of thrombotic microangiopathies

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Mechanisms of Bleeding and Approach to Patients With Axial-Flow Left Ventricular Assist Devices

Axial-flow LVADs have become an integral tool in the management of end-stage heart failure. Consequently, nonsurgical bleeding has emerged as a major source of morbidity and mortality in this fragile population. The mechanisms responsible for these adverse events include acquired von Willebrand disease, GI tract angiodysplasia formation, impaired platelet aggregation, and overuse of anticoagulation therapy. Because of ongoing concerns for pump thrombosis and thromboembolic events, the thrombotic/bleeding paradigm has led to a difficult clinical dilemma for those managing patients treated with axial flow LVADs. As the field progresses, advances in the understanding of the pathological mechanisms underlying bleeding/thrombosis risk, careful risk stratification, and potential use of novel anticoagulants will all play a role in the management of the LVAD patient.

Diagnosis and classification of von Willebrand disease

von Willebrand disease (VWD) is considered to be the most common inherited bleeding disorder. VWD is diagnosed following a clinical and physical review, with personal and familial evidence of (primarily mucocutaneous) bleeding, and confirmed by laboratory testing. The latter typically entails initial plasma testing of factor VIII coagulant, von Willebrand factor (VWF) protein ('antigen') and VWF function which has classically been assessed using the ristocetin cofactor (VWF:RCo) assay. More recent attention has focussed on other functional VWF assays, such as collagen binding and so-called 'VWF activity' assays, as possible replacements to the VWF:RCo, or as supplementary tests of VWF 'function'. Additional laboratory testing can comprise a battery of confirmatory and VWD-type assisting assays, including VWF:multimer and von Willebrand factor VIII binding. This review aims to update knowledge of current VWD diagnostics with a particular emphasis on 'functional' VWF assays.

Von Willebrand Factor Assay Proficiency Testing Continued

To the Editor I was very interested to read the recent report from Chandler and colleagues1 on the issue of von Willebrand factor (vWF) assay proficiency testing from the perspective of the North American Specialized Coagulation Laboratory Association (NASCOLA). This excellent report described the experience from North America, in part confirming previous observations from Europe and Australia.2–4 Among the considered findings, the vWF ristocetin cofactor (vWF:RCo) assay yielded the highest imprecision among all vWF assays, and the vWF collagen binding (vWF:CB) assay yielded best overall usefulness in terms of identifying type 2 von Willebrand disease (vWD). As partly explored in the report,1 proficiency testing in Australasia is largely provided by the Royal College of Pathologists of Australasia (RCPA) Quality Assurance programs (QAPs). Notably, the RCPA Haematology QAP has been investigating the issue of proficiency testing in vWF assays for more than a decade, first reporting in this field in 1999,5,6 and most recently in 2008.7 Notably, more than 65 samples have been distributed to participating laboratories, representing a wide diversity of sample types Table 1. Although an update of our findings is due, this correspondence is not …

Acquired von Willebrand syndrome in patients with extracorporeal life support (ECLS)

Extracorporeal life support (ECLS) is used for patients with refractory heart failure with or without respiratory failure. This temporary support is provided by blood pumps which are connected to large vessels. Bleeding episodes are a typical complication in patients with ECLS. Recently, several studies illustrated that acquired von Willebrand syndrome (AVWS) can contribute to bleeding tendencies in patients with long-term ventricular assist devices (VAD). AVWS is characterized by loss of the high molecular weight (HMW) multimers of von Willebrand factor (VWF) as a result of high shear stress and leads to impaired binding of VWF to platelets and to subendothelial matrix. Since ECLS and VAD share several features, we investigated patients with ECLS for AVWS. We analyzed 32 patients with ECLS and 19 of them without support. To diagnose AVWS, ratios of ristocetin cofactor activity (VWF:RCo) and collagen binding capacity (VWF:CB) to VWF antigen (VWF:Ag) were employed in conjunction with multimeric analysis. Reduced VWF:RCo/VWF:Ag ratios were identified in 28 ECLS patients. Furthermore, VWF:CB/VWF:Ag ratios were decreased in 31 patients. HMW multimers of VWF were missing in the same 31 patients. Thus, 31 of 32 ECLS patients presented with AVWS. Twenty-two of the 32 patients suffered from bleeding complications. Without support, AVWS was not detectable in any analyzed patient. Our data indicate that AVWS is a typical disorder in patients with ECLS. We hypothesize that AVWS could contribute to aggravation of bleeding tendencies in ECLS patients.

