Common Bleeding Disorder Research Articles

144: How do we Treat Acute Immune Thrombocytopoenia (ITP) in the Maritime Provinces?

The diagnosis and management of immune thrombocytopoenia (ITP) of childhood is controversial amongst paediatricians. ITP is the most common bleeding disorder of childhood and is characterized by an isolated low platelet count (<100×109/L). The disorder is usually self-limiting – nearly 80% of childhood ITP spontaneously resolve within a year. However, many patients receive therapy to avoid the rare incidence of clinically significant bleeding. Published guidelines have differed in recommendations for diagnosis and management. This study aimed to explore approaches in managing acute ITP at the IWK Health Centre and across the Maritimes Provinces. A literature review, retrospective chart review of ITP patients followed at the IWK since January 1, 1990, and an online survey of paediatricians' practices were conducted. Published guidelines are converging and encourage paediatricians to treat based on symptoms not absolute platelet counts. Chart review showed that 99 of 144 patients received at least one course of treatment – 60 of 99 with IVIg and 37 of 99 with corticosteroids. The decision to treat 66 of 99 was on platelet count alone. Bone marrow examinations, recommended in an atypical presentation or prolonged course of disease, were performed in 54 patients. IWK paediatricians relied on clinical experience in 135 cases and consultation with colleagues in 36 cases. Twenty-four Maritime paediatricians who responded to our online questionnaire manage ITP. 17completed the survey. Nineteen of 24 were uncertain of recommended diagnostic platelet counts for ITP. Two of 24 reported using published resources to complete the survey. Thirteen of 21 paediatricians reported they would use published guidelines to guide management in provided case scenarios and 17 of 21 would consult colleagues. Actual management of acute ITP differs from published guidelines: patients are treated more for platelet counts and may be over-investigated and over-treated. Lack of adequate documentation may bias results. Maritime paediatricians prefer managing patients collaboratively, suggesting that further education would benefit practice. A prospective pan-Canada survey would explore this further.

Evaluating errors in the laboratory identification of von Willebrand disease in the real world

Introductionvon Willebrand disease (VWD), reportedly the most common bleeding disorder, arises from deficiency and/or defects of von Willebrand factor (VWF). Assessment requires a wide range of tests, including VWF activity and antigen. Appropriate diagnosis including differential identification of qualitative vs quantitative defects has important management implications, but remains problematic for many laboratories and clinicians. MethodsData using a large set (n=29) of varied plasma samples comprising both ‘quantitative’ VWF deficiency (‘Type 1 and 3’ VWD) vs ‘qualitative’ defects (‘Type 2 VWD’) tested in a cross-laboratory setting has been evaluated to assess the ability of real world laboratories to differentially identify these sample types. ResultsDifferent VWF assays and activity/antigen ratios show different utility in VWD and type identification. VWD identification errors were often linked to high inter-laboratory test variation and result misinterpretation (i.e., laboratories failed to correctly interpret their own test panel findings). Thus, moderate quantitative VWF deficient samples were misinterpreted as qualitative defects on 30/334 occasions (9% error rate); 17% of these errors were due to laboratories misinterpreting their own data, which was instead consistent with quantitative deficiencies. Conversely, whilst qualitative VWF defects were misinterpreted as quantitative deficiencies at a similar error rate (~9%), this was more often due to laboratories misinterpreting their data (~50% of errors). For test-associated errors, ristocetin cofactor was associated with the highest variability and error rate, which was at least twice that using collagen binding. ConclusionThese findings in part explain the high rate of errors associated with VWD diagnosis.

Association of autoantibody specificity and response to intravenous immunoglobulin G therapy in immune thrombocytopenia: a multicenter cohort study

Immune thrombocytopenia (ITP) is a common autoimmune bleeding disorder, in which platelet glycoprotein (GP)IIb-IIIa and GPIb-IX are the two most frequently targeted autoantigens. Our previous studies in animal models of ITP demonstrated that intravenous immunoglobulinG (IVIG) could protect against anti-GPIIb-IIIa autoantibody-mediated thrombocytopenia but failed to ameliorate ITP induced by most anti-GPIb-IX autoantibodies. The objective of this human study was to evaluate the association between the specificity of antiplatelet autoantibodies and response to IVIG treatment. In this retrospective study, a cohort of 156 previously untreated adults with severe ITP who underwent IVIG therapy (0.4gkg(-1) day(-1) for 5days) was analyzed. The primary outcome was response defined as platelet counts of ≥30×10(9) L(-1) and a doubling of baseline counts within 7days of dosing, and an absence of bleeding. Among the 66 patients with anti-GPIb-IX autoantibodies, only 24 (36.4%) achieved a response, as compared with 72 of 90 patients (80.0%) who were negative for anti-GPIb-IX autoantibodies (relative risk 2.2; 95% confidence interval 1.6-3.1). This study found no difference in response between patients with anti-GPIIb-IIIa autoantibodies (61.7%) and those without anti-GPIIb-IIIa autoantibodies (61.3%). Logistic regressions, including main effects and the interaction between these two autoantibodies, showed no influence of anti-GPIIb-IIIa autoantibodies on the effects of anti-GPIb-IX autoantibodies with regard to their association with IVIG response. Thus, in adults with ITP, the presence of anti-GPIb-IX autoantibodies is a predictive factor for poor response to IVIG treatment. ClinicalTrials.gov NCT01666795.

