Abstracts of the 58th Annual Meeting of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. June 27-30, 2012.

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ABSTRACTS OF THE 58TH ANNUAL MEETING OF THE SCIENTIFIC AND STANDARDIZATION COMMITTEE OF THE INTERNATIONAL SOCIETY ON THROMBOSIS AND HAEMOSTASIS JUNE 27 – 30, 2012 HOT TOPICS HTB01 The ITIM receptor G6b-B is a critical regulator of megakaryocyte activation, platelet production and function Mazharian A1,*, Wang YJ1, Mori J1, Heising S1, Neel BG2,3, Watson SP1 and Senis YA1 1 Centre for Cardiovascular Sciences, Institute of Biomedical Research, School of Clinical and Experimental Medicine, University of Birmingham, Birmingham, UK; 2Campbell Family Cancer Research Institute, Ontario Cancer Institute, Princess Margaret Hospital, University Health Network; 3Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada Objectives: Platelets are highly reactive cells that adhere to exposed extracellular matrix and prevent excessive blood loss. Paradoxically, megakaryocytes which express the same repertoire of receptors as platelets, including the immunoreceptor tyrosine-based activation motif (ITAM)-containing collagen receptor GPVI-FcR c-chain, remain relatively refractory to the extracellular matrix-rich bone marrow environment. Methods: In this study, we investigate the functional role of the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B in megakaryocytes and platelets through targeted deletion of G6b in mice. Results: G6b-B is highly expressed in mature megakaryocytes and platelets and has been implicated as a negative regulator of GPVI and CLEC-2 signalling. It is unique amongst megakaryocyte/platelet ITIM-containing receptors in that it is constitutively phosphorylated and associated with the non-transmembrane protein tyrosine phosphatases Shp1 and Shp2. We show that mice lacking G6b-B are severely macrothrombocytopenic and exhibit a bleeding diathesis due to aberrant platelet production and function. Platelet clearance is increased and platelet production is reduced in G6b-B-deficient mice, resulting in a dramatic reduction in platelet counts. Furthermore, megakaryocytes are pre-activated leading to enhanced shedding of GPVI and a net loss of GPIba. In addition, G6b-B-deficient megakaryocytes exhibit striking reductions in integrin-mediated functions and proplatelet formation. These functions are partially mediated by Shp1 and Shp2 as megakaryocyte/platelet-specific deletion of Shp1 and Shp2 partially phenocopy G6b conditional knockout mice. Conclusion: Findings from this study establish G6b-B as a major new regulator of megakaryocyte activation, platelet production and function. Disclosure of Interest: None declared. Keywords: Macrothrombocytopenia, Megakaryocytes, Mouse model, Platelets, Proplatelets, Signal transduction. © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

HTB02 Platelet gene therapy corrects the hemophilic phenotype in immunocompromised hemophilia a mice transplanted with genetically manipulated human cord blood stem cells Shi Q*, Kuether EL, Schroeder JA, Fahs SA and Montgomery RR Medical College of Wisconsin, Blood Research Institute, Children’s Research Institute, Milwaukee, WI, USA Objectives: Our previous studies have demonstrated that plateletFVIII (2bF8) gene therapy can improve hemostasis in hemophilia A mice even in the presence of inhibitors. In the present study we evaluated the feasibility for platelet gene therapy of human hemophilia A patients. Methods: Human platelet-FVIII expression was introduced by 2bF8 lentivirus (LV)-mediated transduction of human cord blood (hCB) CD34+ cells followed by xeno-transplantation into immunocompromised NSG mice or immunocompromized hemophilia A mice (NSG/ FVIIInull). Recipients were analyzed by FACS, PCR, FVIII:C assay, electron microscopy (EM), and LAM-PCR. Phenotypic correction in was assessed by tail clip survival test and Rotem analysis. Results: Expression of the 2bF8 product was detected in all recipients that received 2bF8 LV-transduced hCB cells. Platelet-FVIII:C levels in recipients were 0.74 0.45 mU/108 total platelets (n = 6), which corresponded to 17.77 10.36 mU/108 human platelets when converted to human platelets based on chimerism levels measured by FACS. EM determined that FVIII was colocalized with VWF in 2bF8 LV-transduced human platelets from recipients. LAM-PCR analysis showed that no identified insertion sites of 2bF8 LV were located in the area represented in the Retrovirus-Tagged Cancer Gene Database. Tail clip test showed that all NSG/FVIIInull recipients (n = 7) that received 2bF8 LV-transduced hCB CD34+ cells survived tail clipping if animals had greater than 2% (3–57%) of platelets derived from 2bF8 LV-transduced hCB CD34+ cells, while 4 of 6 survived when human platelets were between 0.3% and 2%. Rotem analysis of whole blood confirmed that hemostasis was improved in NSG/FVIIInull mice that received 2bF8 LV-transduced hCB CD34+ cells. Conclusion: Our data demonstrate that 2bF8 LV can efficiently introduce FVIII expression in human platelets and that human plateletFVIII can improve hemostasis in hemophilia A mice, suggesting the great feasibility for platelet gene therapy of human hemophilia A patients. Disclosure of Interest: None declared. Keywords: Gene therapy, Hemophilia A, platelet.

HTB03 Nuclear factor (NF)-jB is a major regulator of blood induced joint damage in a murine model of hemophilia A Sen D1,*, Chapla A1, Walter N2, Daniel V3, Yesupatham S4, Srivastava A5 and Jayandharan GR5 1 Department of Hematology; 2Department of Pathology & Forensic Medicine; 3Department of Orthopedics; 4Centre for Stem Cell Research; 5Department of Hematology and Centre for Stem Cell Research, Christian Medical College, Vellore, India Objectives: The precise molecular pathogenetic mechanism(s) responsible for the development of hemophilic arthropathy are not fully known. We hypothesized that NF-jB or its activation targets are mediators of blood-induced joint damage. Methods: Groups (n = 7) of hemophilia A mice were injured in knee joints (Valentino et al, 2008) to recapitulate the effects of single (1 h/ 3 h/7 h/day14) and multiple (day 30/60/75/90) (re)bleeding episodes. Histological analysis was done to demonstrate synovial inflammation and cartilage damage. The expression of 350 key genes related to NF-

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jB- signal transduction, its responsive inflammatory /immuno-regulatory pathways and micro RNA were profiled in RNA isolated from joint tissue by quantitative PCR and the differential expression of key proteins confirmed further by western blotting and ELISA. Results: Distinct members of the NF-jB (NF-jB1/NF-jB2/RelA/ RelB), NF-jB-responsive inflammatory cytokine genes (IL-1b/IL-6/ IFNc/TNFa) were significantly up-regulated in injured Vs control joints after a single articular bleed (1 h/3 h/7 h/24 h) both at the mRNA (> 2 fold, P < 0.05) and protein level (Fig 1). This indicates that NF-jB driven signaling is a major acute phase modulator of inflammation following articular injury. On multiple injury models, downstream targets of NF-jB, that contribute to hypoxia (HIF-1a, 3.3–6.5 fold), angiogenesis (VEGF-A, 2.5-4.4 fold) and chondrocyte damage (matrix metalloproteinase-13, 2.8–3.8 fold) were significantly (P < 0.05) elevated in injured joints (Fig 1). Many key micro RNAs (miR) playing a role in NFjB activation (miR155, 9), inflammation (miR155, 182, 16) and apoptosis (miR155, 186, 19a) were also differentially expressed following injury ( 4 to +13-fold) indicating that small RNAs could modulate the arthritis phenotype. Image/Graph:

Methods: We have determined the crystal structure of a short 8-residue peptide from domain 6 of high molecular weight kininogen bound to the apple 2 domain of the factor XI zymogen via vapour diffusion protein crystallography and molecular replacement. Results: The HKpeptide binds along a hydrophobic groove on the underside of the A2 domain of FXI. In addition, a hydrogen bond network between the HKpeptide and a number of basic residues on the A2 domain surface also appears important in mediating this interaction. Conclusion: The FXI-HKpeptide complex crystal structure represents the first complex between factor XI and high molecular weight kininogen, confirming a key binding site for domain 6 of HK with the apple 2 domain of FXI and also proposes a similar interaction between domain 6 and the apple 2 domain of the FXI homolog prekallikrein (PK). These molecular insights provide a scaffold for development of novel antithrombotics targeting the intrinsic pathway and point to novel pathways that can contribute to factor XI deficiency (hemophilia C). 1. Tait JF, Fujikawa K (1987) Primary structure requirements for the binding of human high molecular weight kininogen to plasma prekallikrein and factor XI. J Biol Chem 262:11651-11656. 2. Papagrigoriou E, McEwan PA, Walsh PN, Emsley J (2006) Crystal structure of the factor XI zymogen reveals a pathway for transactivation. Nat Struct Mol Biol 13:557-558. 3. Emsley J, McEwan PA, Gailani D (2010) Structure and function of factor XI. Blood 115(13):2569-77. Disclosure of Interest: None declared. Keywords: coagulation factor XI, high molecular weight kininogen, protein structure.

HTB05 Involvement of a2-antiplasmin in dendritic growth during memory formation

Conclusion: These data provide the first evidence that NF-jB pathway is involved in inflammatory and chondro-degenerative processes in hemophilia leading to arthropathy. Disclosure of Interest: D. Sen: None Declared, A. Chapla: None Declared, N. Walter: None Declared, V. Daniel: None Declared, S. Yesupatham: None Declared, A. Srivastava: None Declared, G. R. Jayandharan Grant / Research support from: Bayer Early career investigator 2010. Keywords: haemarthrosis, haemophilia A, inflammation, nf-kb.

HTB04 Structure of factor XI in complex with a high molecular weight kininogen peptide Wong SS1,*, Hall G1, Stennicke H2 and Emsley J1 1 Department of Medicinal Chemistry and Structural Biology, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, UK; 2Department of Protein Biochemistry, Novo Nordisk, M aløv, Denmark Objectives: Factor XI (FXI) and high molecular weight kininogen (HK) are key components of the intrinsic pathway. FXI circulates in complex with HK; an interaction mediated through domain 6 of HK with the apple 2 domain of FXI.1 The interaction of these two proteins appears essential in enhancing the formation of complexes between FXI and various ligands.2 The activation of FXI by factor XIIa (FXIIa), resulting in the enzyme FXIa, has been shown to contribute to fibrin formation and stability by activating FIX.3

Kawashita E1,*, kanno Y1, Asayama H1, Okada K2, Ueshima S3, Kaji H2, Matsuo O2 and Matsuno H1 1 Department of Clinical Pathological Biochemistry, D.W.C.L.A., Kyotanabe; 2Department of Physiology II., Kinki University School of Medicine, Osaka-sayama; 3Department of Food Science and Nutrition, Kinki University, Nara, Japan Objectives: Dendritic development in neurons contributes to memory formation, and its maintenance is critical for the prevention of agerelated cognitive decline. a2-Antiplasmin (a2AP), a principal physiological inhibitor of plasmin, is highly expressed in the hippocampus responsible for memory formation. However, a role of a2AP in the hippocampus still remains unclear. This study investigated the involvement of a2AP in the hippocampus-dependent memory formation and dendritic growth. Methods: We performed the Morris water maze test and light-cued fear conditioning test for WT and a2AP / mice, and examined the effect of a2AP on the expression of microtubule-associated protein 2 (MAP2), a dendritic marker, in the hippocampus and hippocampal neurons. Results: Spatial memory and fear conditioning memory were significantly impaired, and the expression of MAP2 in the hippocampus is reduced in a2AP-/- mice in comparison to WT mice. The deletion of the a2AP gene also caused a reduction of the MAP2 expression and the dendritic growth in the hippocampal neurons. a2AP localized especially in the dendrites, and evidently colocalized with Golgi outpost. Treatment with a2AP enhanced the MAP2 expression and dendritic growth in neurons. In addition, the expression level of a2AP in the hippocampus of WT mice decreased with age. Conclusion: These data suggest that a2AP plays an important role in hippocampus-dependent memory formation by regulating the expression of MAP2 required for dendritic development and maintenance. These findings provide new insights into the mechanism underlying memory formation, and could potentially lead to the development of new clinical therapies for cognitive impairments with age. Disclosure of Interest: None declared.

© 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

ABSTRACTS Keywords: aging, antiplasmin, dendrites growth, memory.

HTB06 Modulation of TAFI-dependent regulation of fibrinolysis by extracellular histones Mosnier LO* and Rozenshteyn D Department of Mol & Exp Med, The Scripps Research Institute, La Jolla, CA, USA Objectives: Extracellular histones in extracellular DNA traps play an important role in the host defense against bacterial infection. Despite their beneficial effects, histones are also prothrombotic and cytotoxic. Since histones contain multiple Lys residues including C-terminal Lys, we investigated the effects of extracellular histones on Thrombin Activatable Fibrinolysis Inhibitor (TAFI)-mediated regulation of fibrinolysis. Methods: Various assays. Results: Using Glu-plasminogen and tPA-induced plasminogen activation, histones H2a and H2b both dose-dependently enhanced plasmin formation, whereas histones H3 and H4 had no effect. Stimulation of plasmin formation by H2a and H2b was effectively inhibited in the presence of activated TAFI (TAFIa). In solid phase binding assays, both H2a and H2b, but not H1, H3 or H4, bound plasminogen with high affinity. Next, we determined the effect of histones on the activation of TAFI compared to protein C by the thrombin (IIa)-thrombomodulin (TM) complex. H4 potently inhibited the activation of both protein C and TAFI, whereas H1, H2a and H3 had no effect on either protein C or TAFI activation. Interestingly, H2b completely inhibited TAFI activation but had no effect on the activation of protein C. Thus, H2b selectively inhibits TAFI activation but not protein C activation. Transient TAFI-H2b complexes could be observed during the activation of TAFI with IIa-TM. This prompted us to test whether H2b acted as a suicide substrate during the activation of TAFI. Indeed, removing the C-terminal Lys from H2b restored TAFI activation by IIa-TM as analyzed by quantitative Western blot. Conclusion: In summary, these results indicate that histones H2a and H2b have profibrinolytic effects that are sensitive to inhibition by TAFIa. The selective inhibition of TAFI but not protein C activation by H2b further contributes to its profibrinolytic effects with potential implications for degradation of NETs and the generation of histonederived anti-microbial peptide fragments. Disclosure of Interest: None declared. Keywords: histones, TAFI.

HTB07 Tissue factor expressing neutrophils initiate coagulation on injured vessel walls Darbousset R1, Thomas G2, Mezouar S1, Renne T3, Mackman N4, Dignat-George F1, Panicot-Dubois L1 and Dubois C1,* 1 Aix Marseille University, Inserm UMR-S1076, Marseille, France; 2 Immune Disease Institute, Harvard Medical School, Boston, MA, USA; 3Department of Molecular Medicine and Surgery and Center of Molecular Medicine, Karolinska Institute and University Hospital, Stockholm, Sweden; 4Department of Medicine, University of North Carolina, Chapel Hill, NC, USA Objectives: Following an injury, activation of the blood coagulation cascade and a rapid accumulation of platelets and leukocytes are critical steps to prevent blood loss and intrusion of microorganisms into the bloodstream. However, their relative contributions in thrombosis are still unclear. Here, we investigate the functions of leukocytes for platelet-rich thrombus formation in mice models. Methods: Using the laser-injury system with a high-definition, highspeed camera, we have explored the involvement of different cellular © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

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partners for experimental thrombosis in living mice. In this model, the subendothelial matrix is not exposed to the blood circulation. Thrombin is locally generated and platelet rich thrombi are formed following activation of the endothelial wall. Results: Neutrophils were detected rapidly following the injury and were identified as the exclusive subpopulation of leukocytes able to accumulate immediately after the injury in vivo. Neutrophil accumulation occurred before platelet adhesion and was dependent on endothelial wall activation by a dye laser. Monocytes roll on a thrombus 3–5 min post-injury and may have bound to it 10–15 min later. Inhibition of neutrophil binding to the activated endothelium prevented laser-injury induced thrombus formation, tissue factor accumulation and fibrin generation. Fibrin generation and thrombus formation were partially restored in low TF mice following infusion of Neutrophils purified from WT mice. The absence of factor XII did not affect thrombus formation and fibrin generation. Conclusion: Our results identify a critical in vivo role of neutrophils and not monocyte for initiating thrombosis in diseased vessels. Disclosure of Interest: None declared. Keywords: intravital microscopy, Neutrophils, thrombus formation, Tissue factor.

HTB08 EGFP-EGF1 protein conjugated PEG-PLGA nanoparticles for cerebral thrombi targeting drug delivery Shi W1,2,*, Hu Y1,2, Mei H1,2, Deng J1,2, Zhang B1, Chen C1,2, Wang FH1,2 and Guo T1,2 1 Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science & Technology; 2 Targeted Biotherapy Key Laboratory of Ministry of Education, Wuhan, China Objectives: EGFP-EGF1 protein-conjugated PEG-PLA nanoparticles, recently developed in our laboratory, have shown cerebral thrombus targeting property by advantage of theTF-targeting property of EGF1-EGFP fusion protein. In this study, EGFP-EGF1 protein-con-

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jugated PEG-PLGA nanoparticle (EGF1-EGFP-NP) was employed as a new vector for cerebral thrombi targeting drug delivery. Methods: At first, the cerebral infarction region delivery property of EGF1-EGFP-NP was evaluated by pharmacokinetics studies. Secondly, Multispectral fluorescent imaging, confocal microscopy and TEM were employed to investigated the distribution of the nanoparticles. The acute toxicity of the nanoparticles were also tested. Results: The biodistribution result in rats revealed the brain drug targeting index of EGF1-EGFP-NP was 2.58 compared with NP. The distribution of EGF1-EGFP-NP to the lung and kidney was increased, and decreased in the liver.Multispectral fluorescent imaging demonstrated that the TF-expressing regions of the model rats injected with EGF1-EGFP-NP exhibited higher fluorescence than those of the rats treated with NP, which was around 2 times more than the NP groups. The EGF1-EGFP-NP was predominantly distributed in the areas adjacent to the illuminated regions, which were restricted to the tissue factor on the vessel wall. TEM of microvaculature proved that it was not only exhibited in the microvilli, but also transfected in the microvesicles and further entered the nuclei of endothelial cell. High dose of EGF1-EGFP-NP didn’t interfere the coagulogram and induce the increase amount of macrophage in cerebrum, heart and liver in, only had transient toxicity to lung, spleen and kidney. Conclusion: EGF1-EGFP-NP is a promising cerebral thrombi targeting drug delivery system with little toxicity. Disclosure of Interest: None declared. Keywords: cerebral thrombosis, coumarin-6, Dir, EGFP-EGF1 fusion protein, tissue factor.

HTB09 Loss of protein C receptors links coagulation and inflammation to parasite sequestration in cerebral malaria Moxon CA1,2,*, Wassmer SC3, Milner DA4, Chisala NV1, Taylor TE5,6, Seydel KB5,6, Molyneux ME1, Faragher B2, Esmon CT7, Downey C8, Toh C-H8, Craig AG2 and Heyderman RS1 1 Malawi-Liverpool-Wellcome clinical research programme, Blantyre, Malawi; 2Liverpool School of Tropical Medicine, Liverpool, UK; 3New York University School of Medicine, New York, NY; 4The Brigham and Women’s Hospital, Boston; 5College of Osteopathic Medicine, Michigan State University, East Lansing, MI, USA; 6Blantyre Malaria Project, Blantyre, Malawi; 7 Oklahoma Medical Research Foundation, Oklahoma City, OK, USA; 8Liverpool University, Liverpool, UK Objectives: Cerebral malaria (CM) is a major cause of mortality in African children but pathogenesis remains incompletely understood. Characteristic petechial lesions in the brain imply a localized coagulopathy but its underlying mechanisms in pediatric CM remain unclear. We sought to test the hypothesis that CM is associated with microvascular thrombosis related to dysfunction in the thrombin- protein C axis. Methods: We investigated the coagulation profile and endothelial protein C activation pathway in Malawian children with CM and comatose and non-comatose controls using post-mortem samples, biopsies and plasma. Results: In fatal CM brain microvessels exhibited marked fibrin deposition (N = 10) and loss of the anticoagulant receptor endothelial protein C receptor (EPCR, N = 5) associated with malaria infected red blood cell (iRBC) sequestration. Loss of endothelial EPCR and thrombomodulin was also seen in children with CM in biopsies of subcutaneous tissue taken on admission (N = 17). Coagulation was activated in children with CM, with raised plasma thrombinanti-thrombin (TAT) complexes (N = 67; geometric mean, 26.7 lg/ mL; 95% CI: 20.5–34.8 lg/mL) when compared with children with uncomplicated malaria (N = 30; 10.6 lg/mL; 95% CI: 7.6–14.8 lg/

mL; P ≤ 0.001) and aparasitaemic febrile illness (N = 30; 12.3 lg/mL; 95% CI: 7.4–20.3 lg/mL; P ≤ 0.01). In CM cases, higher admission TAT levels was associated with fatal outcome (P = 0.02). Prothrombin fragment-to-activated protein C ratio was similar between CM and control groups. Conclusion: In Malawian children with CM there is activation of coagulation. While this is compensated peripherally, there is a local loss of endothelial anticoagulant receptors and thrombosis at sites of iRBC sequestration in the microvasculature. This major endothelial homeostatic pathway defect affects principally the brain, where constitutive expression of thrombomodulin and EPCR is low. Disclosure of Interest: None declared. Keywords: cerebral, endothelial, EPCR, malaria, protein C, thrombomodulin.