Diagnosis of Heydeʼs syndrome by abnormal closure times despite normal von Willebrandʼs activity

Heyde's syndrome is characterized by iron deficiency anemia due to gastrointestinal bleeding and calcific aortic stenosis. Patients with this syndrome have a bleeding diathesis due to a loss of the largest multimers of von Willebrand factor (vWF). Here we present a case of Heyde's syndrome diagnosed with abnormal closure times and normal vWF Ristocetin cofactor activity (vWF:Rco). In this case, a 79-year-old man with known aortic stenosis and recurrent gastrointestinal bleeding was cured of a life-threatening hemorrhage after the replacement of his stenotic aortic valve. Factor VIII activity (1.53 IU/ml), vWF antigen (1.26 IU/ml), vWF:Rco (1.11 IU/ ml), and the ratio of vWF antigen/vWF:Rco (0.88) were all within normal limits. Instead, to prove a defect in platelet aggregation, closure times measured with collagen/ADP and collagen/epinephrine were abnormal (>300 s). Postoperatively, these closure times normalized. What is unique about our current report is that we measured both vWF:Rco and closure times, the two readily available assays in most coagulation laboratories. vWF:Rco is a standard assay for measuring platelet activity but may miss defects in platelet aggregation that are only seen under high shear stress. As the closure times can detect such defects, it is perhaps more representative than traditional assays, and in situations such as our case, closure times may be the only method by which subtle abnormalities in vWF function could be detected.

Type 2B-like acquired von Willebrand syndrome

More than 300 cases of acquired von Willebrand syndrome (AVWS) have been reported, but it is thought that this number reflects only the tip of the iceberg [1]. Data from an international registry has shown that AVWS may be associated with lymphoproliferative (48%) or myeloproliferative (15%) disorders, neoplasia (5%), immunological (2%), cardiovascular (21%) and miscellaneous conditions (9%) [2]. AVWS usually mimics von Willebrand disease (VWD) type 1 or 2A [3, 4]. To our knowledge, only two cases of type 2B-like AVWS have been reported previously [4, 5]. They were both associated with plasma cell dyscrasia. Here we show a new case of type 2B-like AVWS. On 2 November 2000, a 74-year-old man was referred to our hospital for the follow-up of monoclonal gammopathy of undetermined significance (MGUS). Serum immunoelectrophoresis showed two kinds of M protein, IgG (j) and IgG (k). The serum immunoglobulin levels were IgG 2289 mg/dl, IgA 175 mg/dl, and IgM 63 mg/dl. He remained relatively well until 24 July 2008, when he developed a high fever. A diagnosis of hydronephrosis and urinary tract infection due to prostatic hypertrophy was made and after transurethral resection of the prostate, which was performed on 22 August 2008, macrohematuria persisted for 4 weeks despite administration of tranexamic acid. There was no other mucocutaneous bleeding. His serum IgG level had hardly changed up to that point. Routine coagulation tests demonstrated: prothrombin time, 11.4 s (normal 10.0–13.5 s); activated partial thromboplastin time, 48.5 s (normal 25.0–40.0 s); plasma fibrinogen level, 298 mg/dl. Factor VIII activity (FVIII:C) was 17% (normal 50–150%), von Willebrand factor antigen (VWF:Ag) was 11% (normal 50–150%) and von Willebrand factor ristocetin cofactor activity (VWF:RCo) was below 10% (normal 50–150%). Activities of factor IX, factor XI and factor XII were all normal. Bleeding time was 2.0 min (normal 1.0–5.0 min). The platelet count was consistently normal. Platelet aggregation induced by ADP, collagen or epinephrine was increased when compared to that in the control. Ristocetin-induced platelet aggregation (RIPA) was also enhanced, showing 70% aggregation with 0.3 mg/ml ristocetin compared with absent aggregation in a normal control (Fig. 1). His platelet-rich plasma (PRP) aggregated spontaneously without the addition of any agonists. All aggregation studies were performed with an aggregometer (MC Medical, Tokyo, Japan) in siliconized glass cuvettes at 37.0 ± 0.5 C with constant stirring at 1000 ± 100 rpm. Plasma VWF multimeric analysis showed that large multimers were absent and medium multimers were markedly decreased. Since these results T. Shigekiyo (&) E. Sekimoto A. Shirakami H. Yamaguchi H. Shibata S. Ozaki Department of Internal Medicine, Tokushima Prefectural Central Hospital, 1-10-3 Kuramoto-cho, Tokushima 770-0042, Japan e-mail: shigekiyo@tph.gr.jp