Von Willebrand Disease

Von Willebrand Disease

Childhood Immune Thrombocytopenia: Long-term Follow-up Data Evaluated by the Criteria of the International Working Group on Immune Thrombocytopenic Purpura.

Objective: Immune thrombocytopenia (ITP) is a common bleeding disorder in childhood, characterized by isolated thrombocytopenia. The International Working Group (IWG) on ITP recently published a consensus report about the standardization of terminology, definitions, and outcome criteria in ITP to overcome the difficulties in these areas. Materials and Methods: The records of patients were retrospectively collected from January 2000 to December 2009 to evaluate the data of children with ITP by using the new definitions of the IWG.Results: The data of 201 children were included in the study. The median follow-up period was 22 months (range: 12-131 months). The median age and platelet count at presentation were 69 months (range: 7-208 months) and 19x109/L (range: 1x109/L to 93x109/L), respectively. We found 2 risk factors for chronic course of ITP: female sex (OR=2.55, CI=1.31-4.95) and age being more than 10 years (OR=3.0, CI=1.5-5.98). Life-threatening bleeding occurred in 5% (n=9) of the patients. Splenectomy was required in 7 (3%) cases. When we excluded 2 splenectomized cases, complete remission at 1 year was achieved in 70% (n=139/199). The disease was resolved in 9 more children between 12 and 90 months.Conclusion: Female sex and age above 10 years old significantly influenced chronicity. Therefore, long-term follow-up is necessary in these children.

Identification of predictive factors for response to intravenous immunoglobulin treatment in children with immune thrombocytopenia

Acute immune thrombocytopenia (ITP) is a common benign bleeding disorder of variable etiology characterized by isolated thrombocytopenia. Intravenous immunoglobulin (IVIG) treatment is generally given as an initial treatment to pediatric patients with ITP, but markers predictive of the response to IVIG remain poorly defined. We retrospectively evaluated whether clinical and laboratory findings before treatment could predict response to IVIG and progression to chronic ITP in Japanese children with ITP. Between April 1997 and December 2011, a total of 49 children with newly diagnosed ITP were initially treated with IVIG. Their medical records were retrospectively reviewed. In multivariate analyses, lower white blood cell (WBC) count was the only unfavorable factor for response to IVIG and progression to chronic ITP. Patients with WBC count <7.0 × 10(9)/L had a lower probability of thrombocytopenia-free survival (41 vs. 77 %, P = 0.003) and a higher rate of progression to chronic ITP (29 vs. 6 %, P = 0.040) than those with WBC count ≥7.0 × 10(9)/L. These results suggest that ITP with lower WBC count may represent a distinct subgroup requiring initial treatment other than IVIG.

Severe platelet desialylation in a patient with glycoprotein Ib/IX antibody-mediated immune thrombocytopenia and fatal pulmonary hemorrhage

Immune thrombocytopenia (ITP) is a common bleeding disorder characterized by autoantibody-mediated platelet destruction. Antibodies predominantly target platelet glycoproteins GPIIb/IIIa (70–80%) and GPIb/IX (20–40%), with some patients having both. In animal models, anti-GPIb/IX but not anti-

Platelet-Type von Willebrand Disease: Toward an Improved Understanding of the “Sticky Situation”