HTB10 Extracellular histones are eliciting factors for DIC Ito T1,*, Nakahara M2 and Maruyama I1 1 Systems Biology in Thromboregulation; 2Anesthesiology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan Objectives: Histones, abundant proteins in the nucleus, are released into the extracellular space during sepsis. Neutralizing antibodies to histones can rescue mice from lethal sepsis, suggesting that extracellular histones are major mediators of death in sepsis. However, cellular and molecular basis of histone-induced lethality remains to be fully elucidated. Methods: C57BL/6 mice were injected intravenously with purified histones, and the standard laboratory tests, histological examinations, electrocardiogram, and echocardiogram were performed. Prothrombotic effects of extracellular histones were also analyzed in vitro under static and flow conditions. The preventive effects of recombinant thrombomodulin against extracellular histones were then analyzed both in vitro and in vivo. Results: Histones trigger platelet aggregation, leading to thrombotic occlusion of pulmonary capillaries (pictures below) and subsequent right heart failure in mice. These mice displayed signs of disseminated intravascular coagulation (DIC), including thrombocytopenia, prolonged prothrombin time, decreased fibrinogen, fibrin deposition in capillaries, and bleeding symptoms. Recombinant thrombomodulin, a newly approved drug for DIC in Japan, prevented histone-mediated platelet aggregation and protected mice against histone-induced fatal thromboembolism. Image/Graph:

Conclusion: Extracellular histones cause fatal thromboembolism, which is diagnostically-indistinguishable from DIC. Recombinant thrombomodulin can suppress the activity of extracellular histones, and this may contribute to the effectiveness of the drug against DIC. Disclosure of Interest: T. Ito: None Declared, M. Nakahara: None Declared, I. Maruyama Grant / Research support from: Asahi Kasei Pharma, modest. Keywords: DAMPs (damage-associated molecular patterns), Sepsis, Thrombomodulin, Thrombosis


ABSTRACTS HTC01 Prevention of venous thromboembolism after an isolated, non-surgical below-knee injury. Benefit/risk of fondaparinux vs. low molecular weight heparin: the fondacast study Samama CM1,*, Riou B2, Roy P-M3, Sautet A4, Mismetti P5 and FONDACAST study group 1 Department of Anaesthesia and Intensive Care Medicine, HotelDieu University Hospital; 2Department of Emergency Medicine, La Pitie Salp^etriere University Hospital, Paris; 3Department of Emergency Medicine, Angers University Hospital, Angers; 4 Department of Orthopaedic Surgery, Saint Antoine University Hospital, Paris; 5Department of Clinical Pharmacology, SaintEtienne University Hospital, Saint Etienne, France Objectives: Limited data are available about the benefits of pharmacological prophylaxis of venous thromboembolism (VTE) in non-surgical trauma. We have compared the benefit/risk ratio of fondaparinux vs. LMWH in this setting. Methods: Prospective, randomized, open label study in adults requiring 21–45 days of immobilization by cast or brace, for below-knee, isolated, non-surgical injury, with at least one risk factor for VTE. Patients received fondaparinux 2.5 mg (1.5 mg if creatinine clearance 30 to 50 mL/min) or nadroparin 2850 aXa IU (0.3 mL) subcutaneous once daily, until mobilization. Bilateral compression ultrasonography was performed at the end of treatment. The primary endpoint was the occurrence of VTE [asymptomatic or symptomatic deep vein thrombosis (DVT) or symptomatic pulmonary embolism (PE)] or death. Main safety outcomes were major and non-major bleedings. All events were blindly adjudicated by an independent committee. Results: About 1349 patients were randomized and 1170 were evaluable for the primary endpoint (ITT analysis). 91% had a fracture or a ruptured Achilles tendon. The median treatment duration was 31 days in both groups. A primary endpoint occurred in 2.6% (15/584) and 8.2% (48/586) in fondaparinux and nadroparin groups, respectively (P < 0.001; OR = 0.3). There was no difference in the number of patients with symptomatic VTE (3 vs. 7, respectively). There were 2 vs. 3 major or clinically relevant bleeding events, respectively (NS). One death occurred in the fondaparinux group (10 days after the end of treatment, unrelated to treatment). Overall, there was no difference in adverse events. Conclusion: In patients at risk of VTE immobilized following injury to the lower extremity, fondaparinux was more effective than LMWH in the prevention of VTE, with no significant difference in terms of safety. Disclosure of Interest: C. M. Samama Consultant for: AstraZeneca (ximelagatran – ticagrelor) – Bayer (rivaroxaban) – BMS (apixaban) Boehringer-Ingelheim (dabigatran) CSL Behring (PCC) – GSK (nadroparin, fondaparinux) – LFB (PCC) – Leo (tinzaparine) Lilly (prasugrel) – Mitsubishi (argatroban) – Octapharma (PCC) – Organon (danaparo€ıde) – Pfizer (dalteparine, apixaban) – Sanofi-Aventis (aspirin, clopidogrel, enoxaparine, idraparinux), B. Riou Consultant for: GSK, Sanofi, LFB, P.-M. Roy Consultant for: GSK, A. Sautet Consultant for: GSK, Boehringer-Ingelheim, Bayer, Janssen, P. Mismetti Grant / Research support from: Sanofi, Boehringer-Ingelheim, Consultant for: Bayer, BMS, Pfizer, Daichii Keywords: fondaparinux, lmwh, lower limb injury, nadroparin, plaster cast, trauma, venous thromboembolism.


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HTC02 Multiple SNP testing improves risk prediction of first venous thrombosis De Haan HG1,*, Bezemer ID1, Doggen CJ1,2, Le Cessie S1,3, Reitsma PH4,5, Arellano AR6, Tong CH6, Devlin JJ6, Bare LA6, Rosendaal FR1,4,5 and Vossen CY1,7 1 Department of Clinical Epidemiology, Leiden University Medical Center, Leiden; 2Department of Health Technology & Services Research, University of Twente, Enschede; 3Department of Medical Statistics; 4Department of Thrombosis and Hemostasis; 5 Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, The Netherlands; 6 Cardiovascular Department, Celera, Alameda, CA, USA; 7 Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands Objectives: Individual risk prediction for venous thrombosis is currently insufficient. A more accurate identification of high-risk patients is needed to efficiently prevent venous thrombotic events and to reduce treatment-associated bleeding episodes. Our aim was therefore to explore whether inclusion of established thrombosis-associated SNPs in a venous thrombosis risk model improves risk prediction. Methods: We first determined the diagnostic accuracy (i.e., areas under the receiver-operating characteristic curves; AUC) for genetic risk scores based on counting risk-increasing alleles weighted by the literature-derived risk estimates from 31 reported venous thrombosis-associated SNPs in a large case-control study including 2712 patients and 4634 controls (MEGA). Results: Genetic risk scores based on all 31 SNPs or on the 5 most strongly associated SNPs performed similarly (AUCs of 0.70 and 0.68 respectively). For the 5-SNP risk model, the odds ratios showed a 20fold gradient with odds ratios ranging from 0.37 (95% CI 0.25–0.53) for 0 risk alleles to 7.48 (95% CI 4.49–12.46) for ≥ 6 risk alleles. The AUC of a risk scoring system based on non-genetic risk factors was 0.77 (95% CI 0.76–0.78), which improved (P-value < 0.0001) when adding our 5-SNP genetic risk score to 0.82 (95% CI 0.81–0.83). Image/Graph:

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Conclusion: A risk scoring system including five common thrombosisassociated SNPs and non-genetic risk factors for venous thrombosis shows good diagnostic accuracy with an AUC of 0.82 and may aid in the management of high-risk individuals. Disclosure of Interest: H. De Haan: None Declared, I. Bezemer: None Declared, C. Doggen: None Declared, S. Le Cessie: None Declared, P. Reitsma: None Declared, A. Arellano Employee of: Celera, C. Tong Employee of: Celera, J. Devlin Employee of: Celera, L. Bare Employee of: Celera, F. Rosendaal: None Declared, C. Vossen: None Declared. Keywords: epidemiology, Genetics, prediction, venous thrombosis.

HTC03 A randomized-controlled phase II trial of primary thromboprophylaxis with enoxaparin in cancer patients with elevated tissue factor bearing microparticles (the microtec study) Zwicker J1,*, Liebman HA2, Bauer KA1, Caughey T3, Rosovsky R4, Mantha S5, Kessler CM6, Eneman J7, Raghavan V8, Lenz HJ9, Bullock A10, Buchbinder E11, Campigotto F12, Neuberg D12 and Furie B1 1 Division of Hemostasis-Thrombosis, Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, MA; 2 University of Southern California, Los Angeles, CA; 3Mt Auburn Hospital, Cambridge; 4Division of Hematology-Oncology, MGH/ Harvard Medical School, Boston; 5Division of HematologyOncoloy, Lahey Clinic, Burlington, MA; 6Georgetown University Medical Center/Lombardi Comprehensive Cancer Center, Washington, DC; 7York Hospital, York, UK; 8Mt Auburn Hospital/Harvard Medical School, Cambridge, MA; 9University of Southern California-Keck School of Medicine, Los Angeles, CA; 10Division of Hematology-Oncoloy; 11Division of Hematology-Oncology, Beth Israel Deaconess Medical Center/ Harvard Medical School; 12Department of Biostatistics and Computational Biology, Dana Farber Harvard Cancer Center, Boston, MA, USA Objectives: Elevated levels of circulating tissue factor bearing microparticles (TFMP) have been associated with an increased risk of developing venous thromboembolic events (VTE) in cancer patients. Whether primary thromboprophylaxis with enoxaparin prevents the development of VTE in cancer patients with elevated TFMP is not known. Methods: We performed a randomized phase II study to evaluate the cumulative incidence of VTE in cancer patients with high or low levels of circulating TFMP. TFMP were measured by impedance-based flow cytometry. Study subjects with high levels of circulating TFMP were randomized (2:1) to receive enoxaparin 40 mg once daily or observation (without enoxaparin) for 2 months. Individuals with low levels of TFMP were followed by observation alone (without enoxaparin). The observation arms were followed in a double-blinded manner with respect to microparticle status. Lower extremity ultrasounds were performed at baseline and at 2 months. The primary endpoint of the study was the cumulative incidence of VTE at two months. Results: A total of 70 cancer patients were enrolled and four subjects dropped out during screening procedures. The majority of study subjects were diagnosed with advanced pancreatic cancer (45%) followed by non-small cell lung cancer (32%), and colorectal cancer (23%). The cumulative incidence of VTE at 2 months in the high TFMP group randomized to enoxaparin (N = 23) was 5.6% while the high TFMP group-observation (no enoxaparin, N = 11) arm was 27.3% (Gray test P = 0.06). The cumulative incidence of VTE in the low TFMP was 7.2% (N = 32). No major hemorrhages were observed in the enoxaparin arm. The median survival of the high TFMP arms was 360 days compared with 527 days in the low TFMP group (Log rank P = 0.39).

Conclusion: In a prospective randomized trial, increased numbers of circulating TFMP detected by impedance flow cytometry were predictive of a high incidence of VTE. The cumulative incidence of VTE in individuals with high TFMP randomized to enoxaparin was reduced and similar in magnitude to the low TFMP group. Disclosure of Interest: J. Zwicker Grant / Research support from: Sanofi, H. Liebman: None Declared, K. Bauer: None Declared, T. Caughey: None Declared, R. Rosovsky: None Declared, S. Mantha: None Declared, C. Kessler: None Declared, J. Eneman: None Declared, V. Raghavan: None Declared, H. Lenz: None Declared, A. Bullock: None Declared, E. Buchbinder: None Declared, F. Campigotto: None Declared, D. Neuberg: None Declared, B. Furie: None Declared. Keywords: cancer thrombosis, lmwh, tissue factor bearing microparticles.

HTC04 Indirect comparison of efficacy and safety of dabigatran’s RE-LY, rivaroxaban’s rocket-AF, and Apixaban’s Aristotle trial in patients with non-valvular atrial fibrillation Harenberg J1,*, Marx S1, Diener H-C2, Lip G3, Marder V4, Weiss C5 and Wehling M1 1 Clinical Pharmacology, Ruprecht-Karls University Heidelberg, Mannheim; 2Neurology, University of Duisburg, Essen, Essen, Germany; 3Centre for Cardiovascular Sciences, University of Aston in Birmingham, Birmingham, UK; 4Med-Hemat & Oncology, University of California Los Angeles, Los Angeles, CA, USA; 5Biometry and Statistics, Ruprecht-Karls University Heidelberg, Mannheim, Germany Objectives: Dabigatran (two doses), rivaroxaban, and apixaban showed equivalence or superior efficacy and safety compared to warfarin in patients with non-valvular atrial fibrillation (AF). A head-tohead clinical trial comparison of these new oral anticoagulants (NOACs) is unlikely to be performed given the expense of such an investigation. Therefore, the benefits and risks of the NOACs remains to be determined based on the available trial data. Appropriate statistical tools for such an analysis is mixed treatment comparison known as network meta-analysis (NMA). Methods: A NMA of the three new oral anticoagulants was performed extracting the data of the RE-LY-study of dabigatran 110 mg bid and dabigatran 150 mg bid, the ROCKET-trial of rivaroxaban and the ARISTOTLE-trial of apixaban for the composite outcome of ischemic stroke and systemic embolism, and for major bleeding, intracerebral bleeding, mortality and myocardial infarction. The NMA was performed to compare these endpoints using odds ratios and confidence intervals. Results: Dabigatran (150 mg bid) showed superior efficacy in preventing ischemic stroke plus systemic embolism to dabigatran (110 mg bid, P = 0.0364) and rivaroxaban (P = 0.0388). Apixaban had equivalent efficacy with rivaroxaban and dabigatran (both doses). Apixaban induced less major bleeding than dabigatran 150 mg bid (P = 0.036) or rivaroxaban (P = 0.0002). Intracerebral hemorrhage occurred with equal frequency for all agents and regimens except for rivaroxaban (higher risk than dabigatran 110 mg bid, P = 0.0070). Myocardial infarction occurred less frequently with rivaroxaban and apixaban compared to either dose of dabigatran (all P < 0.05). All-cause mortality was not different for any agent or both doses of dabigatran. Conclusion: In the absence of head-to-head comparisons, the NMA suggests that apixaban and dabigatran 110 mg bid may offer the best benefit-risk balance for stroke prevention in AF. Dabigatran 150 mg bid may be preferred for patients with AF and a high risk for embolism. Disclosure of Interest: None declared. Keywords: atrial fibrillation, chromogenic assay, coagulation methods, dabigatran, new oral anticoagulants, rivaroxaban, serum, urine. © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

ABSTRACTS HTC05 Autoantibodies directed against domain I of beta2glycoprotein I indicate an increased risk for thromboembolic events in patients with antiphospholipid antibodies. A prospective cohort study Zuily S1,2,*, De Laat B3,4,5,6, Regnault V2,7, Guillemin F8,9, Kaminsky P10, Albesa R11, Norman G11, De Groot PG3, Lecompte T2,7 and Wahl D1,2 1 Vascular Medicine Division, Nancy University Hospital; 2 INSERM U961, Lorraine University, Nancy, France; 3Clinical Chemistry and Hematology, UMC Utrecht, Utrecht; 4Sanquin Research, Amsterdam; 5Biochemistry, CARIM, Maastricht University; 6Synapse BV, Maastricht, The Netherlands; 7 Haematology Laboratory, Nancy University Hospital; 8INSERM, CIC-EC CIE6, Lorraine University; 9Department of Clinical Epidemiology and Evaluation; 10Orphan disease unit, Nancy University Hospital, Nancy, France; 11INOVA Diagnostics, San Diego, CA, USA Objectives: Our objective was to prospectively determine clinical and laboratory risk factors of thrombotic events, including IgG antibodies directed against domain I of b2-glycoprotein I (b2GPI) in patients with systemic lupus erythematosus (SLE) and/or antiphospholipid antibodies (aPL). Methods: We performed a prospective cohort study in a French University Hospital and tertiary care center. Consecutive patients with SLE and aPL, SLE or aPL alone without ongoing anticoagulant treatment were enrolled. The outcome was the time to a thrombotic event. Blood was drawn at baseline for measuring laboratory factors. Results: Ninety-two patients (median age 40 years [interquartile range IQR: 29–58]; 74 women) were followed-up for a median duration of 35 months (IQR: 26-62; 320 patient-years). Thrombosis during follow-up occurred in 18 patients. Ten patients had levels of anti-domain I IgG antibodies above the cut-off of 25 GPLU. These high levels of antidomain I IgG antibodies were associated with the presence of antib2GPI antibodies (P = 0.016), of lupus anticoagulant (P = 0.006), and of activated protein C (APC) resistance determined by thrombin generation (P = 0.001). During follow-up, thrombotic events occurred in half patients (n = 5) with high levels of anti-domain I IgG antibodies compared to 13 patients (16%) without. The presence of high levels of anti-domain I IgG antibodies was associated with a 3.6-fold higher risk for thrombotic events during follow-up (RR = 3.64 [95% CI, 1.28–10.35], P = 0.016). Clinical variables such as history of hypertension, of superficial venous thrombosis and of arterial thrombosis also did confer an increased risk for incident thromboembolic events. Conclusion: High levels of antibodies directed against domain I of b2GPI are associated with an increased risk for thrombotic events in SLE or aPL patients. In addition, our results suggest that APC resistance is one of the pathogenic mechanisms by which anti-domain I IgG antibodies cause thrombosis. Disclosure of Interest: S. Zuily: None Declared, B. De Laat Grant / Research support from: The Dutch Heart Foundation (2006T053)., V. Regnault: None Declared, F. Guillemin: None Declared, P. Kaminsky: None Declared, R. Albesa: None Declared, G. Norman: None Declared, P. De Groot: None Declared, T. Lecompte Grant / Research support from: Ministere Francßais de la Sante et des Sports (Programme Hospitalier de Recherche Clinique) and Fondation de France., D. Wahl Grant / Research support from: Ministere Francßais de la Sante et des Sports (Programme Hospitalier de Recherche Clinique) and Fondation de France. Keywords: activated protein C resistance, antiphospholipid antibodies, antiphospholipid syndrome, beta2-glycoprotein I, domain I antibodies, lupus, superficial venous thrombosis, thrombin generation test, Thromboembolic.


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HTC06 Whole-exome sequencing identifies novel risk variants for thrombotic storm Ortel TL1,*, Beecham G2, Hedges D2, Whitehead P2, Beecham A2, Hahn SE2, Lawson JW3, Erkan D4, Brandao LR5, James AH6, Manco-Johnson MJ7, Kulkarni R8, Kitchens CS3, PericakVance MA2 and Vance JM2 1 Hematology, Duke University, Durham; 2University of Miami Miller School of Medicine, Miami; 3University of Florida, Gainesville; 4Hospital for Special Surgery, New York, NY, USA; 5 Hospital for Sick Children, Toronto, ON, Canada; 6Duke University, Durham; 7University of Colorado Denver, Aurora; 8 Michigan State University, East Lansing, MI, USA Objectives: Thrombotic storm (TS) is defined by two or more of the following in a short period: (i) more than two acute arterial/venous thromboemboli, or thrombotic microangiopathy, (ii) unusual location, (iii) progressive/recent unexplained recurrence, or (iv) refractory/atypical response to therapy. We hypothesize prothrombotic genetic risk factor(s) trigger an accelerated form of thrombosis following an initial event. Methods: We performed whole-exome sequencing (WES) on two families with TS (one affected, unaffected parents, and unaffected sibs). Another 25 TS cases were sequenced to screen genes identified. DNA was captured using the Agilent 50mb kit and sequenced using Illumina HiSeq2000. Alignment and genotype calling were performed with BWA and GATK. Variants were filtered by putative function, conservation, damaging, quality of sequencing, and frequency. Results: Investigating de novo and autosomal recessive (AR) inheritance patterns revealed 13 de novo and 7 AR variants after filtering. Among these are two nonsense mutations: an AR stop mutation in EGFL8 (tyr74stop) and a de novo nonsense mutation in SLC26A2 (arg178stop). Both variants were validated using Sanger sequencing and were rare in the 5000 sample NHLBI GO Exome Variant Server (EVS). The variant in SLC26A2 was absent in EVS and the EGFL8 variant was rare (MAF < 0.0025). The top 20 genes were screened in another set of 25 TS samples; 12 genes had additional putatively rare, damaging variants in other TS samples. In particular, three genes had three additional variants in the other case samples (GRIK1, LLGL2, TUBGCP6). Conclusion: These are excellent TS candidate loci. In particular, EGFL8 has two EGF domains, a common motif in hemostatic and fibrinolytic proteins, and is, thus, potentially involved in coagulation. We are sequencing our larger collection of TS families. These variants will be further analyzed for frequency in controls and tested in animal models for functional significance. Disclosure of Interest: None declared. Keywords: antiphospholipid syndrome, Catastrophic Antiphospholipid Antibody Syndrome, Gene mutation, Thrombosis, Thrombotic storm.