Value assignment of the WHO 6th International Standard for blood coagulation factor VIII and von Willebrand factor in plasma (07/316).

Value assignment of the WHO 6th International Standard for blood coagulation factor VIII and von Willebrand factor in plasma (07/316).

Validation of an automated latex particle-enhanced immunoturbidimetric von Willebrand factor activity assay.

Laboratory diagnosis of von Willebrand disease (VWD) requires accurate measurement of plasma von Willebrand factor (VWF) activity. To evaluate laboratory characteristics, diagnostic accuracy and testing utilities of an automated latex particle-enhanced immunoturbidimetric VWF assay (VWF:Lx) based on a monoclonal antibody recognizing the VWF-platelet glycoprotein (GP) Ib binding domain. Laboratory characteristics including lower detection limit, linearity, precision, sample stability, and method comparison between VWF:Lx and VWF ristocetin cofactor activity by platelet aggregometry (VWF:RCo) were examined. To assess VWF:Lx diagnostic accuracy, 492 patient plasma samples, including 40 previously characterized VWD patient samples, were tested for VWF antigen (VWF:Ag) and VWF:RCo by either aggregometry or flow cytometry, and VWF:Lx with supplemental VWF multimer analysis when indicated. Based on results of VWF:Ag, VWF:RCo and VWF multimer analysis, and available clinical information, samples were categorized as: normal; VWD types 1, 2A/B, 2M, or severe 1 vs. 2M; or acquired VWF abnormalities (AVWA) due to subtle loss of highest molecular weight multimers. VWF:Lx had excellent laboratory characteristics and linear correlation with VWF:RCo (R(2) = 0.93). VWF:Lx accurately classified virtually all normal and VWD patient samples. Compared with VWF:RCo, VWF:Lx had superior sensitivity and specificity for distinguishing severe type 1 vs. 2M VWD and identifying AVWA. A proposed screening panel comprising VWF:Ag and VWF:Lx had 100% and 83% sensitivity for detecting VWD and AVWA, respectively. VWF:Lx has excellent laboratory characteristics and diagnostic accuracy compared with VWF:RCo, and can be used as part of an initial VWD screening panel and as a supplementary test.

Screening for von Willebrand disease: contribution of an automated assay for von Willebrand factor activity