We are pleased to highlight in this issue of Seminars in Thrombosis & Hemostasis, the study by Wood et al,1 wherein the authors describe a novel mutation in the platelet GP1BA gene creating a hyperresponsive GPIbα protein—a receptor for von Willebrand factor (VWF)—and causing platelet-type von Willebrand disease (PT-VWD). This is a new naturally occurring mutation in a 23-year-old male patient and is considered the sixth reported mutation thus far in patients described with this disease worldwide. Despite being a rare bleeding disorder, PT-VWD represents a significant challenging clinical problem as it may cause life-threatening bleeding, if not appropriately treated, particularly in situations related to surgeries and childbirth. The clinical diagnosis challenge stems from the close similarity of PT-VWD to the more common bleeding disorder, type 2BVWD.2,3 The discrimination and correct diagnosis can only be made after carefully assessing less commonly performed laboratory tests,3–8 and confirmed only after DNA analysis of the binding regions in the two genes VWF and GP1BA9 has been performed. The report by Woods et al1 highlights some important issues that add to our understanding of this rare but potentially life-threatening bleeding disorder. First, the patient presented with severe bleeding symptoms (rather than mild/moderate bleeding symptoms, and unlike previously reported cases) while also showing other typical laboratory phenotypic data known in this disease such as macrothrombocytopenia, mild spontaneous platelet aggregation, absence from plasma of high-molecular-weight VWF multimers, positive ristocetin-induced platelet aggregation (RIPA) at 0.3 and 0.4 mg/mL, VWF ristocetin cofactor (VWF:RCo) T GP1BA gene mutation that predicted the protein change Try246Leu. This mutation was absent in the unaffected mother and also in 100 healthy control subjects. Second, in that report,1 an attempt was made to quantify the bleeding symptoms in a patient with PT-VWD (bleeding score of 13). The knowledge about variation in bleeding symptoms and the ability to objectively assess these symptoms have proven to be important inmanagement of bleeding disorders and can help predict disease outcome and also aid in treatment.10 The only other such attempt was in a small case series reported recently. This series showed considerable variability in bleeding severity among PT-VWD that is independent of age or gender. The phenotypic variability in type 2B VWD has been reviewed in relation to various mutations and different patients’ cohorts.12 However, a systematic analysis of PT-VWD phenotype and the genotype–phenotype relationship remains to be investigated. Third, for the first time, the level of VWF propeptide (VWFpp) and VWFpp/VWF:Ag ratio was reported in a patient with PT-VWD.1 VWFpp is a 741 amino acid portion that gets cleaved from mature VWF by proteolysis. After cleavage, the VWFpp remains in noncovalent association with the VWF multimers, and both are stored together in the α-granules (megakaryocytes/platelets) or Weibel–Palade bodies (endothelial cells). Upon release and under physiologic pH, the VWF multimers and VWFpp dissociate and are secreted in 1:1 stoichiometric amounts.13 Studies have shown a regulatory role for VWFpp as an intramolecular chaperone for the mature VWF protein and an aid to its storage and multimerization.14,15 The VWFpp circulates in the plasma for a short time, with a half-life of approximately 2 to 3 hours and plasma levels of approximately 1 μg/mL, whereas multimeric VWF circulates with a half-life of approximately 8 to 12 hours and plasma levels of approximately 10 μg/mL.16,17 Recent

Clinical studies of 162 patients with von Willebrand disease

To analyse the epidemiological feature, clinical characters and therapeutic regimens for von Willebrand disease(VWD). The clinical data and laboratory tests results of 162 VWD patients in our center were analyzed. There were 76 males and 86 female among these patients with the mean age of 7.2(2.0-41.0) and 20.7(5.0-48.0) years, respectively. 86 patients (53.1%) were identified to be type 1 VWD, 34 patients (21.0%) type 3 VWD and 42 patients (25.9%) type 2 VWD. Among type 2 VWD patients, 33 patients were type 2A, 4 patients type 2M, 5 patients type 2B. Eighty-seven patients (53.7%) had a definite family history of bleeding tendency. The most common and specific bleeding symptoms were easy bruising (61.7%), epistaxis (53.7%), prolonged bleeding after surgery or minor injury (53.1%). Menorrhagia (66.3%) was common in female patients. The analysis of Vicenza bleeding scores in all patients showed that only 56(34.6%) patients had abnormal bleeding scores. FVIII/VWF concentrates and cryoprecipitate were applied to 45 patients (27.8% ), Desmopressin (DDAVP) to 8 patients. Eight female patients need oral contraceptives jointly to control menorrhagia. Hysterectomy had to be performed in 2 female patients with VWD. VWD was a common congenital bleeding disorder with heterogeneous characters, it was necessary to screen, identify, classify accurately this disease in order to supply to effectively individualized treatment.

Arterial Pseudoaneurysm in Neonates with Hemophilia: Successful Treatment with Noninvasive Clotting Factor Replacement and Ultrasound-guided Compression

Vascular cannulation is an invasive procedure that carries the risk of complications such as pseudoaneurysms. Hemophilia, the most common severe bleeding disorder of inheritance, increases the risk of such complications through underlying hypocoagulability. Although surgical ligation has been considered the gold standard treatment, less invasive options are currently available. Here we present 2 hemophiliac neonates for whom clotting factor replacement and ultrasound (US)-guided compression were successfully used. A 3-week-old male infant and a 4-week-old male infant presented with masses in the left antecubital area and the radial aspects of both wrists, respectively, after arterial punctures. The US confirmed the presence of pseudoaneurysms located at the left brachial artery and right radial artery. US-guided compressions with clotting factor administration initially attempted while confirming a thrombus inside the pseudoaneurysm sac indicated successful management. Arterial cannulation and other procedures in hemophiliac neonates must be attempted with caution because pseudoaneurysms or uncontrolled bleeding may occur. If laboratory analyses or invasive procedures are needed for neonates with a bleeding tendency or a suspected hemophiliac disorder, arterial or venous cannulation requires more caution or should be avoided if possible. This case report suggests that US-guided compression and clotting factor administration are suitable modalities for the treatment of small pseudoaneurysms in hemophilia patients.