HTC07 A novel genetic variant of the cytosolic phospholipase A2 alpha associated with bleeding diathesis and duodenal ulcers in an Italian family Faioni EM1,*, Razzari C1, Podda G1, Trinchera M2, Fontana G1 and Cattaneo M1 1 Medicina 3, DMCO, University of Milano, Milano; 2DMCS, University of Insubria, Varese, Italy Objectives: Cytosolic Phospholipase A2 alpha (cPLA2a) releases arachidonic acid (AA) from membrane phospholipids, thereby allowing production of prostaglandins and thromboxane A2. We report a novel inherited defect of cPLA2a identified in a 25 year old man and his family.

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ABSTRACTS

Methods: The cPLA2a gene (PLA2G4A) exons and intron/exon boundaries from the proband were sequenced. The identified genetic variant was searched for in the family members and in 100 normal subjects. Platelet secretion and aggregation induced by collagen (2 lg/ mL) or AA (1 mM) were studied in platelet rich plasma. Thromboxane B2 was measured in serum by ELISA. Results: The variant is in exon 15 (1723G>C), within a catalytic domain, and leads to the substitution of His for Asp in codon 575. Asp575 is highly conserved across species and in the different subtypes of cPLA2. 1723G>C was not identified in 200 alleles from normal subjects. The proband and his twin sister, both homozygous for the variant, are affected by a mucosal bleeding diathesis and duodenal ulcers, while his parents and a sister, heterozygous for the variant, are asymptomatic. Platelets from the proband and the twin sister did not undergo aggregation and secretion when stimulated by collagen, while aggregation and secretion were normal when induced by AA. Serum thromboxane B2 levels were extremely low in the proband and the twin sister, normal in the other studied family members. No other platelet defects were identified. Conclusion: Clinical and laboratory phenotypes co-segregate with the homozygous cPLA2a variant Asp575His. To our knowledge, this is the first family described with a cPLA2a variant associated with bleeding diathesis and duodenal ulcers. A partial cPLA2a defect associated with intestinal ulcers and platelet dysfunction but no bleeding diathesis was described by others (J Clin Invest, 2008). These rare gene defects should be considered in the work-up of patients with GI bleeding and impaired platelet function. Disclosure of Interest: None declared. Keywords: bleeding, genetic defect, phospholipase, thromboxane.

HTC08 Novel MHC peptide- T cell receptor interfaces are necessary for inhibitor formation in mild/moderate haemophilia a secondary to mis-sense mutation genotypes Hart DP1,2,*, Skelton S1,3, Moss DS3, Gomez K4, Sansom C3 and Shepherd A3 1 Haematology, Barts and The London School of Medicine & Dentistry, Queen Mary University; 2Haemophilia Centre, Royal London Hospital; 3Structural & Molecular Biology, Biological Sciences, Birkbeck, University of London; 4Haematology, University College London, London, UK Objectives: We are developing an in silico risk stratification strategy using artificial neural networks, to identify more accurately a person with haemophilia’s inhibitor risk, rather than just a population-based estimate based on the known F8 genotype alone. Missense genotype mild/moderate haemophilia A lends itself particularly well to develop such an analysis, enabling focus on the single, allogeneic amino acid change. Methods: We utilise a validated algorithm to model binding of fVIII derived peptides in the groove of multiple HLA DR alleles. Anti-fVIII antibody responses are T cell dependent. Consequently, we also determined whether a novel interface arises between the antigen presenting cell and CD4 T cell receptor (TCR) as a result of a given fVIII mutation. We hypothesised that a missense mutation resulting in a predicted peptide-interface change with a TCR should be capable of stimulating an anti-fVIII response, and conversely, lack of a novel TCR interface is likely to be protective against anti-fVIII antibody formation. Results: Four hundred and seventy nine different missense mutations causing mild/mod haemophilia A were identified on the HaMSTeRS data base. Each mutation was modelled through all 14 assessable HLA-DR alleles and compared to wild-type fVIII peptide binding. All missense mutations with a reported inhibitor occurrence had a predicted novel interface in at least one of the assessable HLA DR alleles. None of the mutations we predict as having no (or negligible) risk of

developing inhibitory antibodies have had reported development of such antibodies. More generally, our results show that there is a significant correlation between the inhibitor rate associated with a particular missense mutation and the proportion of HLA DR alleles predicted to bind a T-cell epitope spanning the relevant location. Conclusion: Our results are consistent with the hypothesis that an MHC class II response is necessary, but not sufficient, for a patient with mild/moderate haemophilia A to develop an inhibitor. Disclosure of Interest: None declared. Keywords: in silico prediction, interface, MHC, peptide, TCR.

HTC09 Spatial blood coagulation in the presence of anti-TFPI aptamer BAX499 in hemophilia a patients after fVIII infusion Parunov LA1,*, Soshitova NP2, Fadeeva OA2, Balandina AN1,2, Kopylov KG2, Kumskova MA2, McGinness KE3, Panteleev MA1,2, Ataullakhanov FI1,2,4, Schaub RG5 and Gilbert JC5 1 Center for Theoretical Problems of Physicochemical Pharmacology; 2National Research Center for Hematology, Moscow, Russian Federation; 3Baxter Healthcare Corporation, Cambridge, MA, USA; 4Department of Physics, Moscow State University, Moscow, Russian Federation; 5Archemix Corporation, Cambridge, MA, USA Objectives: The aptamer BAX 499 (formerly ARC19499) is a high-affinity, specific tissue factor pathway inhibitor (TFPI) antagonist designed as a bypassing agent to improve hemostasis in hemophilia. Since factor VIII, widely used in replacement hemophilia A therapy, and BAX 499 are both procoagulants, their combined effect on spatial clot formation could be potentially pro-thrombotic. The objective was to investigate drug-drug interaction of BAX 499 and factor VIII in hemophilia A in a spatial, reaction-diffusion experimental in vitro system. Methods: Blood was collected at different time points from hemophilia A patients undergoing prophylaxis (with Haemoctin, Octanate or Kogenate) and supplemented in vitro with BAX 499 at concentrations from 0 to 600 nM. Clotting propagation in recalcified plasma activated by a surface with immobilized tissue factor (TF) was monitored by videomicroscopy. Results: Addition of BAX 499 improves coagulation in a concentration-dependent manner for all hemophilia A plasma samples activated with tissue factor (TF) at 2 pmole/m2 by shortening lag time, increasing initial clot growth velocity, and increasing clot size. In contrast, blood concentration of factor VIII has smaller effects on lag time, but increases spatial clot growth velocity. There is a decrease in the BAX 499 efficiency with increasing factor VIII concentrations. The lag time shortens by 50% if FVIII is < 5%, but the effect is only 25% if FVIII is > 30%; however, due to the initially prolonged lag time in the former case, the final lag times achieved with saturating BAX 499 concentrations are similar for all factor VIII concentrations. Conclusion: The results suggest that patients can be safely treated with BAX 499 without concern for their current factor VIII level, and, vice versa, patients taking BAX 499 during clinical trials can be safely treated with factor VIII concentrates. Disclosure of Interest: L. Parunov Grant / Research support from: Archemix Corp, N. Soshitova Grant / Research support from: Archemix Corp, O. Fadeeva Grant / Research support from: Archemix Corp, A. Balandina Grant / Research support from: Archemix Corp, K. Kopylov Grant / Research support from: Archemix Corp, M. Kumskova Grant / Research support from: Archemix Corp, K. McGinness: None Declared, M. Panteleev Grant / Research support from: Archemix Corp, F. Ataullakhanov Grant / Research support from: Archemix Corp, R. Schaub Employee of: Archemix Corp, J. Gilbert Employee of: Archemix Corp Keywords: BAX499, coagulation, hemophilia A, spatial propagation, tissue factor, tissue factor pathway inhibitor. © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

ABSTRACTS HTC10 The incidence of factor VIII inhibitors in severe haemophilia a following a major product switch in the UK: a prospective controlled study in 1214 previously treated patients Hay CRM1,*, Collins P2, Palmer B3, Liesner R4, Chalmers E5, Hart D6, Rangarajan S7, Ragarajan S8, Talks K9, Talks K10, Talks K11, Williams M12, Talks K13, Talks K14, Williams M15, Williams M15, Williams M15 and UKHCDO Inhibitor Working Party 1 University Department of Haematology, University of Manchester, Manchester; 2University Department of Haematology, National Hospital for Wales, Cardiff; 3National Haemophilia Database, Manchseter Royal Infirmary, Manchester; 4 Department of Haematology, Great Ormond Street Hospital, London; 5Department of Haematology, Glasgow Hospital for Sick Children, Glasgow; 6Department of Haematology, Barts and the London Hospital; 7Department of Haematology, St Thomas’ Hospital; 8Department of Haematology, Guys and St Thomas’ Hospital, London; 9Department of Haematology, Royal Victoria Infirmary; 10Department of Haematology, Royal Victoria Infirmary, Newcastle; 11Department of Haematology, Royal Victoria Hospital, Newcastle on Tyne; 12Department of Haematology, Birmingham Children’s Hospital, Birmingham; 13 Department of Haematology; 14Royal Victoria Infirmary, Newcastle on Tyne; 15Birmingham Children’s Hospital, Birmingham, UK Objectives: Switching of factor VIII products has been suggested to be associated with an increased risk of inhibitor formation. The UK National Tendering Exercise (2009/10) caused half of patients to change rFVIII brands. This presented the opportunity to determine whether switching increased inhibitor risk in PTPs by comparing switchers with non-switchers Methods: Exclusions from analysis included: previous inhibitor history, PUPS < 50 EDs, < 12 mths follow-up before switch date. Patients were locally selected for switching to fulfil contractual agreements on a commercial or random basis, assuming products to be generic. Centres were requested to test all patients for inhibitors prior to the switching date and six-monthly thereafter. All test data was collected to analyse for testing bias. Past UK data led us to expect 5.54 (95% CI: 4.67–6.56) inhibitors/1000 treatment years. Results: About 1214 pts with severe haemophilia A were included in the analysis, 523 who switched (502 to Refacto AF) and 672 who did no (+ 27 treated with multiple products). Although significantly more switchers than non switchers had a pre-switch-date test (50 vs. 38%; P = 0.0005), there was no difference in test frequency after the switchdate (med. 2 [IQR 1–3); P = 0.112). Four new inhibitors were reported amongst PTPs (> 50 EDs), one in a non-switcher using Kogenate and in three who had switched from Kogenate (one) or Advate (two) to ReFacto AF, an incidence of 3.3 per 1000 treatment/years). One inhibitor (non-switcher) persists (53BU). All three inhibitors following switching were transient, disappearing after, 3, 3 and 8 weeks respectively, after 3-weeks ITI in two (peak 0.99, 2.5, 10.8 BU). These displayed type-2 PK and a transient ‘acquired haemophilia’ bleeding phenotype. Conclusion: Switching rFVIII brands was not associated with an increased inhibitor risk. Although this is much the largest study of this type ever conducted, a very much larger sample size would be required to give a conclusive result. Disclosure of Interest: C. Hay Grant / Research support from: The study was supported by Baxter and Pfizer, Speakers Bureau: Baxter Pfizer, Bayer, CSL Behring, Novo, Grifols, LFB., P. Collins: None Declared, B. Palmer: None Declared, R. Liesner: None Declared, E. Chalmers: None Declared, D. Hart: None Declared, S. Rangarajan: None Declared, S. Ragarajan: None Declared, K. Talks: None © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

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Declared, K. Talks: None Declared, K. Talks: None Declared, M. Williams: None Declared, K. Talks: None Declared, K. Talks: None Declared, M. Williams: None Declared, M. Williams: None Declared, M. Williams: None Declared. Keywords: Factor VIII inhibitors, incidence switching, prospective, controlled study.

Antithrombotic Therapy ATT01 XAMOS: a non-interventional study comparing thromboprophylaxis using oral rivaroxaban with conventional regimens after major orthopaedic surgery of the hip and knee Turpie AG1,*, Jamal W2, Schmidt A3, Lassen MR4, Mantovani L5, Kreutz R6 and Haas S7 1 Department of Medicine, McMaster University, Hamilton, USA; 2 Gloabl Medical Affairs, Bayer Healthcare; 3Global Medical Affairs, BayerHealthcare, Berlin, Germany; 4Glostrup Hospital, Spine Center, University of Copenhagen, Glostrup, Denmark; 5 Center of Pharmacoeconomics, Federico II University of Naples, Naples, Italy; 6Charit e-Universit€ atsmedizin, Institut f€ ur Klinische Pharmakologie und Toxikologie, Berlin; 7Institute for Experimental Oncology and Therapy Research, Technical University of Munich, Munich, Germany Objectives: In the RECORD programme, rivaroxaban showed superior efficacy and a similar safety profile to enoxaparin for the prevention of venous thromboembolic events in patients undergoing elective hip or knee replacement surgery. XAMOS, an international, non-interventional, open-label cohort study, assessed rivaroxaban in clinical practice for thromboprophylaxis in patients after major orthopaedic surgery of the hip or knee. Methods: XAMOS assessed adverse events in patients receiving rivaroxaban or conventional thromboprophylaxis regimens. Approximately 200 centres enrolled patients who underwent elective hip or knee replacement surgery and received rivaroxaban or other thromboprophylaxis. The attending physician determined the type, duration and dose of drug. Investigators documented all adverse events, including symptomatic thromboembolic and bleeding events. To address treatment selection bias that can occur in non-randomized trials, further analysis subclassified patients by estimated propensity scores so that patients receiving rivaroxaban and other thromboprophylaxis within each subclass were matched according to baseline variables. Results: Of 17 413 patients enrolled, 8778 received rivaroxaban and 8635 received other thromboprophylaxis (low molecular weight heparin [LMWH], 81.7%). The incidence of any symptomatic thromboemImage/Graph:

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ABSTRACTS

bolic event was lower in patients treated with rivaroxaban compared with the group receiving LMWH and those receiving any conventional therapy. Major bleeding rates were similar in the rivaroxaban and conventional therapy groups (Table), consistent with the findings of the RECORD studies. Conclusion: The clinical benefit of thromboprophylaxis with rivaroxaban demonstrated in the RECORD programme was confirmed in everyday clinical practice in the larger patient population of the XAMOS study. Similar outcomes were observed in the propensity scorebased analysis, providing further evidence that trial results can be translated into clinical practice. Disclosure of Interest: A. Turpie Consultant for: Bayer HealthCare, Janssen Pharmaceutical Research & Development LLC, Astellas, Portola, and Takeda, W. Jamal Employee of: Bayer HealthCare, A. Schmidt Employee of: Bayer HealthCare, M. Lassen Consultant for: Astellas, BayerHealthcare, Bristol-Myers Squibb, Boehringer Ingelheim, GSK, Merck Serono, Pfizer, Protola Pharma and Sanofi-Aventis, L. Mantovani: None Declared, R. Kreutz Consultant for: Bayer HealthCare, S. Haas Consultant for: Bayer Healthcare, BMS, Boehringer Ingelheim, CSL Behring, Novartis, and Sanofi-Aventis Keywords: adverse events, bleeding events, low molecular weight heparin, major orthopaedic surgery, propensity score, rivaroxaban, thromboprophylaxis, venous thromboembolism.

ATT02 Risk of thromboembolism, recurrent bleeding and death after warfarin interruption for gastrointestinal bleeding Witt DM1,*, Delate T1, Garcia D2, Clark N1, Hylek E3, Ageno W4, Dentali F4, Crowther M5 and WARPED Consortium 1 Pharmacy, Kaiser Permanente Colorado, Aurora; 2University of New Mexico, Albuquerque; 3Boston University, Boston, MA, USA; 4University of Insubria, Varese, Italy; 5McMaster University, Hamilton, ON, Canada Objectives: To determine the incidence of thrombosis, recurrent GIB, and death as well as the time to resumption of anticoagulant therapy during the 90 days following a gastrointestinal bleed. Methods: This was a retrospective, cohort study using administrative and clinical databases. Patients experiencing gastrointestinal bleeding (GIB) during warfarin therapy were identified and followed for 90 days after determining if warfarin was resumed following GIB. Variables describing the management and severity of the index GIB were also collected. Kaplan-Meier curves were constructed to estimate the survival function of thrombosis, recurrent GIB, and death between the Resumed Warfarin and Did Not Resume Warfarin groups with Cox proportional hazards modeling to adjust for potentially confounding factors. Results: There were 442 patients with warfarin-associated index GIB included in the analyses. Following the index GIB, 260 patients (58.8%) resumed warfarin therapy. Warfarin resumption post-index GIB was associated with a lower adjusted risk for thrombosis (Hazard Ratio [HR] = 0.08; 95% confidence interval [CI] 0.01 – 0.65) and death (HR = 0.30; 95% CI 0.15 – 0.61), without significantly increasing the risk for recurrent GIB (HR = 1.58; 95% CI 0.65 – 3.80). Conclusion: The decision to not resume warfarin in the 90 days following GIB is associated with increased risk for thrombosis and death. For many patients who have experienced warfarin-associated GIB, the benefits of resuming anticoagulant therapy will outweigh the risks. Disclosure of Interest: D. Witt: None Declared, T. Delate: None Declared, D. Garcia Consultant for: Advisor to Boehringrer Ingelheim, Bristol-Meyers Squibb, Daiichi Sankyo and CSL Behring, N. Clark: None Declared, E. Hylek Consultant for: Advisor to Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Daiichi Sankyo, Johnson and Johnson, and Pfizer, Paid Instructor for: Participated in clinical symposia sponsored by Bayer, Boehringer Ingelheim, and Bristol-

Myers Squibb, W. Ageno: None Declared, F. Dentali: None Declared, M. Crowther Consultant for: Advisor to Leo Pharma, Pfizer, Bayer, Boehringer Ingelheim, Alexion, CSL Behring and Artisan Pharma, Paid Instructor for: Prepared educational materials for Pfizer, Octapharm and CSL Behring Keywords: Discontinuation, Gastrointestinal bleeding, Thrombosis, Warfarin.

ATT03 Despite pharmacological equivalence, generic versions of enoxaparin may differ in their pharmacodynamic actions. Potential therapeutic implications Jeske W1,*, Walenga JM1, McGeehan E2, Harenberg J3, Ramacciotti E4 and Fareed J2 1 Thoracic and Cardiovascular Surgery; 2Pathology, Loyola University Medical Center, Maywood, IL, USA; 3University of Heidelberg, Mannheim, Germany; 4Research and Development, Bristol Myers Squibb, Princeton, NJ, USA Objectives: Most recently available generic enoxaparins are similar to Lovenox (Sanofi Aventis) in terms of potency and molecular profile. This study compared available generic enoxaparins with the branded version in various assays and investigated their pharmacokinetic/pharmacodynamic behavior in a primate model. Methods: Cutenox, Lupenox, enoxaparin Sandoz and Lovenox were compared in terms of molecular weight profiling, NMR profile and in vitro anticoagulant activity. Groups of primates (n = 8–10) were administered LMWH (1 mg/kg subcutaneously) and blood samples were drawn over a 24 h period to measure pharmacodynamic effects. Antithrombotic and bleeding effects were studied in rat models of jugular vein clamping and tail transection, respectively. Complexation studies with PF4 were carried out using a SELDI-MS method. Results: Anti-Xa potency ranged from 86 to 110 U/mg and anti-IIa potency ranged from 27 to 35 U/mg. Mean molecular weight of these LMWHs ranged from 4.2 to 4.5 kDa. All agents showed a comparable NMR profile although some compositional differences of monosaccharides and in the amount of 1,6-anhydromannose were observed. In in vitro anticoagulant studies, thrombin generation inhibition assays and protamine/PF4 neutralization assays, similar differences were noted. The pharmacokinetic profile in terms of anti-Xa and anti-IIa activites were comparable, however, TFPI release and thrombin generation inhibition profiles showed product-based differences. Varying degrees of anti-heparin/PF4 antibody generation was observed. Product-based differences in hemorrhagic and antithrombotic activities were noted. Conclusion: Although the generic enoxaparins are manufactured to be pharmaceutically equivalent, the pharmacodynamic profiles of some of these drugs differ from that of the branded product. Thus, each generic product should be considered as a distinct low molecular weight heparin and individual safety/efficacy data should be considered prior to its clinical use. Disclosure of Interest: None declared. Keywords: enoxaparin, generic.