Measuring von Willebrand factor (VWF) activity is essential to the diagnosis of von Willebrand disease (VWD). The VWF activity is usually assessed based on measurement of the ristocetin cofactor (VWF:RCo). However, that test is technically challenging and has high intra- and inter-assay variabilities. The HemosIL VWF activity (VWF:AC) is a fully automated assay, recently proposed as a good alternative to VWF:RCo for VWD diagnosis. This study was undertaken to assess this new method. First, the analytical performance of VWF:AC on an automated coagulo-meter (ACLTop) was determined, and then this new method was compared with VWF:RCo and the platelet function analyzer (PFA100) for 160 patients referred for VWD screening. The VWF:AC achieved acceptable precision with within-run and between-run coefficients of variation ranging from 2.3% to 14.1%, and linearity from 10% to 100%. Despite some marked differences between VWF:AC and VWF:RCo for 10 plasmas tested, their agreement for VWD diagnosis was good. The VWF:AC had sensitivity similar to that of PFA100 (close to 100%), but better specificity (97.7% vs. 66% or 60%, depending on the cartridge used). The good analytical performance, and the sensitivity and specificity of VWF:AC to detect VWF deficiency renders it a suitable method for VWD screening. Our findings support VWF:AC use for the diagnostic work-up of VWD, paying close attention to concomitant clinical signs and bleeding score, as recommended for VWD.

The Influence of the ABO Blood Type on the Distribution of von Willebrand Factor in Healthy Children With no Bleeding Symptoms

The purpose of this study was to determine the effect of ABO blood groups on von Willebrand factor-ristocetin cofactor activity (vWF-RCo) and on vWF-antigen (vWF-Ag) in children who have no personal or familial history of bleeding. A survey and testing were performed on 200 children with no personal or familial history of bleeding. In all, 100 of them belonged to blood group O, and the remaining 100 belonged to other blood groups. The blood samples were stored at -80°C for a maximum period of 2 weeks to detect vWF-RCo and vWF-Ag levels. The mean vWF-Ag (± 2 standard deviation [SD]) level in children with blood group O was 86% (± 20%); and for those with non-O blood group, it was 98.8% (± 25%). There was a significant difference between the 2 groups (P < .001). The mean vWF-RCo (± 2 SD) level in children with blood group O was 89% (± 23%); and for those with non-O blood group, it was 103% (± 17%). There was a significant difference between those in the 2 groups (P < .001). The lowest value of vWF-Ag and vWF-RCo levels in children with blood group O was found to be 50%. In conclusion, we showed that the selection of normal ranges based on the ABO group might influence the clinical diagnosis of vWD and that while the approach of using ABO group ranges for a vWF-Ag level lower than 50 IU/dL is scientifically sound, it might not be useful to assist a clinician in identifying people at increased risk of bleeding.

Hypercoagulability in Patients With Chronic Noncirrhotic Portal Vein Thrombosis

Although they have normal liver histology and function, patients with chronic noncirrhotic nontumoral portal vein thrombosis (NC-PVT) frequently have abnormal results from coagulation tests. We investigated the significance of these results. We analyzed blood samples collected from 50 stable patients with NC-PVT secondary to a thrombophilic disorder (32%) or local factor (32%), or that was idiopathic (36%). We measured endogenous thrombin potential (ETP) with and without thrombomodulin, prothrombin time, activated partial thromboplastin time, coagulation factors (I, II, V, VII, VIII, IX, X, XI, and XII), antithrombin, proteins C and S, von Willebrand factor (vWF) antigen, vWF ristocetin cofactor, a disintegrin and metalloprotease with thrombospondin type 1 motifs 13 antigen, D-dimer, plasmin-antiplasmin complex, prothrombin fragment F1+2, activated factor VII, and clot lysis time. Samples from 50 age- and sex-matched healthy individuals were evaluated as controls. Compared with controls, patients with NC-PVT had significant increases in prothrombin time and activated partial thromboplastin time; they had significant reductions in levels of procoagulant factors II, V, VII, IX, X, XI, and XII, and the anticoagulants antithrombin, protein C, and protein S. The patients had increased levels of factor VIII and vWF antigen. Irrespective of etiology, patients with NC-PVT had a significant increase in ETP with thrombomodulin and higher levels of factor VIIa, prothrombin fragment F1+2, D-dimer, and plasmin-antiplasmin complex than controls, indicating in vivo activation of coagulation and fibrinolysis. Patients with NC-PVT have hypercoagulability that is independent of the underlying etiology, based on in vitro analyses of thrombin-generation capacity and increased levels of biomarkers in blood samples. Further studies are required to determine if activation of hemostasis increases the risk for thrombotic events.