Gastrointestinal angiodysplasia and bleeding in von Willebrand disease

Von Willebrand disease (VWD), the most common genetic bleeding disorder, is characterised by a quantitative or qualitative defect of von Willebrand factor (VWF). Patients with VWD suffer from mucocutaneous bleeding, of severity usually proportional to the degree of VWF defect. In particular, gastrointestinal bleeding associated with angiodysplasia is often a severe symptom of difficult management. This review focuses on the pathophysiology, diagnosis and treatment of VWD-associated gastrointestinal angiodysplasia and related bleeding.

Von Willebrand Disease: An Update

Von Willebrand disease (VWD) is a common hereditary bleeding disorder, inherited either as an autosomally dominant or recessive trait. It is caused by a quantitative and/ or qualitative defect in the Von Willebrand Factor (VWF) which plays two critical roles in hemostasis i.e., platelet adhesion and aggregation to damaged endothelium, and also as a carrier for Factor VIII (FVIII) molecule which stabilizes FVIII in the circulation. It is a highly heterogeneous disease with bleeding ranging from mild bleeding tendencies to severe life threatening hemorrhage. Patients are classified as type 1, type 2 and type 3, depending on the qualitative and quantitative defects in VWF antigen. Type 3 i.e., severe VWD has been reported to be the most prevalent subtype in Indian population due to two reasons i.e., higher degree of consanguinity in certain parts of the country and the fact that it is a hospital prevalence data and only patients with severe manifestations present themselves in the hospital. The most common bleeding symptoms in VWD reflect the characteristic defect in platelet adhesion and mucocutanous bleeding especially epistaxis, gum bleeding, menorrhagia and ecchymoses. Bleeding symptoms are of mild to moderate severity for patients with type 1 and few variants of type 2 VWD. However, life-threatening bleeding (Central nervous system, gastrointestinal bleed) can occur in type 3 and type 2 VWD patients, and rarely in type 1 VWD patients. Uncommon bleeding manifestations, such as hemarthrosis, and muscle haematomas are more common in severe deficiency, especially those who have type 3 VWD. Diagnosis, genetic counseling, carrier and antenatal diagnosis plays an important role in the comprehensive management of these cases.

Single-Molecule Imaging of Von Willebrand Factor Activation by Flow

Von Willebrand Factor (VWF) is a long multimeric blood protein that plays a central role in platelet adhesion at sites of elevated hydrodynamic stresses, such as in injured or stenotic vessels. The function of VWF is closely related to thrombogenesis in cardiovascular diseases and the most common hereditary bleeding disorder von Willebrand disease. The effectiveness of the current therapies is limited partly due to the poor understanding of the activation mechanism of VWF, which is regulated by the hydrodynamic stresses in the blood stream. VWF only binds platelets under high shear or elongational flow. Moreover, the length of VWF is regulated by the ADAMTS13 protease cleavage under high shear flow. Studies suggest a hypothesis that the elevated hydrodynamic stresses exert tensile forces on VWF to unfold the tertiary and secondary structures and expose the binding sites for the platelet membrane receptor GP1b and the protease ADAMTS13.This study elucidates the mechanism of VWF function regulation at single-molecule level under tethered shear flow and elongational flow, which are more effective at activating VWF than pure shear flow according to molecular dynamics simulation. We infuse a FRET tension sensing module in each VWF monomer to report the shape and the tensile force of the multimers, and image them under flow with a microscope system that simultaneously records the length and the fluorescence spectrum of a single VWF multimer. For elongational flow, real-time feedback is applied to trap the fast moving single VWF molecules within the high hydrodynamic stress region and the field of view. By adding fluorescently labeled GP1b and ADAMTS13, the rates of binding and cleavage activities in VWF is also measured. These results provide experimental basis for quantitative modeling of the intra-molecular interactions that are responsible for VWF function regulation.

Changes Of Coagulation Parameters During The Menstrual Cycle

Introduction Von Willebrand disease is the most common bleeding disorder with a prevalence of 1-2% of the population. Nevertheless diagnosis of a von Willebrand Syndrom Typ 1 is still challenging. In a newer publication (1), 30 studies about the haemostatic variables during the menstrual cycle were compared. 11 studies were focused on the von Willebrand parameters but only in one study these parameters in patients with von Willebrand disease were observed. We investigated possible cyclic variations in women with menorrhagia, which can lead to a diagnosis of a coagulation disorder. Samples and Methods We conducted a laboratory workup in 122 women sent to our lab for menorrhagia. The following tests were conducted: Blood count, VWF:RCo, VWF:Ag, VWF:CB, Fibrinogen (Clauss), activities of FII, FV, FVII, FVIII (clotting and chromogenic), FIX, FX, FXI, FXII, FXIII during the menstrual cycle on predefined time points (day 1-6, day 7-11, day 12-18, day 19-23, day 24-28). Results In 51 (40%) patients a von Willebrand disease could be detected, 37% hat other coagulation disorders like p. e. factor-VII-deficieny and factor-XIII-deficiency. 11.5% had an iron deficiency. In 11.5% no coagulation disorder could be found. In patients with von Willebrand disease we found cyclic variations especially in the VWF:Ag (p = 0.02). They showed the lowest level during the ovulation. For other coagulation parameters no significance for variations during the menstrual cycle were found. Conclusion There are cyclic variations in von Willebrand antigen. To investigate women on predefined time points during the menstrual cycle can be useful to diagnose a von Willebrand disease particularly in mild cases in which no other suspicious bleeding symptoms exists. Literature Knol H.M., Kemperman R.F.J., Kluin-Nelemans C., Mulder A.B., Meijer K., Haemostatic variables during normal menstrual cycle, Thrombosis and Haemostasis 107.1/2012 Disclosures: Halimeh: Octapharma AG: Investigator Other, Research Funding.