ATT04 Perceived stress reduces the stability of vitamin Kantagonist treatment Skov J1,*, Leppin A2, Bladbjerg E-M1, Sidelmann JJ1 and Gram J1 1 Unit for Thrombosis Research; 2Unit for Health Promotion, University of Southern Denmark, Esbjerg, Denmark Objectives: In order to maximise the benefits and reduce the risk of complications for patients treated with vitamin K-antagonists (VKA),


ABSTRACTS it is crucial to maintain the intensity of anticoagulation within therapeutic range. We investigated the hypothesis that higher levels of perceived stress compromise the stability of VKA treatment. Methods: A prospective observational study of 250 consecutive patients from a Danish anticoagulant clinic. The patients, the majority suffering from atrial fibrillation or deep venous thrombosis, were interviewed about socio-demographics, health-related behaviour, medications and psycho-sociological factors. Various biomarkers of haemostasis and inflammation were determined, in addition to polymorphisms known to influence VKA treatment. Subsequently, the patients were followed for one year. The main outcome measure was time in range (TTR) of the international normalized ratio (INR). Results: The average TTR was 73.8% (SD = 15.7%). Higher scores on the 10-item questionnaire Perceived Stress Scale (PSS), lower age and shorter duration of VKA treatment at baseline all significantly decreased TTR during follow-up. The strongest univariate correlation was observed for PSS (P = 0.03). In a linear regression model including potential confounding variables, PSS remained the most significant predictor of TTR, which differed significantly among quintiles of PSS (P = 0.011), being 78.5% (SD = 12.4%) in the lowest quintile and 69.6% (SD = 19.6%) in the quintile with the highest PSS score. Conclusion: The study demonstrated that perceived stress was the strongest determinant of stability of VKA treatment in a well-regulated cohort of unselected anticoagulant clinic patients. These results suggest that clinicians responsible for VKA treatment should include perceived stress or other indicators of psycho-social functioning in their assessment of patients. Disclosure of Interest: None declared. Keywords: anticoagulant clinic, anticoagulant treatment, perceived stress, time in range of INR, vitamin K antagonist, Warfarin.

ATT05 One week vs. four week heparin prophylaxis after laparoscopic surgery for colorectal cancer. The pro-laps pilot feasibility study Becattini C1, Vedovati MC1,*, Rondelli F2, Boncompagni M3, Camporese G4, Balzarotti R5, Mariani E6, Flamini O7, Natalini G3, Donini A8, Giustozzi M1 and Agnelli G1 1 Internal and Cardiovascular Medicine and Stroke Unit; 2 Oncologic Surgery, University of Perugia; 3General Surgery, S. Maria della Misericordia Hospital, Perugia; 4Unit of Angiology, University Hospital of Padua, Padua; 5General Surgery, Niguarda Hospital, Milan; 6General Surgery; 7Angiology, S. Giovanni Battista Hospital, Foligno; 8General and Emergency Surgery, S. Maria della Misericordia Hospital, Perugia, Italy Objectives: Four-week heparin prophylaxis reduces the incidence of venous thromboembolism (VTE) in patients undergoing open abdominal surgery for cancer. Whether extended prophylaxis is required after laparoscopic surgery for cancer is unknown. Methods: A complete compression ultrasonography (cCUS) of the lower limbs was performed in consecutive patients at day 8 2 after laparoscopic surgery for colorectal cancer. Patients with no evidence of VTE were randomized to withdraw or to continue heparin for three additional weeks. A cCUS was repeated at day 28 2 by operators blinded to treatment allocation. The study primary end-point was the incidence of VTE at day 28 2. Results: As for January 20th 2012, 270 patients were evaluated for inclusion in the study and 200 were randomized. No difference was observed between the two treatment groups concerning the prevalence of risk factors for VTE. VTE occurred at day 28 2 in 11 out of 98 patients randomized to withdraw and in none of the 102 patients randomized to continue heparin prophylaxis. The incidence of VTE at 3 months was 1% and 11% in patients randomized to continue or to withdraw heparin prophylaxis, respectively (RRR 91%, 95% CI 34– 99%). No major bleeding event was observed. © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

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Conclusion: This study shows that extended heparin prophylaxis is safe and reduces VTE as compared with one week prophylaxis after laparoscopic surgery for colorectal cancer. A randomized double-blind placebo-controlled trial has been planned and is about to start in this indication. Disclosure of Interest: None declared. Keywords: cancer, laparoscopic surgery, prophylaxis, Venous thromboembolism.

ATT06 The safety and efficacy of inferior vena cava filters: the experience of the Ottawa hospital Duffett LD1,*, Cheung A2, Bose G3, Forster A4 and Wells P1 1 Department of Medicine, Hematology; 2Medical School; 3 Faculty of Science; 4Department of Medicine, University of Ottawa, Ottawa, ON, Canada Objectives: To audit the use, and evaluate the safety and efficacy of inferior vena cava (IVC) filters. Methods: A retrospective cross-sectional study was conducted on consecutive inpatients with insertion of an IVCF over a 4 year period at two tertiary care hospitals. Administrative billing data was used to identify patients and medical records were reviewed for: patient characteristics, indications for IVCF, clinical course including use of systemic anticoagulation, IVCF retrieval, and complications associated with the IVCF. Results: Three hundred and thirty six patients had 338 attempted IVCF insertions. Two attempted insertions were unsuccessful due to anatomical reasons. Median age was 64 years, 205 (61%) patients were male and 161 (48%) patients had active cancer. Indications for insertion were: acute venous thromboembolic disease with a need to discontinue anticoagulation in 324 (96%) patients and prophylaxis in 14 (4%) patients. The most frequent IVCF model was Bard (Recovery or G2) (n = 218, 65%) and 332 (98%) were retrievable models. Prophylactic doses of anticoagulation were used in 196 (58%) patients after IVCF insertion. The filter was removed in 140 (42%) patients with the most common reasons for failing to remove: death or palliative status (n = 102, 30%), continuing indication for IVCF (n = 16, 16%), and unsuccessful retrieval (n = 17, 5%). Not removing the filter was significantly associated with active cancer (P < 0.0001) and age ≥60 (P < 0.0001). At least one adverse event associated with the IVCF occurred in 68 (20%) patients. The most frequent events were failed retrieval (n = 28, 8.4%), filter thrombosis (n = 25, 7.5%), new or progression of lower extremity DVT (n = 15, 4.5%), new PE (n = 10, 3%), and bleeding or hematoma at insertion site (n = 6, 1.8%). Conclusion: The results suggest that IVC filters are associated with significant adverse events such as failure to retrieve, filter thrombosis and development of lower extremity DVT. Further prospective studies are needed to evaluate the risks and benefits of using such devices. Disclosure of Interest: None declared. Keywords: deep vein thrombosis, inferior vena cava filter, intravascular devices, pulmonary embolism.

ATT07 Evaluation of a community pharmacy anticoagulation management service utilising point-of-care testing and online computerised decision support Harper P1,*, Shaw J2, Harrison J2 and Harrison J2 1 Clinical Haematology, Palmerston North Hospital, Palmerston North; 2Pharmacy, Auckland University, Auckland, New Zealand Objectives: To determine if accredited pharmacists using point of care (PoC) INR testing and decision-support software could provide safe,

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ABSTRACTS

and effective anticoagulatant management acceptable to patients and other healthcare providers. Methods: Fifteen community pharmacies in New Zealand each recruited up to 50 patients for anticoagulant monitoring between December 2010 and August 2011. INR testing was performed using the CoaguChek XS Plus (Roche Diagnostics) linked to online decision support software (INR Online). Pharmacists advised the dose and date for the next test supported by the software recommendation. All results were electronically linked to the patient’s medical records. The proportion of time in the therapeutic range (%TTR) was used to evaluate anticoagulant control. Patients, pharmacists, family doctors and practice nurses were surveyed by questionnaire about the service. Results: Six hundred and ninty three patients were enrolled. The mean %TTR across all sites was 78.6% (range 71.4–84.1%); rising to 80.2% for patients with over 26 weeks tests. The mean interval between tests was 14.4 days, rising to 16.7 days in months 4–6. Compliance with appointments showed 83.1% were on or before the due date, and only 6.5% were more than 3 days overdue. The prior six months INR data were obtained for 154 patients; in this cohort the%TTR for “standard care” was 60.4%, whereas it was 77.5% in the study. Adverse events and hospitalisations were no greater than reported events associated with standard care. Conclusion: The%TTR correlates with the incidence of anticoagulant complications. The mean%TTR in our study (>80%) demonstrates that a pharmacy based service can achieve safe and effective management with better control than “standard care”, based on the prestudy values of a cohort of patients. Our survey results showed the majority of patients found the service convenient and accessible, preferable to the previous system. Pharmacists were overwhelmingly positive about the service. Disclosure of Interest: P. Harper Shareholder of: Director of INR Online: Warfarin decision support software, J. Shaw: None Declared, J. Harrison: None Declared, J. Harrison: None declared. Keywords: Decision support software, Near patient testing, pharmacy management, Warfarin.

ATT08 Is there a greater role for thrombolysis in the acute management of deep vein thrombosis (DVT)? Review of the management and outcome of patients presenting with proximal DVT to a single UK centre over a 2-year period Jayakody Arachchillage D*, Chattree S, Robinson B, Rivett L, Kesteven P and Talks K Haematology, Newcastle Upon Tyne Hospitals NHS foundation Trust, Newcastle Upon Tyne, UK Objectives: The CaVenT study found at 2 years, 55%of patients with acute deep vein thrombosis (DVT) treated with conventional anticoagulation versus 37% treated with additional catheter directed thrombolysis developed post thrombotic syndrome (PTS). We reviewed the management and outcome in a consecutive cohort of young patients (age19–60 years) presenting to our centre June 2009 to May 2011 with an acute DVT (symptoms < 14 days). Methods: The presence of recurrence and PTS at their last clinic review was ascertained. A total of 83 patients were diagnosed over this period of which 10 were excluded as symptoms had been present for > 14 days. Follow up ranged from 6 to 30 months Results: Of the 73 patients (male: female 48:25) no identifiable risk factors for DVT were found in 34.3% (25) and transient risk factors in 49.3% (36). The remaining 16.4% (12) had permanent risk factors. Three patients had thrombolysis at presentation in addition to anticoagulation for 6 months. The other 70 patients had conventional anti-

coagulation for at least 6 months, 10 patients were lost to follow up. All patients were advised to wear graduated elastic compression stocking for 2 years; five patients had poor compliance.32 patients developed some degree of PTS (54%). Using the Villalta scale this was mild in 14, moderate in 11 and severe in seven patients. Of the 3 patients who had thrombolysis, two patients had severe PTS and the other patient did not develop PTS. Over this follow up period 15/63(23.8%) patients continued on long term anticoagulation due to either ongoing risk factors, previous DVT or patient preference. Of the 48 remaining patients, six patients developed a recurrent DVT whilst off anticoagulation (12.5%). Conclusion: PTS is a significant cause of morbidity following DVT; the incidence in our cohort with conventional anticoagulation was similar to that in the CaVenT study. The number of patients receiving thrombolysis was very low; nevertheless, the trend observed does not support the widespread use of this treatment. Disclosure of Interest: None declared. Keywords: Deep vein thrombosis, Post thrombotic syndrome, Proximal, Thromolysis.

ATT09 Endogenous thrombin potential in patients with low intensity vitamin K antagonists treatment Shmeleva V1,*, Namestnikov Y1, Matvienko O1, Papayan L1 and Soldatenkov V2 1 Laboratory of blood coagulation, Russian Research Institute of Haematology and Transfusion; 2Surgery department, Russian Research Institute of Haematology and Transfusion, St-Petersburg, Russian Federation Objectives: Low intensity treatment with vitamin K antagonists (VKA) i.e. international normalized ratio (INR) 1.5–1.9 is still of interest for some patients with venous thromboembolism (VTE) in the extended-treatment phase, though the ninth edition of American College of Chest Physicians Guidelines recommend therapeutic INR range of 2.0 to 3.0. Aim of our study was to compare thrombin generation in VTE patients with stable INR of 1.5–1.9 and of 2.0–3.0. The study involved 42 patients with VTE (M/F 22/20, mean age 54.6 15.8 years), duration of VKA 1.5–3 y, and 28 controls. None of the patients had recurrent VTE during the observational period. Methods: The endogenous thrombin potential (ETP) was measured directly using a fluorogenic assay (ThrombinoscopeTM, The Netherlands) according to Hemker et al, both in the presence and in the absence of thrombomodulin (TM). STATISTICA 6.1 was used, data are given as mean SD. Results: ETP (nMmin) and peak (nM) in patients with INR 1.5–2.0 were significantly higher than in those with INR 2.0–3.0 (in the absence of TM: 642.8 129.7 vs. 358.1 210.4 and 118.4 22.1 vs. 66.5 39.2, respectively, P < 0.0001; in the presence of TM: 481.2 106.8 vs. 293.2 167.4 and 108.1 23.0 vs. 60.9 37.6, P < 0.0001). Importantly, low intensity VKA patients had significantly lower ETP and peak than controls (in the absence of TM: 642.8 129.7 vs. 1731.4 253.6 and 118.4 22.1 vs. 292.3 50.0, respectively, P < 0.0001; in the presence of TM: 481.2 106.8 vs. 932.9 272.6 and 108.1 23.0 vs. 196.2 58.1 respectively, P < 0.0001). None of the patients with INR 1.5–1.9 had increased ETP, i.e. above 90th percentile measured in controls (> 2061 nM min in the presence of TM and > 1353 nM min in the absence of TM). Conclusion: Our data suggest that in some clinical cases low intensity VKA in aims of reduced risk of bleeding and reduced frequency of monitoring is possible without higher risk of recurrent VTE. Disclosure of Interest: None declared. Keywords: endogenous thrombin potential, low intensity VKA, venous thromboembolism.


ABSTRACTS ATT10 Prophylaxis of postoperative venous thromboembolism: pursuit of a rational systematic approach Vardanyan AV1,*, Mumladze RB1, Roitman EV2, Dolidze DD1, Patrushev LI3 and Shieh M1 1 Department of Surgery, Russian Medical Academy of Postgraduate Education, Ministry of Health and Social Development of the Russian Federation; 2Hemostasis Laboratory, Federal Research and Clinical Center on Children’s Hematology, Oncology and Immunology, Ministry of Health and Social Development of the Russian Federation; 3ShemyakinOvchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russian Federation Objectives: An analysis of the efficacy of low molecular weight heparin (LMWH) prophylaxis of postoperative venous thromboembolic complications (VTEC) was carried out with the goal of formulating a rational systematic approach for prophylaxis. Methods: We analyzed the results of VTEC prophylaxis in 274 average and high risk patients, who had undergone various general surgery procedures from 2005 to 2011 at Botkin Hospital. LMWH was given to: 1 76 (27.7%) patients (group 1) from 12 h before surgery until recovery of full activity (7–10 day); 2 82 (29.9%) patients (group 2) starting 6–12 h after surgery. 3 116 (42.3%) patients (control) did not receive LMWH prophylaxis. In all patients, venous blood flow was monitored using ultrasound scanning and coagulation status monitored with conventional lab tests; in 12 patients with suspicion of pulmonary embolism (PE), ventilation-perfusion (V/Q) lung scanning was performed; in 29 patients who developed deep vein thrombosis (DVT), DNA testing for thrombophilia was performed. Results: Group 1: DVT was not detected. Hemostasis tests revealed an increase in the activity of Antithrombin III (AT III) and Protein C (PC). In group 2, DVT was detected in 2 (2.4%)* patients and PE in 1 (1.2%)* patient. In the control group, DVT was detected in 32 (27.5%)* patients and PE in 4 (3.4%)* patients. Differing trends and discordance of AT III and PC were noted in the controls. *P < 0.05 for DVT & PE rates between group 2 & control. DNA tests revealed a eterozygous FV Leiden mutation in 2 (6.8%) patients. One of these, along with 3 others, (13.7%) had a homozygous C677T mutation in the MTHFR gene; 12 (41.3%) had a heterozygous mutation. Heterozygous 4G/5G mutation in the plasminogen activator inhibitor 1 gene PAI-1 was detected in 7 (63.6%) of 11 tested patients who had DVT. Of 12 patients with DVT, V/Q scanning revealed PE in 3 (25%) patients. Conclusion: The use of LMWH for VTEC prophylaxis shows maximum efficacy, without an increase in adverse effects, when begun 12 h prior to surgery. Disclosure of Interest: None declared. Keywords: C677T MTHFR, deep vein thrombosis, hemostasic disorders, Leiden, lmwh, low molecular weight heparin, plasminogen activator inhibitor 1 PAI-1, postoperative DVT prophylaxis, pulmonary embolism, ultrasound scanning.

ATT11 Safety and efficacy of bridging with low molecular weight heparins: a systematic review Eijgenraam P1,*, ten Cate H1,2 and ten Cate-Hoek A1,2 1 Laboratory for Clinical Thrombosis and Haemostasis, Maastricht University; 2Department of Internal Medicine, Cardiovascular Research Institute Maastricht, Maastricht University Medical Center+, Maastricht, The Netherlands Objectives: Every treating physician faces a challenge when a patient on chronic vitamin K antagonists (VKA) has to undergo surgery; a © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

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balance must be found between the risk of thromboembolism (TE) and bleeding. The risk of bleeding has been clearly established, it is not clear whether the risk of TE is high enough to warrant bridging with low molecular weight heparins (LMWH). Methods: We performed an Embase and Medline search for studies that compared bridging anticoagulation with continuation or cessation of VKA without bridging; with perioperative TE and bleeding as outcomes. We identified 878 articles and finally selected 17 articles. The quality of the studies was assessed with the Newcastle Ottawa scale; results of individual studies were pooled using the random effects model. Results: None of the included studies reported significant differences in incidence of TEs between the bridging group and the comparator group; four studies reported zero TEs. Heparin was identified as a risk factor for bleeding in multivariable adjusted analyses in three studies on pacemaker/implantable cardioverter defibrillator (PM/ICD) surgery. In five studies with unadjusted analyses, bridging was compared to warfarin cessation: three studies (different types of surgery) reported null results for bleeding; two studies (different types of surgery) identified bridging as a risk factor. Two studies report that full dose postoperative heparin as well as restart of VKA therapy within 24 hours after surgery augments bleeding risk in several procedures. We pooled a subset of six studies regarding postoperative bleeding after PM/ICD surgery and found an increased risk for bleeding with a RR of 3.03 (95% CI: 1.86–4.95) when we compared bridging to continuation of VKA. Image/Graph:

Conclusion: While the antithrombotic efficacy of perioperative bridging with LMWH has not been demonstrated, increased bleeding risk is observed in different types of surgery. PM/ICD surgery can be safely performed without the need of bridging anticoagulation. Disclosure of Interest: None declared. Keywords: Bridging anticoagulation, low molecular weight heparin, oral anticoagulation, perioperative management, unfractionated heparin.

ATT12 Variance growth rate: a superior method to monitor oral anticoagulation therapy Sharda AV1,2,*, Johnson G3,4 and Kuskowski M5 1 Hematology and Oncology, Beth Israel Deaconess Medical Center; 2Harvard Medical School, Boston, MA; 3Hematology, Oncology and Transplant, University of Minnesota; 4Hematology and Oncology; 5VA Health Care System, Minneapolis, USA Objectives: Oral anticoagulation therapy (OAT) with vitamin K antagonists is monitored by the International Normalized Ratio (INR). Prior studies of the adequacy of OAT used various methods to estimate time spent in the target INR range (TTR), or percentage of INR values within the target range (ITR). To compare these methods with variance growth rate (VGR), a measure of the coefficient of variation

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of sequential INRs (Van Leeuwen Y et al JTH 2007), we performed a retrospective cohort study of patients on stable, long-term, OAT. Methods: Subjects (mean age 72.8 years, 98.6% male, 74% atrial fibrillation, 85.6% 2–3 target range) on warfarin OAT continuously between September 2007 to September 2009 were included if they had at least one INR measurement per month. Indication, target range, INR values, and major hemorrhagic and thromboembolic events were recorded. TTR, ITR, and VGR (three methods) were calculated for all subjects. Hemorrhagic and thromboembolic events were combined for analysis. The significance of TTR, ITR, and VGR, with respect to composite endpoints was studied by t-tests, and logistic regression adjusting for clinical characteristics (age, hypertension, congestive heart failure, atrial fibrillation, cancer). Results: Two hundred and seventy eight patients had a total of 12 389 INR measurements over 556 patient-years of follow up. The mean TTR was 70.3%, with16.8% time spent below and 12.9% above the range. The corresponding values for ITR were 62%, 22.2% and 15.8%, respectively. The rate of major adverse events (13 hemorrhagic, 10 thromboembolic) was 4.13/100 patient-years. Neither TTR, nor ITR was statistically associated with major adverse events in univariable or multivariable analyses. However, all three VGR estimates were significantly associated with risk of major adverse events (P < 0.05) in univariable or multivariable analyses. Conclusion: VGR, a measure of INR variability over time, is superior to TTR and ITR to monitor the efficacy of OAT in patients on longterm anticoagulation. Disclosure of Interest: None declared. Keywords: International Normalized Ratio, Oral Anticoagulation Therapy, Time-in-range, Variance Growth Rate, Warfarin.

Table. Adjudicated bleeding, thrombotic events and deaths in patients with AF. Patients with AF (events per 100 patient years)

Not previously on oral anticoagulation

Established on oral anticoagulation

Events Minor Bleeds Major Bleeds Thrombotic Deaths Fatal Bleeds Fatal Thrombotic

361 (5.2) 189 (2.7) 61 (0.9) 55 (0.8) 56 (0.8) 14 10

108 (4.6) 58 (2.5) 19 (0.8) 17 (0.7) 14 (0.6) 2 1

Image/Graph:

ATT13 European action on anticoagulation (EAA) evaluation of the UK national health service (NHS) improvement guidelines for ‘safety indicators’ in anticoagulation of atrial fibrillation (AF) Poller L1,*, Ibrahim S1, Baglin T2 and Jespersen J3 1 Central Facility, European Action on Anticoagulation, Faculty of Life Sciences,University of Manchester, Manchester; 2 Addenbrooke’s NHS Trust, Cambridge, UK; 3Hospital of South West Denmark, University of Southern Denmark, Esbjerg, Denmark Objectives: The EAA prospective study (JTH 2008;6:935–43) results on computer-assisted anticoagulant dosage have been used to evaluate “safety indicators” in the UK NHS Anticoagulation for AF Commissioning Support Document (UK NHS Improvement 2011;157:1–6). EAA experience with oral anticoagulation in AF has been used to assess the proposals in this NHS Document (figure). The “safety indicators” for patients initiating oral anticoagulation and those already established on warfarin were studied. All “clinical events” were assessed centrally by an independent Adjudication Committee without knowing patients’ identity or participant centres. Methods: EAA clinical results are given in the table. About 5839 patients were followed over 9000 patient-years. There were 469 clinical events of which 73% were bleeding (major, minor or fatal). The incidence of events in patients not previously on oral anticoagulation was 5.2 per 100 patient-years but in patients who were already receiving oral anticoagulation was 4.6. Results: New patients who developed an INR < 5.0 on at least one occasion had significantly greater numbers of clinical events compared with patients whose INR never exceeded 5.0 (P < 0.001). The overall TIR in patients already on anticoagulation was 69.4% (SD = 1.1%). New patients achieved 60% TIR within 3 months of therapy and achieved a TIR similar to patients already on oral anticoagulation after 6 months.

Conclusion: The EAA computer-dosage study provides quantitative support for the “safety indicators” in anticoagulation of AF listed in the NHS Improvement Document and shows these give a reliable basis for its conclusions. Disclosure of Interest: None declared. Keywords: Atrial fibrillation, oral anticoagulation, ‘safety indicators’, INR, bleeding.

ATT14 Infrequent removal of retrievable IVC filters Furmark L*, El Amm J, Mobarek D, Faselis C, Rickles F and Aggarwal A Internal medicine, VA medical center, Washington, DC, USA Objectives: The incidence of retrievable IVC filter placement has increased significantly. Given that the beneficial effect of IVC filters has been found only in the short term, the option to remove a filter that is no longer necessary is appealing. U.S. FDA guidelines state that filters should be removed to reduce long-term complications including increased incidence of deep venous thrombosis. Despite the retrievability of these filters, we hypothesized that a very small percentage are actually removed. We performed a retrospective chart review to gather data on retrieval rates of IVC filters placed from 1/1/2006 to 9/ 1/2011 at the Veterans Administration (VA) Medical Centers in DC and MD. Methods: The study was conducted using the electronic medical record system, which is standardized at all VA Medical Centers in the U.S. © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

ABSTRACTS Patients were excluded if they had a permanent IVC filter placed. Demographics and all clinical data including IVC filter indication, date of placement and removal of IVC filter, duration of IVC filter, complications, anticoagulation use and follow-up were retrieved. Results: Of the 85 retrievable filters placed, only 6% were removed. IVC filter placement was documented in the problem list in only 21% of records. Retrieval rate was 100% successful and all attempts were made prior to 60 days after placement. Conclusion: The use of retrievable IVC filters has significantly increased over the past decade. Unfortunately, the number of veterans with IVC filters who failed to have their filters removed when still appropriate and technically feasible has also significantly increased. Although the computerized chart system at the VA should be ideal for the development of an electronic alert system, no such system currently exists. In view of the low retrieval rate we established as a baseline, we propose a prospective trial evaluating the utility of an electronic alert system to improve IVC filter retrieval rates, where appropriate. The alert system is currently in development. Disclosure of Interest: None declared. Keywords: IVC filters.

ATT15 Prediction of optimal warfarin maintenance dose using advanced artificial neural networks Pugliano M1,*, Grossi E2, Podda GM1, Gabba S2, Carpani G3, Verri A2, Buscema M4, Faioni EM1 and Cattaneo M1 1 Medicina 3, DMCO, University Of Milano; 2Centro Diagnostico Italiano; 3S.I.M.T., Azienda Ospedaliera San Paolo, Milano; 4 Semeion Research Centre, Roma, Italy Objectives: The individual response to vitamin K antagonists (VKA) is highly variable, being influenced by genetic variants of enzymes that are involved in drug and vitamin K metabolism (CYP2C9 and VKORC1) and interference by clinical variables. In the last years, mathematical algorithms were developed for estimating the appropriate VKA dose, based on different mathematical approaches working on clinical and genetic data. Artificial Neural Networks (ANN) are computerized algorithms resembling interactive processes of human brain, which allow to study very complex non-linear phenomena like biological systems. The objective was to evaluate the performance of new generation ANN on a large database of patients on chronic VKA treatment. Methods: Clinical and genetic data from 377 patients (186 men; 191 women) treated with a VKA (warfarin) average weekly maintenance dose of 23.67 mg (11.42 SD) were used to create a dose algorithm. Forty-eight variables, including demographic, clinical and genetic data (5 CYP2C9 and 3 VKORC1 genetic variants) were entered into Twistâ system, which can select fundamental variables during their evolution in search for the best predictive model. The final model expressed a functional approximation of the actual dose within a validation protocol based on a tripartite division of the data set (training, testing, validation). Results: In the validation cohort, the pharmacogenetic algorithm, based on 23 variables, reached high accuracy with an average absolute error of 5.7 mg. The most accurate prediction was achieved in the subset of patients requiring 21–49 mg warfarin, 73% being correctly identified by the algorithm. Conclusion: Our results suggest that ANN can be applied successfully for VKA maintenance dose prediction and represent a robust basis for a prospective multicentre clinical trial of the efficacy of genetically informed dose estimation for patients who require VKA. Disclosure of Interest: None declared. Keywords: artificial neural networks, maintenance dose, warfarin.


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ATT16 Emergency reversal of vitamin K antagonists with prothrombin complex concentrate: the everyday practice Toth P1,*, Robinson K1, Laidlaw S1, Maclean R1, van Veen J1, Hampton K1,2 and Makris M1,2 1 Department of Haematology, Sheffield Teaching Hospitals NHS Foundation Trust; 2Department of Cardiovascular Science, University of Sheffield, Sheffield, UK Objectives: Although guidelines give clear recommendations for emergency reversal of vitamin K antagonists (VKA) there are only limited data how these are applied in practice.The aim of this study was to show how real life emergency reversal works in our center including compliance with guidelines,indication,timing,dosing,efficacy and safety of the Prothrombin Complex Concentrate (PCC) used. Methods: Retrospective observational study.Data collection was based on notes review,Blood Bank records and results from the Information Technology system.Between November 2010 and November 2011, 131 patients required emergency reversal of VKA anticoagulation.All received a licenced four-factor PCC. Results: The commonest indications for immediate VKA reversal were emergency procedures 29.8%,gastrointestinal bleed (GI) 27.5% and intracranial haemorrhage (ICH) 26.7%.In 97% of the cases the indication was in line with current guidelines.The median time from door to intervention was 4 h in case of Vitamin K and 5.6 h with PCC administration.In subgroup analysis,the median time from presentation to PCC administration was 3 h in ICH patients,7.2 h in patients with GI bleed and 16.7 h in patients waiting for emergency procedures.The dose of PCC was complient with the current guidelines in 65.8%. The mean INR was 4.54 before and 1.27 after reversal. In 84% of the cases the INR was less than 1.5 and in 98.2% it was less than 2 after PCC Vitamin K administration.The mortality of patients receiving PCC was 25.2%. The cause of death was bleeding in 30.3% and thromboembolic complication in 3%.In total 3.8% of the patients were diagnosed with a thromboembolic event after receiving PCC. Conclusion: Patients with life threatening bleed should have their anticoagulation reversed without delay. In certain indications early administration of Vitamin K could supersede the use of the potentially prothrombotic PCC. Appropriate indication, timing and dosing are essential to ensure optimal clinical effect and to avoid potential adverse effects. Disclosure of Interest: P. Toth: None Declared, K. Robinson: None Declared, S. Laidlaw: None Declared, R. Maclean: None Declared, J. van Veen: None Declared, K. Hampton: None Declared, M. Makris Consultant for: CSL Behring Keywords: emergency reversal, Prothrombin Complex Concentrate, Vitamin K antagonists.

ATT17 Biosimilar enoxaparins available for clinical use in Brazil: structure and haemostatic effects Glauser BF1,*, Vairo BC1, Oliveira SN1, Cinelli LP2, Pereira MS1 and Mour~ ao PS1 1 Institute of Medical Biochemistry, Federal University of Rio de Janeiro, Rio de Janeiro; 2Federal University of Rio de Janeiro, Maca e, Brazil Objectives: Patent protection for LMWHs has expired. Biosimilar preparations of enoxaparins are approved for clinical use. There is an intense debate about the possibility of obtaining preparations similar to the original drug, because of the complexity of the process involved in generating LMWHs. Differences between the physicochemical and biological properties of biosimilar and original enoxapa-

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rins may have significant clinical consequences. Here, a careful analysis of the biosimilar enoxaparins available in Brazil is reported. Methods: We have analyzed biosimilar enoxaparins available for clinical use in Brazil, from six different suppliers. Those preparations were assessed regarding their chemical structure and molecular size distribution by NMR spectroscopy and gel permeation chromatography. The in vitro anticoagulant effect based on anti-Xa and anti-IIa activity, pharmacokinetics and pharmacological effects in animal models of thrombosis and bleeding were also determined. Results: Biosimilar versions and original drug showed similar NMR spectra and molecular size distribution, similar anticoagulant activities, equivalent responses in animal models of experimental thrombosis and bleeding and achieve similar plasma concentrations. Three batches of the active ingredient showed differences from the original drug in molecular size distribution but still attend the recommendations of the American and European Pharmacopeias. Three final pharmaceutical biosimilar enoxaparin from one supplier contained lower amounts of the active ingredient than that declared by the suppliers. Conclusion: Our results suggests that biosimilar enoxaparins are similar to the original drug and a valid therapeutic alternative, which are, however, in need of appropriate regulation to ensure compliance with regulatory requirements. The results obtained in the present study may help to establish guidelines for the regulation of biosimilar LMWHs and may establish more rigorous criteria to assess their purity and efficacy. Disclosure of Interest: None declared. Keywords: anticoagulants, antithrombotic agents, biosimilar drugs, enoxaparin, low molecular weight heparin.

ATT18 Dabigatran experience in New Zealand Stephens M, Pollock D and Harper P* Clinical Haematology, Palmerston North Hospital, Palmerston North, New Zealand Objectives: On July 1st, 2011 dabigatran became available in New Zealand for the prevention of stroke in atrial fibrillation. There were no restrictions on prescribing and it was available free to patients through government funding. The impact of the introduction was monitored by measuring the incidence of bleeding episodes and the effect on the existing anticoagulant service. Methods: Three parameters were monitored. 1. Details of bleeding episodes collected informally by Haematologists and from the Centre for Adverse Reaction Monitoring (CARM). 2. The number of patients changing from warfarin to dabigatran through an anticoagulant clinic audit. 3. The number of dabigatran prescriptions issued over 6 months collected from NZ Pharmaceutical Management Agency (PHARMAC). Ethics approval was obtained. Results: In New Zealand about 40 000 patients take warfarin. In the first week of July, 1935 scripts for dabigatran were issued and approx 7000 patients were taking the medication at 2 months. Subsequently the number of new cases fell to approx 150/week and at 6 months around 8000 patients remained on dabigatran. 44 cases of bleeding were reported by haematologists and a further 34 identified through CARM in the first 2 months. The commonest site of bleeding was the GI tract presenting as rectal bleeding. Other bleeds included haematuria, haemoptysis, intracerebral and subdural haematoma. The number of patients on warfarin in the anticoagulant clinic fell by 8.5% from 2758 to 2523 over 6 months. Conclusion: The initial uptake of dabigatran was rapid but the high incidence of bleeding reported during the first 2 months raised concerns and led to the distribution of additional safety advice to medical staff. Cases of bleeding were also reported in the National media. This resulted in a fall in new cases starting dabigatran. The impact on the anticoagulant service was less than expected with only an 8.5% fall in numbers. It is assumed that many of the patients starting dabigatran were not previously on warfarin.

Disclosure of Interest: None declared. Keywords: anticoagulant clinic, anticoagulant treatment, bleeding events, dabigatran, new anticoagulants.

ATT19 Role of sulfated galactans on thrombosis and antithrombin interaction De Oliveira SNM*, Fonseca RC and Mourao PDS Medical Biochemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Objectives: We compared sulfated galactans from two species of red algae, G crinale and B. occidentalis. These polysaccharides have an identical saccharide structure and the same size chain, but with slight differences in their sulfation patterns. Methods: We used a specific coagulation assays and experimental models of thrombosis like venous and arterial thrombosis in rats, exvivo recalcification time, bleeding time in rats and fluorescence assays. Results: As a consequence of these differences, the two sulfated galactans differ in their anticoagulant and venous antithrombotic activities. Sulfated galactans from G. crinale exhibits procoagulant and prothrombotic effects in low doses (up to 1.0 mg/kg body weight), but in high doses (> 1.0 mg/kg) this polysaccharide inhibits both venous and arterial thrombosis in rats and prolongs ex-vivo recalcification time. In contrast, sulfated galactans from B. occidentalis are a very potent anticoagulant and antithrombotic compound in low doses (up to 0.5 mg/ kg body weight), inhibiting venous experimental thrombosis and prolonging ex-vivo recalcification time, but these effects are reverted in high doses. Only at high doses (> 1.0 mg/kg) the sulfated galactans from B. occidentalis inhibit arterial thrombosis. Similar with heparin, sulfated galactans from G. crinale not activate factor XII, while the polysaccharide from B. occidentalis activates factor XII in high concentrations, which could account for its procoagulant effect at high doses on rats. Despite their structural similarities both molecules appear to interact differently with AT in fluorescence assays. Both sulfated galactans do not modify bleeding time in rats. Conclusion: These results indicate that slight differences in the proportions and/or distribution of sulfated residues along the galactan chain may be critical for the interaction between proteases, inhibitors and activators of the coagulation system, resulting in a distinct pattern in anti- and procoagulant activities and in the antithrombotic action. Disclosure of Interest: None declared. Keywords: Antithrombin Interaction, Bleeding Tendency, Fluorescence, Sulfated Galactans.

ATT20 Developing an iphone app for the management of the anticoagulant dabigatran Harper P* Clinical Haematology, Palmerston North Hospital, Palmerston North, New Zealand Objectives: Aim: In order to prescribe dabigatran safely three factors should be considered; appropriate dosing based on age and renal function, appropriate guidelines to manage the complication of bleeding and a safe procedure to adjust the dose at the time of surgery. The aim of this project was to develop a computer program combining these three factors in the form of an iphone application allowing easy access to this advice for medical staff. Methods: Prescribing information was collated for each country where dabigatran is available. Literature relating to anticoagulant reversal and available guidelines was reviewed. Algorithms were included to calculate the creatinine clearance, the half-life of the drug (from published pharmacokinetic studies) and the interpretation of coagulation results (based on published reports). A guideline was developed to © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

ABSTRACTS manage bleeding. All advice was referenced with a brief explanation and links to the original articles. Guidelines recommending when to stop dabigatran prior to elective and emergency surgery were developed based on renal function and the drug’s half-life. The program was linked to a website to receive feedback. Results: The first version of “Managing Dabigatran” was released as a free application through the App store in January 2012. The application was downloaded 300 times in the first 2 weeks. A second version including more detailed international prescribing was released February 2012. Feedback has been positive. Conclusion: Prescribing information and guidelines can be presented in a convenient accessible way using an iphone application. It has the advantage of being easily updated to ensure that the most recent guidelines are available, allows immediate calculation of creatinine clearance and plasma half-life and interpretation of coagulation results to enable safe treatment. Disclosure of Interest: P. Harper Shareholder of: HealthObs Ltd. Keywords: anticoagulant treatment, dabigatran, iphone application, new oral anticoagulants, reversing anticoagulants.

ATT21 The role of LMWH in prevention of deep veins thrombosis and pulmonary emboism in patients with severe multiple trauma

Coagulation Factors: Basic Science CFBS01 Fibrin formation under flow on biomimetic tissue factor rich surfaces Onasoga A* and Neeves K Chemical and Biological Engineering, Colorado School of Mines, Golden, CO, USA Objectives: Most coagulation assays do not account for role of flow, which influences coagulation and fibrin formation by carrying zymogens to and enzymes away from a growing thrombus. The objective of this work was to evaluate fibrin formation in a flow-based plasma assay. Methods: Silica microparticles (1 lm) were coated with lipid bilayer containing tissue factor (TF). These microparticles were patterned into 100 lm spots to mimic an aggregate of TF bearing cells. Normal pooled plasma (NPP) or FXI deficient plasma was perfused over the spots at wall shear rates of 100–1000 s for 5 min. Fibrin formation was measured by epifluorescence (Fig. 1) and reported in relative fluorescence units (RFU). Fibrin fiber diameter was measured by scanning electron microscopy. Data is reported as the average and standard deviation of five repeats. Image/Graph:

A

Danchev D1,*, Petrov N2 and Zidarova D2 1 Department of Haemostasis, Clinical Laboratory and Immunology; 2Department of Anesthesiology and Critical Care, Military Medical Academy, Sofia, Bulgaria Objectives: DVT and PE are common and important complications, observed among patients with severe multiple trauma. When such complications occurs, the mortality rate rises significantly. Although antithrombotic prophylaxis lessens the overall incidences of DVT, and PE especially, the data are still not enough to prove which combination of antithrombotic therapy is the most convenient one. Aims: To compare the mortality rate due to PE in these two groups, and to establish if it is in any relations with thrombprophylaxis used. Methods: We randomized retrospectively 1214 patients with severe multiple trauma, during the years 2003–2011. They were divided into two groups: 1 Group A – 548 patients, during the period 2003–2008. Antithrombotic prophylaxis in this group is realized using combination of LMWH and heparin in doses 5000 UI LMWH s.c. and 5000 UI heparin i.v respectively. 2 Group B – 666 patienys during the next three years. In all of them DVT and PE were prevented using LMWH alone (5000 UI s.c.). Results: In the first group overall mortality rate was 31% (170 incidence of death). 53 patients died due to PE. In other terms, the mortality, due to PE was 9% for the group at all and it represented 31% of the mortality at all. In the second group the results were: overall mortality – 16%(107 incidence of death), mortality due to PE – 22 persons, which correlates to mortality rate due to PE – 3%. It represents about 20% of overall mortality in patients with severe multiple trauma during this period of time. Conclusion: These results are, more or less, significant to prove that there are some advantages using LMWH alone. But it is important to emphasize, that the treatment provided to the patients in the first group differs, in some aspect, from the methods used in the second group. Nevertheless, there since is some distinct between the mortality rate and the mortality pattern in the two groups, which can be explained by the different methods of antithrombotic prevention used. Disclosure of Interest: None declared. Keywords: DVT, PE, LMWH.


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B

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Results: As the shear rate was increased (100, 250, 500 and 1000 s) the initiation times to visible fibrin formation was prolonged (38 8, 72 10, 150 13 and 216 35 s). The rate of fibrin formation (1719, 1367, 351 and 97 RFU/s) and the final fibrin density (26415 3613, 19299 973, 6475 2525 and 2263 277 RFU) decreased with increasing shear rate. Similarly, the diameters of the fibrin fibers decreased (162 49, 99 20, and 58 15 nm). The appearance of fibrin was delayed in the absence of FXI (480 64 s) at low surface TF concentration (5 molecules/lm2) compared to NPP (150 38 s). We observed no difference in fibrin formation between NPP and FXI-deficient plasmas at shear rates > 250 s or at high surface TF concentration (20 molecules/lm2). Conclusion: These results show that an increase in wall shear rate prolongs the initiation of fibrin formation, decreases the rate of fibrin formation and fibrin fiber diameter. Experiments with FXI deficient plasma suggests that the FXI contribution to fibrin formation is limited to wall shear rates of less than 250 s and low TF concentrations. Disclosure of Interest: None declared. Keywords: fibrin polymerization, Tissue factor.