Identification Of Factors That Can Predict a Response To Intravenous Immunoglobulin Treatment In Children With Immune Thrombocytopenic Purpura

Immune thrombocytopenic purpura (ITP) is a common benign bleeding disorder characterized by isolated thrombocytopenia caused by the immune-mediated destruction of platelets. The 2004 Japanese Guidelines for Childhood ITP recommended intravenous immunoglobulin (IVIG) as first-line therapy for children with ITP, because this strategy results in more rapid increases in platelet counts when compared with oral steroid therapy. However, predictive markers of the response to IVIG have not been well defined. The present study therefore evaluated whether clinical and laboratory findings before treatment could predict the response to IVIG and progression to chronic ITP.We reviewed the clinical data of 49 newly diagnosed patients (< 18 years) with ITP who were given IVIG as initial treatment at Japanese Red Cross Nagoya First Hospital from 1997 to 2011, and evaluated the short-term response to IVIG as outcomes at 2 weeks after IVIG and the long-term response to IVIG as thrombocytopenia-free survival (TFS). TFS was defined as the probability of survival without treatment failure (death, relapse, disease progression requiring other therapeutic intervention, or progression to chronic ITP) after initial IVIG and was estimated by the Kaplan-Meier method.Short-term response to IVIG was observed in 40 patients (82%), and long-term treatment response, defined as 1-year TFS, was 65%. At 6 months after initial IVIG treatment, seven patients (14%) progressed to chronic ITP. In multivariate analysis, lower white blood cell (WBC) count at diagnosis was identified as the only independent risk factor for the short- or long-term response to IVIG (P=0.007 or P=0.043, respectively) and the progression to chronic ITP (P=0.034). Patients with WBC count<7.0×109/L had a lower probability of TFS (41% vs. 77%, P=0.003) and a higher rate of progression to chronic ITP (29% vs. 6%, P=0.040) than those with WBC count ≥7.0×109/L. None of the other variables, including gender, age at diagnosis, season of onset, initial platelet count, initial hemoglobin level, preceding infection, fever at diagnosis, H. pylori infection, total and daily dose of IVIG, product of IVIG, and laboratory findings including CRP, TP, T-bil, LDH, AST, ALT or BUN, were significantly associated with the response to IVIG and the progression to chronic ITP in multivariate analysis.In conclusion, this study showed that WBC count at onset of ITP is a strong predictor of short- and long-term treatment response to IVIG and the development of chronic ITP in pediatric patients with ITP. For patients with low WBC count, the addition of other agents, such as steroids, to IVIG at the time of initial diagnosis might improve the treatment outcomes. Disclosures:No relevant conflicts of interest to declare.

Assessment Of The Validation and Performance Of The Cone and Plate(let) Analyzer (CPA) In The Diagnosis Of Von Willebrand's Disease