CFBS02 Pre-analytical routines in coagulation testing: are guidelines followed? Kristoffersen A-H1,*, Stavelin AV2, Vannes S1 and Kristensen GB3 1 Laboratory of Clinical Biochemistry, Haukeland University Hospital; 2Norwegian Quality Improvement of Primary Care Laboratories (NOKLUS), Department of Public Health and Primary Health Care, University of Bergen; 3Norwegian Clinical Chemistry EQA Program (NKK), Bergen, Norway Objectives: It has been documented that as much as 70% of errors in laboratory medicine occur in the pre-analytical phase. External quality assessment (EQA) organisations have mainly assessed the quality of the analytical phase, but in the recent years more focus has been put on the pre-analytical phase. The aim of our study was to conduct a survey of how pre-analytical conditions of the most common coagulation analyses are taken care of in the different laboratories in Norway, and to compare this with current guidelines. Methods: Hospital laboratories in Norway (n = 59) regularly participating in the EQA for routine coagulation assays (INR, APTT, Fibrinogen, D-dimer) were asked to fill in a web-based questionnaire which consisted of 49 questions regarding sample handling routines for coagulation analyses. The first part focused on method information (instruments and reagents) for the different coagulation analyses used in the laboratories. In the second part, we asked about routines regarding venipuncture (needle gauge, citrate concentration, use of stasis, filling volume), handling of the sample before analysis, as well as routines for detection of sample clot, samples with high or low hematocrit and/or hemolysis, bilirubinemia or lipemia, and the consequences of detecting any of this (for example rejection, comment on the results or changing the protocol for analysis). The third part focused on storage and stability and the handling of blood samples received from primary care laboratories or other hospital laboratories. It was asked about type of acceptable sample material (citrated blood or citrated fresh or frozen plasma) and the stability of the analytes for the different materials. Results: The study is ongoing. So far, 57 of 59 (97%) have replied to the questionnaire. Conclusion: Results and conclusion will be presented at the congress. Disclosure of Interest: None declared. Keywords: APTT, D-dimer, External Quality Assessment, Fibrinogen, INR, Pre-analytical errors, Questionnaire.

CFBS03 The atomic force microscopy a suitable technique to characterize procoagulant vesicles in the nanometer scale Hardij J1,*, Dogn e J-M1, Chatelain B2 and Mullier F1,2 1 Pharmacy, FUNDP, Namur, Belgium; 2Hematology Laboratory, UCL, Mont Godinne, Namur, Belgium Objectives: Circulating microvesicles (MVs) are potential biomarkers for the thrombotic risk encountered by a variety of patients. No device currently allows the size and expression analysis in the nanometer scale. Yuana and coworkers first used the Atomic Force Microscopy (AFM) in analysis purposes in 2010. Three major information are brought with AFM: a number of particles, an estimation of their volume and a selection regarding to an antigenic expression. Our objective was to validate the use of AFM to quantify Tissue Factor (TF) MVs. Methods: (i) Mica sheets are activated with ehanoalmine (55% in DMSO) and glutaraldehyde (7% in PBS). They are abundantly rinsed in PBS and water and than coated with a drop of 10 lg/lL antibody (anti CD142 = TF and anti CD41) and rinsed. MVs are added on the surface that is than washed and dried under nitrogen before AFM observation. (ii) Twelve millions of MDA-MB-231 breast cancer cells are suspended in 1 mL PBS and placed at 37 °C for 45 min. Cells are eliminated with a 5 min centrifugation at 500 g. Whole blood is centrifuged twice at 2500 g for 15 min. Results: From AFM images the number of CD142 antibodies on the surface was estimated to approximately 2200/lm2, which is 100-fold larger than obtained by Yuana et al. The molecular diameter was about 20 4 nm. The mean size of MVs released by MDA-MB-231 cells, as estimated from 1200 MVs, was 38 9 nm. The TF-MVs concentration produced by 12 9 106 MDA-MB-231/ml was between 420 9 1010 and 420 9 1011MVs/mL. When imaging the healthy donor-PFP on TF coated surfaces, only four particles (60–100 nm) among whole platelet derived MVs were found. Conclusion: The AFM is suitable to analyse TF-MVs. The use of AFM to detect TF-MVs in human plasma should be validated in order to use MVs as biomarker of the prothrombotic risk in different diseases such as cancer. Disclosure of Interest: None declared. Keywords: cancer biomarker, microvesicles.

CFBS05 Influence of dabigatran on coagulation assays, an ‘in vitro’ study Samama MM1,*, Guinet C1, Gerotziafas G2 and Le Flem L1 1 BIOMNIS, Ivry sur Seine; 2Tenon hospital, Paris, France Objectives: Dabigatran etexilate has been approved by Health Authorities for the prophylaxis of VTE in major orthopedic surgery and for stroke prevention in non valvular atrial fibrillation without laboratory monitoring. This in-vitro study evaluated the possible interference of thrombin inhibition induced by dabigatran on routine coagulation assays. Methods: Normal platelet poor plasma (PPP) has been spiked with increasing concentrations of dabigatran up to 0.5–1.0 lg/mL and assessed for prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), antithrombin (AT), fibrinogen (Fg), clotting factors (II, V, VII, X, VIII, IX, XI and XII), protein C (PC), protein S (PS) and activated PC resistance (APCR). Results: Addition of dabigatran in PPP had a more pronounced effect on aPTT than on PT. There was a concentration dependant decrease in factor level determination in the presence of dabigatran, with intrinsic factor levels and FII decreased to a greater extent than factors V, VII and X. The presence of dabigatran resulted in overestimation of PS activity when assessed with clotting based assay. Dabigatran did © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

ABSTRACTS not significantly influence chromogenic or ELISA assays (PC, total and free PS). The TT was very sensitive at low concentrations of dabigatran and should be measured with dilute TT (HemoClot DTI). Dabigatran also influenced the clotting assay for the measurement of Fg Clauss as well as the APCR assay and anti-IIa based chromogenic assay for the measurement of AT. Conclusion: The presence of clinically relevant concentrations of dabigatran in normal human plasma interferes with clotting based assays and with chromogenic anti-IIa activity based assays. Consequently it is expected that treatment with dabigatran induces an error in the measurement of some clotting factors activity, Fg Clauss and protein S. The interference of dabigatran with clotting based and anti-IIa based assays should be taken into account when these tests are performed in patients treated with dabigatran. Disclosure of Interest: None declared. Keywords: coagulation assays, dabigatran, thrombophilia diagnosis.

CFBS06 Recommendations for laboratory testing of new anticoagulants in The Netherlands Stroobants AK1,* and Working Group New Anticoagulants of the Vereniging Hematologische Laboratoria (VHL) 1 Clinical Chemistry, AMC, Amsterdam, The Netherlands Objectives: In general one of the advantages of direct thrombin inhibitors (DTI) and direct Xa inhibitors is that no monitoring is required. However, there are situations, like unexpected bleeding/thrombosis or aberrant water homeostasis, in which clinicians need information on the coagulation status of a patient. Therefore, we collected information on laboratory evaluations and wrote a recommendation for Dutch clinical chemical laboratories on which tests to use for monitoring the new anticoagulants. Methods: We investigated the laboratory evaluations performed in the Netherlands and the ones presently mentioned in the literature. Samples were spiked with the DTIs Lepirudin, Argatroban, Bivalirudin, Dabigatran or the direct Xa inhibitor Rivaroxaban. The assays tested were aPTT, PT/INR, diluted Thrombin Time (dTT), Ecarin Clotting Time, Ecarin Clotting Assay, Endogenous Thrombin Potential (ETP), and anti-Xa. The Prothrombinase Induced Clotting Time (PICT) was not yet available. The assays were calibrated using specific standards per anticoagulant. The tests were compared on analytical (reproducibility, precision, linearity, dose-response) and practical properties (costs, availability, user friendliness). The data were presented to clinical chemists from hematology laboratories and their ideas were used to formulate the recommendation. Results: For monitoring DTIs the dTT scored best on a combination of analytical and practical properties, for direct Xa inhibitors this was the anti-Xa assay. Other tests were less suitable because of poor doseresponse, costs, small measuring range and/or difficulties with standardization. Conclusion: We recommend clinical chemical laboratories to use a dTT assay for monitoring the effect of DTI, an anti-Xa assay for direct Xa inhibitors, and an overall anticoagulation test (possibly PICT). The next step is the evaluation of these tests with samples from patients or volunteers receiving the new anticoagulants. Disclosure of Interest: None declared. Keywords: direct thrombin inhibitor, direct Xa inhibitor, laboratory assay, recommendation.


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CFBS07 Development of a novel imaging technique for the quantification of mass, volume, and density of thrombus formation Baker SM1,*, McCarty OJ1, Phillips KG1 and Rees KJ1 1 Biomedical Engineering, Oregon Health & Science University, Portland, OR, USA Objectives: We present two label-free imaging techniques to characterize mass, volume and density of blood cells, platelet aggregates and thrombus formed under shear. Methods: We have developed two novel optical measurement tools: (i) tomographic bright field imaging (TBFI), that enables the quantification of cellular refractive index, spatial mass density, total mass, and total volume using bright field microscopy, and (ii) a label-free technique utilizing DIC microscopy to measure cell volume and to quantify sub-cellular mass-density variations. Results: We validated TBFI by measuring the refractive index, mass density, total mass, and total volume of red blood cells (RBCs). As measured by TBFI, RBCs were found to have an average refractive index of 1.40 0.01 (mean SD), in keeping with reported Hb associated refractive index values. RBCs were measured to have an average dry mass of 27.2 5.3 [pg], volume 100.7 17.9 [fL], and density 27.1 3.1 [pg/fL]; all within physiological norms. We next employed these techniques to characterize the physical parameter of platelet aggregates and thrombi that were formed under shear on a surface of fibrillar collagen. We determined that platelet aggregates and thrombi formed at lower shear rates had similar surface area coverage and volume, while at higher shear rates, volume and surface area coverage of platelet aggregates were significantly larger than the formed thrombi in the presence of coagulation. Inhibition of the coagulation factor X or XI eliminated fibrin formation and resulted in the formation of large platelet aggregates at high shear. Conclusion: Utilization of these simple imaging techniques and calculations can allow for insights into the composition of thrombi and the ability to determine volume and density fluctuations of thrombi under various conditions. Disclosure of Interest: None declared. Keywords: coagulation, imaging, thrombus formation.

CFBS08 ADAMTS13 in von Willebrand factor (VWF) concentrates: is an intact VWF triplet structure important for VWF function? Kannicht C1, Solecka B1, Kroening M1 and Fuchs B1,* 1 Octapharma R&D, Molecular Biochemistry Berlin, Berlin, Germany Objectives: The characteristic multimeric pattern of plasmatic VWF results from asymmetric cleavage by ADAMTS13. Regulation of VWF multimer distribution is critical for its physiological function. In human plasma, VWF multimer gels reveal species of various multimeric sizes with flanking satellite bands (triplets). The faster and slower migrating bands encompassing a VWF multimer lack one N-terminal fragment or possess an additional N-terminal fragment, respectively. Defects in VWF secretion, impaired assembly of multimers, or increased proteolysis can cause von Willebrand Disease (VWD). Distribution of VWF triplet bands is significantly altered in some plasmaderived VWF concentrates. The impact of triplet structure on VWF function has not been investigated so far. Methods: Four commercially available VWF concentrates were analyzed for ADAMTS13 content as well as VWF multimer- and triplet structure using agarose gel electrophoresis. ADAMTS13 activity was quantified by fluorescence resonance energy transfer (FRET) assay. Samples composed of different VWF triplet distribution but comparable multimers were obtained by heparin affinity chromatography.

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Platelet adhesion under flow was determined using a flow-chamber model. Results: VWF concentrates markedly differed with respect to their content of ADAMTS13 antigen and activity. A higher content of ADAMTS13 correlated with an altered triplet structure reflected by an increased presence of the faster migrating triplet band, indicating VWF proteolysis. VWF-mediated platelet adhesion under flow over time was increased using a VWF fraction predominantly containing the slower migrating triplet band. Conclusion: These findings suggest that an intact triplet structure has an impact on platelet adhesion at physiological high arterial shear rate conditions. The relevance of VWF N-terminal domains for platelet binding and potential clinical consequences of enhanced proteolysis in commercial concentrates has to be further evaluated. Disclosure of Interest: None declared. Keywords: ADAMTS13, von Willebrand factor concentrates, VWF concentrates, VWF multimer, VWF triplet function, VWF triplet structure.

CFBS09 SMIA: a new approach in FVIII inhibitor measurement and standardisation Raut S* Haemostasis Section, NIBSC, Potters Bar, UK Objectives: Previous studies have shown high variability between laboratories when measuring FVIII inhibitors in patient samples, with CVs ranging from 40 to 200%. The Nijmegen Modification is currently the gold standard inhibitor assay, in which patient’s inhibitor titres are measured relative to a ‘Control’ mixture consisting of equal volumes of buffered normal pooled plasma and FVIII-deficient plasma (FDP). We questioned the need for FDP, which was introduced into the assay as a like-for-like diluent for the Reference ‘Control’ and we hypothesised that this would actually introduce a variant in the assay whose variability can be exacerbated by the many different FDPs now commercially available. More importantly, as the Inhibitor titre is based on% of FVIII in the Reference (‘Control’), we should be able to substitute the FDP with a more like-for-like diluent such as Buffered Normal Plasma. The unknown inhibitor titre in this assay (South Mimms Inhibitor Assay - SMIA) would now be expressed relative to 200% FVIII in the Reference ‘Control’ (rather than 100% FVIII previously). Furthermore, this approach would remove the variant and more importantly, significantly reduce the cost of an inhibitor assay. Methods: Inhibitor samples, over a wide range of concentrations, were used to test the above hypothesis by comparing Nijmegen inhibitor assay with the SMIA test. Results: Results showed that, for high titre samples (5–40 BU/mL), equivocal inhibitor titres could be obtained using the two methods. For low titre (1.0–0.15 BU/mL) samples, Nijmegen assay was able to detect inhibitor titres down to approximately 0.6 BU/mL, whilst SMIA was able to detect inhibitor titres down to approximately 0.2 BU/mL. Conclusion: SMIA can produce equivocal results, without the use of FDPs, and is more sensitive to low titre inhibitors (approximately 0.2 BU/mL) than the Nijmegen assay. Furthermore, the removal of the FDPs (SMIA) will significantly reduce assay costs and will be a very simple but a welcome modification to the assay by clinical testing laboratories. Disclosure of Interest: None declared. Keywords: Bethesda Assay, Factor VIII, FVIII-deficient plasma, Haemophilia, Inhibitor, Nijmegen Assay, SMIA.

CFBS10 Thrombin inhibition activity and in vitro stability in human plasma of two aptamers Gribkova I1,*, Spiridonova V2, Gorbatenko A1 and Sinauridze E1 1 National Research Center for Hematology; 2Belozersky Institute of Physico-Chemical Biology at Lomonosov Moscow State, Moscow, Russian Federation Objectives: Comparison of the properties of two thrombin inhibitors – single-stranded DNA-aptamers - (well-known 15TBA – 5′-GGTTGG TGTGGTTGG-3′ and it’s modification - 31TBA - 5′-CACTGGTA GGTTGGTGTGGTTGGGGCCAGTG-3′), which can be used as anticoagulants. Methods: Thrombin inhibition activity was measured experimentally as effects on fibrin polymerization in vitro in a pure buffer system. Buffer contained human fibrinogen (4 mg/mL) and varying concentrations of aptamers. The reaction of polymerization was triggered by thrombin (1 nM) and kinetics was studied at 37 °C using spectrophotometer for at least 40 min. The stability of aptamers was investigated in vitro by measuring activated partial thromboplastin time (APTT) in human plasma containing aptamer (1 lM). Samples for analysis of coagulation were withdrawn after 1, 2, 3 and 4 h of incubation. Results: 15TBA and 31TBA inhibited polymerization of fibrin in dosedependent manner. Concentrations of aptamers which decreased rate of polymerization by 50% (IC50) were equal 10 nM and 3 nM for 15 TBA and 31 TBA, correspondingly. Thus 31TBA was three times more effective than 15TBA. In human plasma in vitro both aptamers were stable for at least 4 h. Conclusion: It was shown that 31TBA was more effective than 15TBA. And both aptamers were stable in human plasma in vitro. The work was supported by RFBR grant N11-04-01530. Disclosure of Interest: None declared. Keywords: aptamer, fibrin polymerization, thrombin inhibitor.

CFBS11 Technoclotâ DTI and Technoviewâ dabigatran evaluation of assay performance monitoring the direct thrombin inhibitor dabigatran Wagner L1,*, Kaufmann V1, Mager J1, Geiter S1 and Leitner M1 1 Technoclone GmbH, Vienna, Austria Objectives: Dabigatran etexilate is a new orally direct thrombin inhibitor which is administered for prevention of VTE in patients after orthopedic surgery. The increasing use of the new oral direct thrombin inhibitors (DTI) such as dabigatran creates the need of their measurement in the clinical routine. Technoclotâ DTI is a modified thrombin time based assay which can be used for measurement of thrombin inhibitors using lyophilized calibration plasmas for assay calibration and controls for assay performance control. The aim of this study was to evaluate the performance of the Technoclotâ DTI assay with lyophilized Dabigatran calibrators and controls on coagulation analyzers. Methods: Technoclotâ DTI is a clotting assay based on the inhibition of a constant and defined concentration of thrombin. Clotting times measured are directly related to thrombin inhibitor concentrations. Calibration of the assay was performed using the lyophilized Technoviewâ Dabigatran calibrators with assigned values. Lyophilized Technoviewâ Dabigatran controls with assigned values were measured to calculate recovery and precision. The assay was performed automated using the coagulation analyzers Ceveronâ alpha, Coasys Plus C, STA Compact with adapted pipetting schemes for each analyzer. Results: Calibration curves with R2 = 1.0 0.1 were obtained for dabigatran in the range of 0–500 ng/mL on all analyzers. The recovery of control was 10% of target value for all controls on all analyzers. Precission data of the assay are summarized below:


ABSTRACTS Image/Graph:

Dabigatran ng/mL intra-assay Mean CV%

103 4.71

inter-assay 300 2.33

103 5.40

300 2.6

Conclusion: Our data demonstrate that Technoclotâ DTI is suitable for monitoring Dabigatran by using lyophilized calibrators for assay calibration and optimised pipetting schemes for the different analyzers in order to obtain results with good precision. Disclosure of Interest: L. Wagner: None Declared, V. Kaufmann Employee of: Technoclone GmbH, J. Mager Employee of: Technoclone GmbH, S. Geiter Employee of: Technoclone GmbH, M. Leitner Employee of: Technoclone GmbH. Keywords: dabigatran, direct thrombin inhibitor, Technoclot DTI.

CFBS12 Technochrom anti-Xa and technoview rivaroxaban evaluation of assay performance on different analyzers Wagner L1,*, Kaufmann V1, Mager J1, Geiter S1, Leitner M1, Siegemund A2 and Lindhoff-Last E3 1 Technoclone GmbH, Vienna, Austria; 2Labor Reising Ackermann, Leipzig; 3Internal Medicin, University Hospital Frankfurt Main, Frankfurt am Main, Germany Objectives: The increasing use of the new oral direct Factor Xa (FXa) inhibitor rivaroxaban creates the need of its measurement in the clinical routine. Technoview Rivaroxaban Calibrators and the Technochrom anti-Xa assay are available, but to be suitable for the routine clinical laboratory a validated system of calibrators, assay and analyzer protocols is necessary. The aim of this study was to establish application protocols for Technochrom anti-Xa and Technoview Rivaroxaban on the main coagulation analyzers and evaluate their performance. Methods: TECHNOCHROM anti-Xa is a one stage chromogenic assay for the determination of Xa inhibitor activity in human citrated plasma. The assay is based on the inhibition of activated factor X (FXa) by indirect or direct Xa inhibitors like Rivaroxaban as measured by a chromogenic FXa substrate. Calibrators and Controls: Technoview Rivaroxaban calibrators were used for assay calibration. The assay was performed on coagulation analyzers: Ceveron alpha, ACL-TOP, STAR, Thrombolyzer XRC and BCS with adapted settings for each analyzer and for two measurement ranges. Controls in three ranges were measured to calculate recovery and precision. Results: Calibration curves with R2 = 1.0 0.1 were obtained for rivaroxaban in the range of 0–150 ng/mL, as well as for the range of 0– 500 ng/mL on all analyzers. The recovery of control was 10% of target value on all analyzers and precision was very good with intra-assay variations between 0.9% > 4.2% and inter-assay variations between 1.8 and 4.9%. The detection limit for rivaroxaban was 10 ng/mL. Conclusion: Our data demonstrate that Technochrom anti-Xa and TECHNOVIEW Rivaroxaban can be used with adapted settings on different analyzers with very good results. Disclosure of Interest: L. Wagner: None Declared, V. Kaufmann Employee of: Technoclone GmbH, J. Mager Employee of: Technoclone GmbH, S. Geiter Employee of: Technoclone GmbH, M. Leitner Employee of: Technoclone GmbH, A. Siegemund: None Declared, E. Lindhoff-Last: None Declared. Keywords: anti-Xa assay, rivaroxaban, rivaroxaban calibrators, rivaroxaban controls.