BackgroundVon Willebrand disease (VWD) is the most common significant bleeding disorder in the western world. VWD presents with a wide spectrum of clinical manifestations, ranging from totally asymptomatic individuals to severe, occasionally life-threatening bleeding episodes and hence early and accurate diagnosis is crucial for starting treatment.Diagnosis of VWD requires specialized laboratories with skilled personnel and the testing process itself is time and labor consuming, and is costly. In recent years several methods for VWD diagnosis were developed including several flow bedside instruments which test whole blood coagulation following shear stress activation of clotting. One such tool is the cone and plate(let) analyzer (CPA). In the CPA test whole blood is activated by a rotating conical disc on a polystyrene surface at high shear rate. The test result is expressed as the % of the plastic surface covered with adherent platelets (SC- surface coverage %) and the average size of the platelet aggregates (average size- AS μm2).The aim of the current study was to test the properties of the CPA as a diagnostic tool for VWD in a non-selected group of patients, to compare its diagnostic abilities with current methods of diagnosis, and to test for presence of (patient and physician) bias in the diagnostic process. MethodsPatients referred for evaluation of bleeding symptoms, post-surgical bleeding, abnormal coagulation tests or with a known familial bleeding disorder were compared to a group of healthy controls and warfarin treated patients. Diagnosis of VWD and all other bleeding disorders was achieved by standard clinical and laboratory methods and in the final analysis VWD patients were compared to all other non VWD groups. The CPA was tested in all patients and controls, first on an inception cohort (268 subjects) and results from that sample were then applied to a validation cohort (221 subjects). The optimal SC and AS values were determined by ROC analysis in each sample and both samples overall. Sensitivity, specificity and likelihood ratios (likelihood of having (LR+) or not having (LR-) VWD given a specific cut-off value) were computed for each cut-off value. ROC method was also applied to test whether age, gender, severity of VWD, presence of bleeding symptoms, genetic/familial diagnosis of VWD and comorbidity of patients had any effect on CPA's testing accuracy. ResultsDuring a 10 year period 489 consecutive patients referred to Sheba medical Center were studied. Following a complete workup we diagnosed (in both cohorts) 149 patients with VWD (type 1-72, type 2-42, type 3+acquired VWD- 35), that were compared to 217 healthy controls, 35 VKA treated patients, and 88 patients with other bleeding disorders (hemophilia A/B – 39, Factor II, VII, XI deficiencies – 49). The surface coverage was found as the best CPA diagnostic parameter for diagnosis of VWD, with an optimal cut-off value of 8.3% in the inception cohort and 6.9 % (but with better sensitivity) in the validation cohort (table and figure). In all of the study cohorts SC optimal cut-off maintained its high specificity with an improved sensitivity and a cut-off value of 7.1%. Age, sex, severity of VWD, or additional genetic/familial diagnosis of VWD had no effect on CPA's testing accuracy. However, patients tested soon after a bleeding event or during hospitalization (originated mainly from the validation cohort) were older, had higher levels VW activity and higher levels of SC and therefore different SC cut-off value with different test characteristics (table).CohortSC %Sensitivity (%)Specificity (%)LR+LR-Inception8.397888.30.03Validation6.9749617.30.27Both7.1849622.10.16 ConclusionIn this relatively large and representative cohort of healthy volunteers and patients with various bleeding disorders the CPA test was found sensitive and highly specific in the diagnosis of VWD. The test was robust to patient's age, sex, and VWD severity but a mild spectrum bias cannot be ruled out (as found in the validation cohort) deriving mainly from patient's comorbidity and recent bleeding. This highly specific test may prove its utility in preliminary or urgent diagnosis of VWD before a complete clinical and laboratory workup is done. [Display omitted] Disclosures:Savion:Mattis medical: Equity Ownership.

Biologic Response and Adverse Events To Stimate In Patients With Von Willebrand Disease

von Willebrand factor (VWF) is a large multimeric glycoprotein secreted from platelet α-granules and Weibel-Palade bodies of endothelial cells. VWF mediates the initial adhesion of platelets at sites of vascular injury and binds to and stabilizes blood coagulation factor VIII in the circulation to protect it from inactivation and clearance. von Willebrand disease (VWD) is the most common hereditary bleeding disorder and results from the deficiency or dysfunction of VWF leading to mucocutaneous bleeding, including epistaxis, menorrhagia, and excessive bleeding after trauma or surgery. Bleeding in patients with VWD is treated with infusion of plasma-derived VWF containing FVIII concentrates or 1-desamino-8-D-arginine vasopressin (DDAVP, desmopression). DDAVP is an analog of vasopressin antidiuretic hormone which can stimulate the exocytosis of VWF from storage sites and increase VWF and FVIII levels. DDAVP can be administrated by intravenous and intranasal routes. There are several reports of the safety and efficacy of intravenous DDAVP, but few with concentrated intranasal DDAVP (Stimate®). Here we report on the efficacy and safety of Stimate® in patients with VWD MethodsHospital records for 72 patients with VWD who received Stimate® from 1998 to 2012 were reviewed. The primary endpoint was the patients' biological response to Stimate®, defined as at least a 3 fold increase compared to baseline of both ristocetin cofactor activity (VWF:RCo), factor VIII procoagulant activity (FVIII:C), or both VWF:RCo and FVIII:C are over 100% after Stimate®. Individuals not meeting the response criteria were deemed to be nonresponse. The adverse events were analyzed between different VWD types, response, age, gender, and race. ResultOf those 72 VWD patients, 45 have type 1 (62%), 7 have type 2 (10%), and in 20 cases the subtype was unclear (28%). Responsive to Stimate® was observed in 43 (95.6%) of type 1, 4 (57.1%) of type 2, and 19 (95%) in which the diagnosis was unclear. In total, 16 patients (22.2%) had adverse events: 1 – allegoric response, 8 - mild headache, 4 - mild hyponatremia, and 4 - blood pressure (BP) reduced more than 20 mmHg. ConclusionPatients with VWD 1 have higher response rates to Stimate® than patients with VWD 2 in our test. The Stimate® is effective and safe for treatment of VWD. Disclosures:Valentino:Baxter, Bayer, Biogen Idec, GTC Biotherapeutics, Inspiration Biopharmaceuticals, Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees.

CD8+ T Cells Are Predominantly Protective In a Murine Model Of ITP and Is Required For Effective Steroid Therapy

BackgroundImmune thrombocytopenia (ITP) is a common bleeding disorder characterized by autoantibody mediated destruction of autologous platelets. The predominant autoantibody detected in ITP patients target the platelet Glycoprotein (GP)IIbIIIa, however autoantibodies can only be detected in 50-70% of patients. Cytotoxic CD8+ T-cells (CTL) may therefore also be a significant contributing factor for thrombocytopenia, either through direct cytotoxicity against platelets, or decreasing platelet production through interaction with megakaryocytes in the bone marrow. Nevertheless, whether CD8+ T cell mediated cytotoxicity significantly contributes to thrombocytopenia in ITP is controversial. Interestingly, CD8+ regulatory T cells have been demonstrated to play significant roles in other autoimmune diseases, but their function in the context of ITP has not been adequately examined. Methods and ResultsWe developed both passive and active murine models to investigate mechanisms of pathogenesis and steroid treatment of ITP. In the passive model, we injected anti-b3 or anti-GPIb antibodies to induce thrombocytopenia; this causes transient antibody-mediated thrombocytopenia. We found that a single intraperitoneal (IP) injection of steroids post-antibody injection was effective at rescuing platelet counts. We also adapted an active model of ITP whereby wild-type (WT) BALB/c mice were transfused with splenocytes from WT platelet immunized β3-/- mice to induce thrombocytopenia. This model encompasses both antibody and cell-mediated ITP and causes sustained thrombocytopenia in the mice. In this model, we found steroid treatment administered either orally or through IP-injection were equally efficacious at ameliorating thrombocytopenia. The successful use of steroids to treat thrombocytopenia in these animal models is representative of the therapeutic effects of steroid treatment seen in human ITP patients.To study the role of CD8+ T-cells in the response to steroid treatments in ITP, we depleted CD8+ T-cells from splenocytes prior to transfusion into WT mice. Unexpectedly, we found CD8+ T cell depleted splenocyte (lacking in CTL cells), engrafted mice had lower, but not higher, platelet counts. They were also less responsive to dexamethasone treatment compared to non-depleted engrafted mice. Furthermore, transfusion of splenocytes from immunized β3-/- mice in conjunction with antigen-primed CD8+ T-cells (isolated from immunized β3-/- splenocytes) was able to rescue platelet counts in WT mice. Co-transfusion of non-primed CD8+ T cells from β3-/- mice could not rescue platelet counts. These results indicate that platelet-antigen specific CD8+ Tregs play a dominant protective role in attenuating platelet clearance. In further support of our observations, we detected significantly increased CD8+CD25+Foxp3+ Treg percentages in the blood, thymus and spleen of immunized β3-/- mice. Further in vitro splenocyte cultures demonstrated putative regulatory mechanisms of CD8+CD25+ Tregs from immunized β3-/- splenocytes. We found CD8+CD25+ Tregs significantly inhibited CD4+ T and CD19+ B cell proliferation, platelet apoptosis, and platelet associated IgG production in the presence of platelet-antigens, but increased the secretion of the anti-inflammatory cytokine IL-10. ConclusionTo the best of our knowledge, these are the first reported animal models of steroid treatment of ITP. We demonstrated that steroid therapy was effective and CD8+ T cells are required for this efficacy. We further unveiled a population of CD8+ regulatory T-cells in the CD8+ T cell populations, which were able to rescue platelet counts. This suggests that CD8+CD25+Foxp3+Treg may impart a predominantly protective role, overcoming the cytotoxic function of CD8+ T-cells in ITP. These data provides significant insights into the understanding of immunopathogenesis of ITP, which may be important in designing effective therapy including the potential usage of CD8+ Tregs as a cellular therapeutic method against ITP. Disclosures:No relevant conflicts of interest to declare.

Binding Of VWF To Type IV Collagen: An Additional Collagen Binding Mechanism Beyond Types I, III, and VI Collagen?