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CFBS13 Staphylothrombin-mediated fibrin facilitates s. aureusplatelet interactions Vanassche T1,*, Hoylaerts M1 and Verhamme P2 1 Center for Molecular and Vascular Biology; 2Department of Cardiovascular Medicine, University of Leuven, Leuven, Belgium Objectives: Staphylococcus aureus (S.aureus)-mediated platelet activation contributes to the pathogenesis of intravascular infections such as infective endocarditis and sepsis. S.aureus generates thrombin activity through the secretion of staphylocoagulase (coa) and von Willebrand factor binding protein (vWbp), which bind to prothrombin to form the active staphylothrombin complex. Although staphylothrombin has been linked to S. aureus virulence, its role in S.aureus–platelet interaction has not yet been studied. Methods: We compared platelet activation in the presence of thrombin and staphylothrombin, and measured the effect of staphylothrombin inhibition by dabigatran on aggregation induced by S. aureus Newman and a coa-,vWbp- mutant strain in platelet-rich plasma (PRP). The role of staphylothrombin-mediated fibrin on platelet-bacteria association was studied under arterial shear stress in a parallel flow chamber. Results: Although staphylothrombin did not directly activate platelets, staphylothrombin-mediated fibrin generation led to platelet trapping and secondary activation. In PRP, S. aureus-induced aggregation was delayed by dabigatran (lag time of 16.9 8.3 min vs. 4.6 2.0 min), and could be completely abolished when staphylothrombin inhibition was combined with inhibition of platelet-antibody binding (lag time > 30 min vs. 4.2 1.7 min). Both staphylothrombin inhibition (Fig. 1, middle panel) and absence of staphylothrombin (lower panel) led to a reduced platelet binding to S. aureus under arterial shear stress compared to control (upper panel) (46.6 2.6% and 18.2 4.9% vs. 100%). Conclusion: Staphylothrombin did not directly activate platelets, but staphylothrombin-mediated fibrin facilitated S. aureus–platelet interaction. The inhibition of staphylothrombin by dabigatran reduced S. aureus-mediated platelet activation and decreased platelet binding to S. aureus under arterial flow conditions, suggesting a potential therapeutical role for staphylothrombin inhibition in intravascular infections. Disclosure of Interest: T. Vanassche Grant / Research support from: This work was in part supported by a research grant from Boehringer Ingelheim, M. Hoylaerts: None Declared, P. Verhamme Consultant for: Peter Verhamme is co-holder of the Pfizer Chair in Atherothrombosis, KU Leuven, and received research funding from BoehringerIngelheim, Sanofi-Aventis, and Leo Pharma. He is a member of a scientific advisory board and/or speaker’s bureau of BoehringerIngelheim, Bayer, Sanofi-Aventis and Daiichi-Sankyo. Keywords: dabigatran, infective endocarditis, staphylocoagulase, Staphylococcus aureus, staphylothrombin, von willebrand factor binding protein.

CFBS14 Acute phase proteins reduce histone-induced platelet aggregation and coagulation activation Abrams ST1,2,*, Manson J3, Liu T1, Baluwa F1, Barrera V1, Brohi K3, Thachil J1, Wang G1 and Toh CH1,2 1 Institute of Infection and Global Health, University of Liverpool; 2 National Institute of Health Research Biomedical Research Centre, Royal Liverpool University Hospital, Liverpool; 3Trauma Sciences, Barts and the London School of Medicine & Dentistry, Queen Mary University of London, London, UK Objectives: Coagulation activation, acute phase responses and multiorgan failure (MOF) are common phenomenon in critically ill patients. However, the pathophysiology of these patients is still not

fully understood. In this study, we aim to determine the pathological role of toxic circulating histones in this process. Methods: A mouse model for severe trauma and patients with severe blunt trauma are studied. Results: Here, we established a direct relationship between circulating histones and the extent of coagulation activation, as reflected by elevated thrombin-antithrombin (TAT) levels both in the mouse model and in trauma patients. More importantly, the levels of circulating histones were significantly higher in trauma patients with organ failure. We demonstrated that circulating histones directly triggered the acute phase response by stimulating cytokine release. Furthermore, the acute phase proteins C-reactive protein (CRP) and fibrinogen play an important role in the acute phase response to injury by reducing the toxicity of histones released into the circulation after extensive cell death. This response blocked histone-induced platelet aggregation and significantly reduced coagulation activation in mice as reflected by lowered TAT levels and prevention of thrombosis, which ultimately increased their resistance to a lethal dose of histones. Conclusion: Circulating histones cause coagulation activation and contribute to the development of organ failure in critically ill patients. They also initiate the acute phase response as an endogenous anti-histone mechanism to protect the host. However, this response to injury lags behind the histone surge in patients. Therefore, we propose a time-critical period for potential clinical interventions using anti-histone therapy to reduce the incidence of coagulation activation, MOF and mortality in critically ill patients. Disclosure of Interest: None declared. Keywords: Acute phase proteins, Acute phase response, Circulating histones, coagulation, C-reactive protein (CRP), fibrinogen, mouse model, Platelet aggregations, thrombin-antithrombin (TAT), trauma.

CFBS15 CTP - a clinically validated technology for elongating the half life of coagulation factors, enabling a prolonged haemostatic activity in hemophilic animal model Hart G1,*, Zakar M2, Monahan PE3, Bar-Ilian A1, Hershkovitz O2 and Fima E1 1 R&D, Prolor Biotech, Nes Zionna; 2CMC, Prolor Biotech, Nes Zionna, Israel; 3Division of Hematology/Oncology, University of North Carolina, Chapel Hill, NC, USA Objectives: Prolor Biotech Inc. is a clinical stage public company developing long acting versions of existing therapeutic proteins utilizing a technology called CTP. The technology involves fusion of the C terminus peptide of hCG to the target protein. This is a clinically validated technology and was shown to be safe and effective, increasing the half life of proteins like FSH-CTP (ELONVAâ; Merck) and hGHCTP (PROLOR-Biotech).Study objectives were to evaluate the in-vitro activity and in -vivo long lasting hemostatic effects of FVIIaCTP and FIX-CTP in hemophilic animal models. Methods: FVIIa-CTP and FIX-CTP were expressed in CHO cells transfected with the proper genes and purified utilizing CTP specific steps. FVIIa-CTP and FIX-CTP in vitro activity was evaluated and purified proteins were administered to FVIII / and FIX / mice, PK and PD profiles were determined and the hemostatic effect was evaluated following bleeding challenge as compared to commercial products. Results: FVIIa-CTP pharmacokinetic parameters, as assessed by a clotting assay, were superior to those of rFVIIa. Its half-life and AUC were 5 and 3.5 fold higher, respectively. Improved thrombin generation and recovery were also observed. In a TVT study, FVIIa-CTP had a profound survival effect, which was maintained for a significantly longer time as compared to rFVIIa. Following CTP fusion, FVIIa specific activity was comparable. FIX-CTP half life (aPTT) was 4–5 times longer than rFIX in FIX / mice and displayed a signifi© 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

ABSTRACTS cant reduction in bleeding duration and intensity following a tail vein bleeding challenge. Fusion of CTP significantly improved FIX recovery by folds, and its specific activity was slightly reduced. Conclusion: Fusion of CTP to FVIIa and FIX has a markedly enhanced pharmacokinetics, increased exposure, increased recovery and a prolonged hemostatic effect in hemophilic mice. Our data suggest that CTP fused coagulation factors might be useful for improving prophylactic and on demand therapy in patients with hemophilia. Disclosure of Interest: None declared. Keywords: CTP, haemostatic activity, half life, recovery, TVT study.

CFBS16 Influence of crystalloid diluents concentration on thrombin generation: comparison of nacl and mannitol Ogweno GO1,*, Gikonyo NK2, Mwanda W3, Bukachi F4 and Peters T5 1 Department of Medical Physiology, Kenyatta University, Nairobi, Kenya; 2Department of Pharmacy and Complementary /Alternative Medicine, Kenyatta University, Nairobi, Kenya; 3 Xxxxxxxx; 4Xxxxxxxx; 5Xxxxxxxx Objectives: Crystalloid haemodilution induces a hypocoagulable state. However, direct effects of certain diluents concentration on coagulation measured by thrombin generation remains undetermined. We therefore determined the influence ofcrystalloid diluents’on thrombin generation both in the extrinsic and intrinsic systems in vitro, comparing NaCl and mannitol solutions. Methods: Solutions that were tested are NaCl at (w/v%) 0.9, 2.5, 5, 7.5,10, 20,; D-Mannitol: 2.5, 5, 10, 15, 20, and Ringer’s lactate as control. Platelet poor plasma was diluted 1:5 with the different concentrations of the diluents (diluted in Ringer’s lactate) diluents. Two different activators were used, 1pM TF and Kaolin (predominantly intrinsic), and 5 pMTF (extrinsic). Thrombin generation was determined by Calibrated Automated Thrombogram according to Hemker in a 96-well plate. Results: Increasing concentrations of mannitol (upto 20%) had no influence on thrombin generation either on the intrinsic or extrinsic systems. Using 1 pM TF, increasing NaCl concentration marginally prolonged lag time below 5%, more pronounced at 7.5% and completely inhibiting at 10% & 20% with 1 pM TF. Using kaolin as activator, NaCl progressively prolonged lag times at each concentration without affecting velocity of thrombin generation with complete inhibition from 5% NaCl concentration. Conclusion: At 1:5 plasma dilution, mannitol upto 20% concentration preserves thrombin generation both on the extrinsic and intrinsic system. In contrast, the impairment by NaCl is concentration and trigger dependent. The mechanisms on thrombin generation may be related to strong ionic influence of NaCl compared to mannitol alluded to by Ferry. These results likely have an impact on the choice of either hypertonic saline or mannitol in management of elevated intracranial pressure in traumatic brain injury, who are often coagulopathic. Further tests are necessary to define which coagulation factor(s) is affected in the intrinsic pathway of the coagulation cascade. Disclosure of Interest: None declared. Keywords: Thrombin generation, crystalloid concentration, Nacl and Mannitol.


Image/Graph:

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CFBS17 Spatial fibrin clot formation in plasma induced by surface-immobilized tissue factor: assay development and standardization Korotina N1,*, Khrapkova O1, Vuimo T1, Balandina A2,3, Lipets E1,3, Karamzin S1, Fadeeva O1, Spiridonov I1,2, Panteleev M1,2,4,5 and Attaulakhanov F1,2,3,4,5 1 LLC ‘HemaCore’; 2Center for Theoretical Problems of Physicochemical Pharmacology RAS; 3Centre of Pediatric Hematology, Oncology and Immunology; 4National Research Center for Hematology; 5Moscow State University, Moscow, Russian Federation Objectives: Blood coagulation i is spatially heterogeneous: diffusion of activated factors propagates 3-dimensional fibrin clot growth. Previously, we designed an experimental in vitro model of locally activated fibrin clot growth for basic research and drug design purposes [J Thromb Haemost 2005;3:321-31; J Thromb Haemost 2011;9:1825–34] in an attempt to mimic conditions of clotting in vivo. Here, we report further development of this approach into a reproducible, userfriendly global coagulation laboratory assay called Thrombodynamics that can be used for all-purpose coagulation diagnostics. Methods: Fibrin clot formation in a thin layer of nonstirred platelet free plasma was initiated by a surface-immobilized tissue factor and monitored by light scattering videomicroscopy. The acquired series of images were processed by specially designed software, and numerical parameters of spatial clot formation were calculated: lag time, rate of fibrin clot growth, etc. Results: To mimic conditions of clotting in vivo several essential problems were resolved. pH of plasma sample, which is shifting during experiment in vitro due to CO2 evaporation, was stabilized at a physiological level by HEPES buffer. To combine minimal dilution of the sample with convenience-to-use and avoidance of small volume addition, we developed stabilized lyophilized forms of reagents needed to perform the assay. The air bubbles arising in the system due to temperature gradients were successfully removed by carrying out experiments in a pressurized chamber. The technique of tissue factor immobilization on a plastic surface provides high reproducibility of the test (for protein density >50 pmol/m2). To validate the system functioning, reference plasma was developed: the rate of clot growth was 30 lm/min, CV = 7% (n = 120). Conclusion: We developed a new global coagulation assay that takes into account spatial heterogeneity of the coagulation process and meets typical laboratory requirements of usability, reproducibility, and standardization. Disclosure of Interest: N. Korotina Employee of: LLC ‘HemaCore’, O. Khrapkova Employee of: LLC ‘HemaCore’, T. Vuimo Employee of: LLC ‘HemaCore’, A. Balandina Grant / Research support from: LLC ‘HemaCore’, E. Lipets Employee of: LLC ‘HemaCore’, S. Karamzin Employee of: LLC ‘HemaCore’, O. Fadeeva Employee of: LLC ‘HemaCore’, I. Spiridonov Employee of: LLC ‘HemaCore’, M. Panteleev Employee of: LLC ‘HemaCore’, F. Attaulakhanov Employee of: LLC ‘HemaCore’. Keywords: spatial clot growth, thrombodynamics, assay standardization, coagulation diagnostics.

CFBS18 Interim results from a german prospective postmarketing surveillance of treatment of von Willebrand’s disease with a new generation VWF/FVIII concentrate Scharrer I1, Depka MV2, Alesci S3, Feddern J4, Hegener O5,*, Kadar J6 and Miesbach W7 1 University Medicine Mainz, Haemostaseology, Mainz; 2Werlhof Institute Hannover, Hannover; 3Haemophilia Centre, University Clinic Frankfurt, Frankfurt; 4Octapharma Langenfeld, Langenfeld, Germany; 5Octapharma AG, Lachen (SZ), Switzerland; 6Practice €ln, Ko €ln; 7University Clinic Frankfurt, for Transfusion Medicine Ko Haemophilia Centre, Frankfurt, Germany Objectives: With the marketing authorisation in 2005, a prospective post-marketing study (SET = Surveillance of Efficacy and Tolerability) with a double virus inactivated VWF/FVIII concentrate (Wilate) was initiated in Germany. As pre-marketing clinical trials with coagulation factors usually include only a limited number of cases, a post-marketing study is an excellent tool to enhance safety surveillance and to provide evidence on efficacy and safety in routine clinical use. By October 2010, data from 120 patients had been collected. Current interim results are presented. Methods: Besides demographic and anamnestic data, details of all injections for treatment of bleeding episodes, surgeries and prophylactic treatment were documented. Clinical efficacy and tolerability were rated using a four point verbal scale. All data undergo a pre-defined data management process including a plausibility check at the sponsor’s and the statistician’s site. Data entry and statistical analysis are performed by an external statistical institute. Results: Twenty-four treatment centers provided data of 120 patients suffering from von Willebrand’s disease (VWD) reflecting the broad spectrum of disease severity. Seventy-three patients had type 1, 37 type 2, 6 patients type 3 and 4 patients acquired VWD. The age ranged from 1 to 84 years (median 37). 69% of patients were female. Haemostatic efficacy and tolerability in 85 patients that underwent 65 surgeries (43 major procedures) was assessed ‘excellent/good’. In total, 121 bleeding episodes were treated with a median dose of 35 IU/kg per day. Efficacy was rated as ‘excellent/good’ in 96.5% of all cases. The tolerability was assessed as ‘very good/good’ in 99% of rated injections. Conclusion: Results of the interim analysis of the ongoing wilate SET study, reflecting the experience in routine clinical use further confirm the excellent data on efficacy and tolerability demonstrated by the premarketing clinical trials. Disclosure of Interest: None declared. Keywords: prospective post-marketing surveillance, VWD, VWF/ FVIII concentrate, Willebrand’s disease.

CFBS19 impact of thrombin and FXA inhibitors (daigatran, apixaban, and rivaroxiban) on PT/APTT assays on IL instrument/reagent systems Hammelburger J1,*, Bottenus R1 and Triscott M1 1 R & D, Instrumentation Laboratory (IL), Orangeburg, USA Objectives: Direct FXa (Apixaban [APIX] and Rivaroxiban [RIV]) and thrombin (Dabigatran [DAB]) inhibitors are new oral anticoagulants that do not require routine laboratory monitoring. Their impact on IL PT/APTT assays was evaluated. Methods: RIV, APIX, and DAB, were spiked into normal pooled (NPP) and coumadin plasma with international normalized ratio ([INR] 2–3) in the range of 0–1000 ng/mL. PTs were acquired using HemosIL RecombiPlasTin 2G (RPT 2G), ReadiPlasTin, PT HS Plus, and PT Fibrinogen, while APTTs were determined using HemosIL © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

ABSTRACTS SynthASil, APTT-SP, and SynthAFax. Results are in ratio for interference (1.25), peak plasma levels (Cmax = 200 ng/mL), and half-life ( 100 ng/mL) for steady-state. RIV/APIX INRs were calculated from the PT ratios and assigned ISI against RPT 2G (ISI = 1) per the WHO ISI Protocol. Results: RIV/APIX induced a linear concentration-dependent prolongation of the PT/APTT.DAB exhibited a similar but curvelinear response with a lag-time for PT. The PT response was amplified 1.5- to 3-fold when spiked into OAC plasma. The results were reagent-dependent, but conversion to RIV/APIX INR reduced their differences. RIV interfered in PT at 75 ng/mL with a PT ratio of 1.7 at Cmax, which was reduced to 1.4 at the half-life of 9 h. DAB interfered in APTT at 25 ng/mL with a ratio of 2 at the Cmax, which was reduced to 1.5 at the half-life of 12 h. RIV interfered in the APTT at 125 ng/mL with a ratio of 1.4 at Cmax. APIX interfered in the PT/APTT and DAB in the PT at ≥ 200 ng/mL. Conclusion: RIV impacts the PT with ratio 1.7 and APTT 1.4 at the Cmax, with sensitivity to 75 ng/mL and 125 ng/mL over the half-life of 9 h, respectively. DAB impacts the APTT, with ratio 2 at the Cmax and is sensitive over 24 h to trough levels (37 ng/mL). APIX has no impact on the PT/APTT and DAB on PT Rivaroxaban>Apixaban. In comparison to the anti-Xa inhibitors, dabigatran was found to be relatively weaker inhibition of thrombin and Xa generation in various systems. Dabigatran also produced a strong inhibition of the generation of protein C. Conclusion: While each of the newer oral anticoagulants are potent inhibitors of hrombin and Xa, the anti-Xa agents are relatively potent inhibitors of thrombin generation. Furthermore, Dabigatran may compromise thrombins regulatory functions such as activation of protein C, TAFI and Factor XIII. Disclosure of Interest: None declared. Keywords: Thrombin.

CFBS25 Comparison of the anticoagulant and antithrombotic activities of fucosylated chondroitin sulfate with the direct thrombin inhibitor dabigatran etexilate ~o PADS1 Fonseca RJC1,*, Cortez R1 and Moura 1 Institute of Medical Biochemistry, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Objectives: Fucosylated chondroitin sulfate (fucCS) is a potent anticoagulant polysaccharide extracted from sea cucumber. In the present work, we compare the effect of fucCS with the direct thrombin inhibitor dabigatran etexilate after oral administration to rats in coagulation, thrombosis and bleeding. Methods: Wistar were randomly divided into several groups and received different doses of fucCS or dabigatran etexilate by gavage. Plasma samples were collected with a cannula in the carotid artery in different periods of time after oral administration and assayed for aPTT and thrombin time. Antithrombotic activity was investigated in rats with the vena cava model using thromboplastin as the thrombogenic stimulus. Bleeding tendency was evaluated using the bleeding time model.


ABSTRACTS Results: After oral administration of fucosylated chondroitin sulfate (50 mg/kg) to rats, we observed an increase in the plasma anticoagulant activity, as assessed by aPTT and TT (about 3 and 5 fold, respectively) and by anti-IIa activity. Thrombus formation was inhibited by 87% and no alterations of bleeding time were observed. Dabigatran etexilate altered aPTT and TT values at 1 mg/kg (1.93 and 5 fold, respectively), but completely inhibition of thrombus formation was observed only at 18 mg/kg. Moreover, intense blood loss was observed in rats that received the 18 mg/kg dose of dabigatran. Conclusion: These results indicate that fucCS is absorbed through the gastrointestinal tract. Preparation of new compounds by chemical synthesis is limited, partly for economic reasons. On the other hand, a natural sulfated polysaccharide such as fucosylated chondroitin sulfate, with anticoagulant and antithrombotic actions after oral administration, could be an interesting alternative. Disclosure of Interest: None declared. Keywords: Fucosylated Chondroitin Sulfate, Oral Anticoagulants, Sulfated Polysaccharides.

CFBS26 Standardization of rotation thromboelastometry (Rotemâ): evaluation of recombinant factor viii products in a multi-national clinical trial Driessler F1,*, Li X2, Liu T3, Zhang X4, Yang B1, Jiang H3, Luk A4, Pierce GF5 and Sommer J1 1 Bioanalytical Development Hemophilia, BIOGEN IDEC, Waltham; 2Biostatistics, BIOGEN IDEC, Boston; 3Hemophilia Pre-clinical & Clin Pharm, BIOGEN IDEC, Waltham; 4Hemophilia Clinical Operations; 5Hemophilia Development, BIOGEN IDEC, Weston, USA Objectives: Estimation of the in vivo factor VIII activity for the treatment of hemophilia A has been performed with the standard one-stage clotting assay using plasma samples. Global hemostasis assay such as rotation thromboelastometry (ROTEM) using whole blood samples may provide additional and more relevant information about in vivo function of novel FVIII products. Due to lack of assay standardization, ROTEM has not been routinely used in hemophilia care. Methods: We standardized the ROTEM procedure for use at multiple clinical sites in support of a study on the long-lasting rFVIII-Fc product. To ensure consistent assay performance across multiple sites, Biogen Idec provided all necessary reagents, supplies and training to 15 selected clinical sites. We present here the analysis of frozen plasma controls that were performed at each site as part of the QC procedures. Results: A mixed-effect model was fitted for the ROTEM global QC data (the response variable) with ‘sites’ and ‘plasma FVIII levels’ as covariates. The results of clotting time (CT) and maximum clot formation (MCF) showed the least variance (10–18% CV). The ‘sites’ covariate had no statistically significant effect on CT, MCF and a-angle. An increase in FVIII level was shown to significantly reduce the CT but not MCF or a-angle (P < 0.05). To further examine the inter-site variance, two parameters, operator and instrument, were also evaluated. Comparison of the global data to a replicate analysis at a single site indicated equivalent variability with an overall ANOVA P-value of 0.69 for CT and a comparable MCF and a-angle. Conclusion: We conclude that the inter-site variance is not a significant factor in our ROTEM study. Assuming the pre-analytical variables for the blood collection are also minimized in our procedure, we expect the major differences in the ROTEM parameters to be a meaningful indicator of a subject’s hemostatic potential rather than assay variability between individual testing sites. Disclosure of Interest: None declared. Keywords: factor FVIII, hemophilia, rotation thromboelastometry.