Von Willebrand disease (VWD) is a common bleeding disorder characterized by defects in the interaction of von Willebrand factor (VWF) with its ligands factor VIII, platelet glycoprotein Ib, and/or collagen. Nearly all assessment of collagen binding is performed with types I or III collagen. Type VI collagen, which is formed as a tight triple helix, has also been reported to interact with VWF. In contrast, type IV collagen lacks this tight helical structure and is located in the basal lamina. Type IV collagen may therefore serve as another important ligand for VWF. We examined this interaction in samples from the Zimmerman Program for the Molecular and Clinical Biology of VWD, a large US study of subjects with all types of VWD.Subjects were enrolled based on a pre-existing diagnosis of VWD and compared to a cohort of healthy controls. VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and type III collagen binding using 1 mcg/mL human placental type III collagen (VWF:CB3) were performed in the clinical laboratory at the BloodCenter of Wisconsin. Type IV and type VI collagen binding (VWF:CB4 and VWF:CB6) were performed in the research laboratory by ELISA using 1 mcg/mL human placental type IV or type VI collagen captured on amine binding plates. DNA sequencing was performed on all subjects to identify VWF sequence variations. Bleeding scores were calculated according to the scoring system used in the MCMDM-1VWD study.For this analysis, 251 healthy controls, 535 type 1, 49 type 2A, 16 type 2B, 18 type 2M, 11 type 2N, and 18 type 3 VWD were included. The mean VWF:CB4/VWF:Ag ratio for the healthy controls was 0.96, with a standard deviation of 0.24. This was similar to the mean VWF:CB6/VWF:Ag ratio (mean 0.95, p=NS), but slightly lower than the mean VWF:CB3/VWF:Ag ratio (mean 1.05, p<0.01). The mean VWF:CB4/VWF:Ag ratio was 0.87 for the type 1 VWD subjects. The mean VWF:CB4/VWF:Ag ratio was significantly lower in type 1 subjects as compared to healthy controls (p<0.01). This was attributed in part to the presence of several A1 domain sequence variations, including p.R1399H, p.F1369I, and p.L1382P. While the latter sequence variations were isolated findings in single subjects, p.R1399H was present in 1% of the healthy controls, 1% of the type 1, and 6% of the type 2M VWD subjects.Mean VWF:CB4/VWF:Ag ratios were low for both type 2A and type 2B VWD subjects, at 0.54 and 0.52 respectively. This was not significantly different from the VWF:CB3/VWF:Ag data (p=NS for both type 2A and 2B VWD), consistent with the loss of high molecular weight multimers in these subjects and the concomitant reduction in collagen binding. As expected, VWF:CB4/VWF:Ag ratios were normal in the type 2N subjects, with a mean of 1.04. The type 2M subjects, however, displayed a significant reduction in type IV collagen binding, with a mean VWF:CB4/VWF:Ag ratio of 0.79. One subject with an 11 amino acid deletion (from p.R1392 through p.Q1402) had absent binding to type IV collagen yet normal binding to type III collagen with a VWF:CB3/VWF:Ag ratio of 0.82. Another subject with p.I1425F and p.R1399H had a VWF:CB4/VWF:Ag ratio of 0.48, and VWF:CB3/VWF:Ag ratio of 1.10. Type IV collagen binding did not correlate with VWF:RCo (R squared less than 0.1). The type 2M subjects with a mean VWF:CB4/VWF:Ag ratio less than 0.7 had higher bleeding scores, with a mean bleeding score of 9 compared to a mean of 6 for type 2M subjects with a mean VWF:CB4/VWF:Ag ratio greater than 0.7. Not surprisingly, the subjects with type 3 VWD and undetectable VWF:Ag also had undetectable VWF:CB4.These data suggest that type IV collagen binding reflects an independent function of VWF apart from platelet binding, and distinct from binding to type III collagen. In our study, more variation was seen between type IV and type III collagen than between type IV and type VI collagen. Neither VWF:CB4 nor VWF:CB6 are currently tested as part of the clinical workup of VWD. Measurement of this function may assist in identification of variant VWD, particularly in the subgroup of patients with type 2M VWD. Disclosures:No relevant conflicts of interest to declare.

Rare Bleeding Disorders in Children: Identification and Primary Care Management

Bleeding symptoms are common in healthy children but occasionally may indicate an underlying congenital or acquired bleeding diathesis. The rare bleeding disorders (RBDs) comprise inherited deficiencies of coagulation factors I (congenital fibrinogen deficiencies), II, V, VII, X, XI, and XIII and combined factor deficiencies, most notably of factors V and VIII and of vitamin K-dependent factors. These disorders often manifest during childhood and may present with recurrent or even serious or life-threatening bleeding episodes, particularly during the neonatal period. Accordingly, primary care and other nonhematologist pediatric providers should be familiar with the clinical presentation and initial evaluation of these rare disorders. Bleeding manifestations generally vary within the same RBD and may be indistinguishable from 1 RBD to another or from other more common bleeding disorders. Serious bleeding events such as intracranial hemorrhage may be heralded by less serious bleeding symptoms. The results of initial coagulation studies, especially prothrombin time and activated partial thromboplastin time, are often helpful in narrowing down the potential factor deficiency, with factor XIII deficiency being an exception. Consultation with a hematologist is advised to facilitate accurate diagnosis and to ensure proper management and follow-up. The approach to bleeding episodes and invasive procedures is individualized and depends on the severity, frequency, and, in the case of procedures, likelihood of bleeding. Prophylaxis may be appropriate in children with recurrent serious bleeding and specifically after life-threatening bleeding episodes. When available, specific purified plasma-derived or recombinant factor concentrates, rather than fresh frozen plasma or cryoprecipitate, are the treatment of choice.