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CFBS27 The condensed MCMDM-1 VWD bleeding questionnaire as a predictor of bleeding disorders in women with unexplained menorrhagia Azzam H1,*, Ghoneim HR1, Elsadeik A1, azmy E2, hassan M3 and elsharawy S1 1 Clinical Pathology, Mansoura University, Mansoura; 2Clinical Hematology, Mansoura University, Mansoura; 3Gynaecology & Obestetrics, Mansoura University, Mansoura, Egypt Objectives: Menorrhagia is a common clinical problem and is unexplained in more than 50% of women. Many studies have suggested that underlying bleeding disorders are prevalent in menorrhagic women. However, the assessment and quantifying of hemorrhagic symptoms are still limited and not widely used. The present study was conducted to assess the diagnostic utility of the condensed MCMDM1VWD bleeding questionnaire as a predictive of bleeding disorders in women with unexplained menorrhagia. Methods: Thirty women aged 11-31 with a clinical diagnosis of unexplained menorrhagia were investigated to assess the diagnostic utility of the condensed MCMDM-1VWD bleeding questionnaire (BQ) as a predictive of bleeding disorders in these women. In addition to administration of the questionnaire, comprehensive hemostatic testing was performed to all women. Results: The incidence of inherited bleeding disorders among this group was 66.6% (20/30). 8 patients had von Willebrand disease (VWD) and 7 had possible Glanzmann’s thrombasthenia. Rare bleeding disorders including hemophilia A carrier, Afibrinogenemia, Factor V deficiency and combined FV&FVIII deficiency were also identified.. The receiver operator characteristic (ROC) analysis of the condensed MCMDM-1 VWD BQ in menorrhagic women showed that the cut off, sensitivity, specificity, positive and negative predictive values were 3.5, 85%, 90%, 89%, 86% respectively. Bleeding score (BS) was strongly correlated to bleeding time in women with possible Glanzmann’s thrombasthenia. In VWD, a significant inverse correlation between the BS and the VWF levels was detected with a significant increase of BS in type 3 VWD compared to type 1. Conclusion: Bleeding disorders are common in women with unexplained menorrhagia and the condensed MCMDM-1VWD BQ can distinguish between menorrhagic women with and without bleeding disorders and help assess their severity. Disclosure of Interest: None declared. Keywords: VWD, Platelet function defect, bleeding score, Bleeding questionnaire, unexplained menorrhagia.

CFBS28 Genetic defects in von Willebrand disease Type 3 in South West Iran Keikhaei B1,*, Nasiri M2, Galehdari H3 and Yavarian M4 1 Thalassemia, AJMUS,Research Center for Thalassemia and Hemoglobinopathy, Ahvaz; 2Azad University, Fars; 3Shahid Chamran University of Ahvaz, Ahvaz; 4Shiraz University of Medical Sciences, Shiraz, Iran, Islamic Objectives: Von Willebrand disease (VWD) is the most common inherited bleeding disorder. VWD is due to quantitative (type 1, 3) and qualitative (type 2) defects of von Willebrand factor (VWF). VWD inherited autosomally (Dominant or Recessive) depends on its type. VWF is a glycoprotein involve in primary homeostasis and as carrier of factor VIII in the circulation. Type 3 VWD, is a rare but severe form of disease. Most of the reported mutation cause of VWD Type 3 is concentrated in exon 28. Although the mutation causes may be distributed over the entire VWF. Purpose: To assess the mutation type of VWD Type 3 from South west Iran.

28

ABSTRACTS

Methods: Blood samples were collected from 36 patients of 29 families with type 3 VWD. DNA was extracted by QIAGEN extraction kit. The DNA for the exon 28 was amplified by PCR and the PCR products were analyzed by sequencing. The results were analyzed by chromas software. Samples also examine by PCR-RFLP technique for proving the pathogenicity of mutations. Results: Sequencing results showed that two point mutations were present within the exon 28 in almost all patients at codon 1311 and 1659 with changes Glutamine and Arginine to stop codon respectively. All patients were heterozygous for Q1311X mutations expect one. 14/36 also was heterozygous for R1659X mutation. 3/36 patients were homozygous for R1659X mutation. The mutation was reconfirmed by using PCR-RFLP with Restriction enzymes FspBI and DdeI respectively. Conclusion: The two point mutations at codon 1311 and 1659 are the most mutational causes of VWD Type 3 in South West Iran. These two point mutations have previously been reported in other ethnic groups but in different pattern. In other studies, all patients were homozygous but we found in compound heterozygous. Three homozygous patients for R1659X also complicate our result interpretation. Disclosure of Interest: None declared. Keywords: None.

CFBS29 Octanate shows low inhibitor incidence in treatment of previously untreated patients with haemophilia A Klukowska A1, Komrska V2, Jansen M3, Bichler J4,* and Laguna P1 1 Medical University of Warsaw, Warsaw, Poland; 2Motol University Hospital, Prague, Czech Republic; 3Octapharma Vienna, Vienna, Austria; 4Octapharma AG, Lachen SZ, Switzerland Objectives: octanate is a human, plasma-derived, VWF-stabilized FVIII product with demonstrated haemostatic efficacy in patients with HA. All coagulation FVIII present in octanate is bound to its natural stabiliser VWF in a VWF:RCo/FVIII:C ratio of approximately 0.4. The manufacturing process comprises two virus inactivation steps, namely solvent/detergent treatment and terminal dry heat treatment at 100 °C for 30 min. octanate has now been approved in 75 countries and is extensively studied in GCP clinical trials. Between August 1998 and February 2011, approx. 3.5 billion IUs of octanate have been safely infused worldwide. Methods: Five prospective GCP studies with octanate were conducted in 77 previously treated patients (PTPs) with severe haemophilia A (< 2% FVIII:C) for the observational period of at least 6 months. None of these 77 PTPs treated with octanate developed an inhibitor. No inhibitor development in PTPs has been notified to Octapharma’s Drug Safety Unit. To assess the immunogenicity of octanate in previously untreated patients (PUPs) during prophylaxis, on-demand treatment and surgical procedures, an ongoing prospective clinical trial has been initiated in 2000. Results: Two of 39 subject (5.1%) developed clinically relevant inhibitor titres over the course of the study. Another two displayed inhibitors that disappeared spontaneously without octanate dose change. All inhibitors developed under on-demand treatment and before exposure day (ED) 50. Of 39 subjects, 30 had exceeded 50EDs at the time of this analysis. octanate was well-tolerated and the adverse event profile was consistent with the population studied. The haemostatic efficacy of octanate in prophylaxis and treatment of bleeding were generally rated as ‘excellent’, and no complication was reported for surgery. Conclusion: These interim results indicate octanate to be an efficacious, well-tolerated human FVIII product for management of haemophilia A in PUPs, associated with a minimal risk of inhibitors. Disclosure of Interest: None declared. Keywords: Haemophilia A, low inhibitor incidence, octanate, plasmaderived von Willebrand factor, previously untreated patients, PUPs, VWF stabilized FVIII.

CFBS30 The effect of CPB on FXIII levels in children Cini C1, Attard C1, Monagle P1,2,3,* and Ignjatovic V1,2,3 1 Haematology, Murdoch Childrens Research Institute; 2 Paediatrics, The University of Melbourne; 3Clinical Haematology, Royal Childrens Hospital, Melbourne, Vic., Australia Objectives: Cardiopulmonary Bypass (CPB) is often associated with haemostatic defects such as consumptive coagulopathy which contributes to excessive, prolonged bleeding and hemodynamic instability post-surgery. This involves activation of thrombin, which indirectly causes the exhaustion of certain circulating coagulation factors, such as Factor XIII (FXIII). In adults, a decrease in FXIII levels post-CPB has been associated with an increase in blood loss. Although this effect has not been sufficiently studied in children, it is likely that haemodilution and therefore bleeding is more pronounced in this population. This may be due to the greater interface between the circulating blood and the artificial surface of the CPB circuit. As bleeding is considered one of the significant causes of hemodynamic instability and morbidity post-CPB in children, it is important that the mechanism behind these events are investigated further. This study aimed to determine the changes in FXIII levels pre and post CPB surgery in children. Methods: Blood samples were collected from 10 children aged up to 5 years undergoing CPB. Samples were collected prior to (baseline) and immediately post CPB administration (following chest closure). FXIII levels were measured using a commercially available automated kit (HemosILTM Factor XIII Antigen, Instrumentation Laboratory) on the STA-R coagulation analyser. Results were analysed using a paired t-test. Results: The results demonstrate a significant decrease (P-value = 0.0046) in FXIII levels between baseline samples (Mean = 102 25%) and post CPB samples (Mean = 65 19%). We hypothesise that this significant reduction in FXIII levels may contribute to the post-CPB bleeding observed in children. Conclusion: Further studies of this mechanism are warranted in the paediatric population in order to improve clinical outcomes post-CPB. Disclosure of Interest: None declared. Keywords: Cardio Pulmonary Bypass, Children, Factor XIII.

CFBS31 Dysfibrinogenemia caused by two mutations present in a single chromosome Jiang L1,* and Wang X2 1 Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine; 2Ruijin Hospital, Shanghai Jiaotong University School of Medicine, shanghai, China Objectives: Congenital dysfibrinogenemia is a rare disease characterised by inherited abnormality in fibrinogen molecule, which may result in defective fibrin function and/or structure. The clinical manifestations diverse between different patients. Two chinese pedigrees with inherited dysfibrinogenemia were studied. Methods: The activity and antigen of plasma fibrinogen were analyzed by Clauss and immunoturbidimetry methods, respectively. The dynamic process of blood coagulation was evaluated by the Thrombelastography (TEG). The molecular weight of fibrinogen was assessed by western blot. The function of abnormal fibrinogen was evaluated by fibrinogen clottability measure, fibrinogen dynamic polymerisation measure and fibrinolysis measure. The sequences of all the exons and exon-intron boundaries of the three fibrinogen genes were amplified by PCR and analyzed by direct sequencing. Results: Two probands had reduced plama fibrinogen activity levels and normal antigen levels. Bleeding and thrombosis occurred in the two pedigrees, respectively. Hypocoagulability state of the whole © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

ABSTRACTS blood was found by TEG test. The assays of western blot showed no abnormal molecular weight of fibrinogen. Fibrinogen clottability was reduced. Fibrinogen dynamic polymerisation and fibrinolysis of the two pedigrees have different characteristics. Both Aa chain Arg289Gln and c chain Arg275Cys mutations were identified in the two probands and these two mutations were both localized to the same chromosome. Conclusion: The two probands with dysfibrinogenemia were caused by Aa chain Arg289Gln and c chain Arg275Cys mutations localized to the same chromosome. The same mutation may lead to different clinical manifestations in different pedigrees. Disclosure of Interest: None declared. Keywords: Dysfibrinogenemia, fibrinogen, function research, hemorrhage, missense mutation, Thrombosis.

CFBS32 Determination of fibrinogen level to guide replacement therapy in patients undergoing cardiopulmonary bypass with direct thrombin inhibitors Macdonald SG1,*, Bannister N2, Ortmann E3, Jenkins D4, Falter F3, Gray S3, Luddington R5, Whitbread J6, Colah S6 and Besser M5 1 Department of Haematology, Addenbrooke’s Hospital; 2Blood Sciences Department; 3Cardiothoracic Anaesthesiology; 4 Cardiothoracic Surgery, Papworth Hospital; 5Haematology Department, Addenbrooke’s Hospital; 6Cambridge Perfusion Services, Papworth Hospital, Cambridge, UK Objectives: Low fibrinogen levels are observed commonly following heparin based cardiopulmonary bypass (CPB) and respond to administration of cryoprecipitate or fibrinogen concentrate. We present the case of a 74 year old male requiring emergency triple valve replacement with the direct thrombin inhibitor (DTI) Bivalirudin due to Heparin induced Thrombocytopenia. In the presence of DTI determination of fibrinogen by the Clauss method may be inaccurate and the best method to measure fibrinogen levels is unclear. Methods: CPB was performed as described by Koster et al. in the EvolutionON trial. Intrabypass monitoring was performed with whole blood Ecarin Clot time, Activated Clotting Time (ACT) and Anti IIa levels (Biophen DTI)).Retrospectively, comparison of fibrinogen levels at time points during and following CPB by Clauss method, PTDerived and an automated latex based immunoassay (Liaphen Fibrin-

Image/Graph:


29

ogen) assessed which method was able to guide treatment strategies in this group of patients. Results: Despite good clearance of DTI, the patient bled excessively. Clauss fibrinogen and PT-Derived fibrinogen suggested levels below the limits of detection of the assay ( A) predicted to lead to c p.Ala353Asp in the Fibrinogen D domain. A different substitution at the same position (Ala353Thr) has previously been reported in association with a prothrombotic hypodysfibrinogenemia and this variant has been shown to be resistant to degradation by tissue plasminogen activator (tPA). We investigated whether the novel mutation in our patient also resulted in tPA resistance by measuring tPA-induced fibrinolysis in whole blood thromboelastography. Methods: Blood samples were taken from a 52 year old female patient with hypo-dysfibrinogenemia (Fib Ag and Fib Clauss < 0.5 g/dL) and a past history of a sagittal sinus thrombosis, menorrhagia and excessive haemorrhage after dental extraction, parturition and hysterectomy. Samples were also taken from a female, age-matched control. Whole blood thromboelastography was carried out to assess clot stability after activation with tissue factor (TF) 0.5pM and the addition of tPA at varying concentrations. Results: Whole blood thromboelastography activated with TF and tPA 1.5 nM (fig. 1) showed a longer Clot Formation Time (324s vs. 103s), reduced a angle (52° vs. 69°) and lower Maximum Velocity (6 mm/min vs. 12 mm/min) in the patient compared to the control. Image/Graph:

35

The Lysis Index at 30 mins was higher (85% vs. 2%) and the Lysis Time (LT) was longer (1801s vs. 875s) in the patient. Similar results were gained with tPA 1.25 nM. Clot lysis parameters were similar with tPA 1.5 nM and 1 nM in the patient (LT 2332s vs. 2515s) compared to a quicker clot lysis time with tPA 1.5 nM compared to 1 nM in the normal control (LT 1181s vs. 2004s) (Fig. 2). Conclusion: The reduced clot formation and reduced tPA-induced clot lysis explains the haemorrhagic and thrombotic phenotype of this patient. We propose that the novel mutation leading to c p.Ala353Asp results in tPA resistance and therefore a prothrombotic tendency as seen with Ala353Thr. Disclosure of Interest: None declared. Keywords: hypofibrinogenemia, thromboelastography, tissue plasminogen activator.

FVB04 Effect of dabigatran and rivaroxiban on thrombomodulin mediated activation of protein c and thrombin activated fibrinolysis inhibitor (TAFI) Hoppensteadt D1,*, Cunanan J1, Fareed J1 and Lewis B2 1 Pathology, Loyola University Medical Center, Maywood, IL; 2 Cardiology, Loyola University Medical Center, Maywood, IL, USA Objectives: The new oral anticoagulant agents Dabigatran and Rivaroxiban target thrombin and factor Xa to mediate their anticoagulant effects perspectively. This study was designed to investigate the effects of active form Dabigatran and Rivaroxiban on the activation process of protein C and TAFI by thrombin-thrombomodulin complex. Methods: The active form of Dabigatran was synthesized. While Rivaroxiban was extracted from commercially available tablets. Both agents were dissolved in appropriate solution matrices at a stock concentration of 100 lg/mL. Thrombin-thrombomodulin mediated activation of protein C and TAFI were measured using specific chromogenic substrate based methods at a concentration of 0–10 lg/ mL in different matrices. Results: Dabigatran produced a strong inhibition of the generation of both the activated protein C and TAFI (IC50 < 1.0 lg/mL) whereas Rivaroxiban did not produce any inhibition of the activation of either of these proteases. Dabigatran also inhibited the amidolytic actions of thrombin-thrombomodulin complex whereas Rivaroxiban did not produce any effect. Neither Dabigatran nor Rivaroxiban produced a direct inhibition of activated protein C or TAFI at concentrations of up to 10 lg/mL. Conclusion: The persistant inhibition of thrombin and its regulatory effects by Dabigatran may differentiate its pharmacologic profile from Rivaroxiban. Disclosure of Interest: None declared. Keywords: protein C, thrombomodulin.

FVB05 The anticoagulant effect of bromelain: an in vitro study using thromboelastography Toukh M, Elbatarny HS and Othman M Biomedical and Molecular Sciences, Queens University, Kingston, ON, Canada Objectives: Bromelain is a crude extract from the pineapple that has a wide array of pharmacological effects including protein digestion, fibrinolytic and anti-immune inflammatory effects. However, its clinical applications are still very limited. The aim of this study was to investigate the anticoagulant effect of Bromelain on whole blood samples using thromboelastography.


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ABSTRACTS

Methods: Following several optimization trials, we identified 0.4 U/ mL as the minimum concentration of Bromelain that results in modification of a normal TEG tracing. We next studied the effects of this dose on 10 blood samples (seven normal and three hypercoagulable). We incubated 1 mL citrated whole blood with 0.4 U of Bromelain for 10 min at room temperature before TEG analysis and we compared TEG parameters before and after incubation. Results: A significant elongation of the R time, reduction of the alpha angle, and coagulation index (CI) was observed in normal (P = 0.02, 0.01 and 0.04) and hypercoagulable blood samples (P = 0.01, 0.002, 0.06) incubated with Bromelain compared to native ones, indicating an anticoagulant effect. The average values of TEG parameters in normal samples before incubation with Bromelain were as follows: R time: 10.6 2.0 min, alpha angle: 27.6 8.0, MA: 45.7 3.5 and CI: 0.4 1.2 compared to 17.2 5.2, 18.8 5.7, 45.0 6.9 and 1.2 2 following incubation with Bromelain respectively. In the hypercoagulable samples, the average values of TEG parameters before addition of Bormelain were: R time: 4.3 3.5 min, alpha angle: 77.0 7.8, MA: 65.7 5.7 and CI: 3.2 1.4 compared to 9.6 4.2, 61.6 7.8, 61.2 2.0 and 1.4 0.6 respectively following incubation with Bromelain. Conclusion: Bromelain 0.4 U/mL can reduce coagulability of normal whole blood and results in normalization of TEG tracing of hypercoagulable samples. The findings from this small in vitro study is a proof of principle of the anticoagulant effect of Bromelain and indicates a potential for it as a future anticoagulant drug that may be monitroed using TEG. Disclosure of Interest: None declared. Keywords: pinapple, TEG, anticoagulant prophylaxis, phytomedicine.

FVB06 D-dimer level, factor XII activity and XIIA-dependent fibrinolysis in normal pregnancy Silina N1,*, Golovina O1, Nikolaeva A2 and Papayan L1 1 Russian Research Institute of Haematology and Transfusiology; 2 Antenatal Clinic #22, Saint Petersburg, Russian Federation Objectives: Normal pregnancy is associated with hypercoagulation state and activation of fibrinolysis. Elevated level of factor XII (f. XII) assists both activation of coagulation and fibrinolysis. Prolongation of the time of XIIa-dependent fibrinolysis gives evidence of imbalance between coagulation and fibrinolysis in favour of former during pregnancy. It is important to establish normal deviations of D-dimer, f. XII levels and XIIa-dependent fibrinolysis for physiological pregnancy. Methods: The citrate plasmas of 117 healthy pregnant women at the age from 19 till 40 years were evaluated. D-dimer level was measured by ELISA (‘Asserachrom D-DI’, Diagnostica Stago, France), f. XII activity – with ‘Helena’ coagulometer, England, XIIa-dependent fibrinolysis was tested by G. Eremin and A. Archipov, 1985. STATISTICA 6.0 was used. Results are given as median (Me) with 95% confidence intervals (CI) for each gestational period. P < 0.05 was considered statistically significant. Results: Table D-dimer level, factor XII activity and XIIa-dependent fibrinolysis in normal pregnancy (Me; 95% CI)

Trimesters I (n = 44) II (n = 51) III (n = 22)

D-dimer, ng/mL FEU

F. XII, %

320.0; 172.4–1109.8 540.0*; 244.4–1268.0 885.0**; 512.2–1891.5

125.0; 65.0–220.0 140.0*; 91.0–208.0 165.0**; 119.0–231.0

XIIa-dependent fibrinolysis, min 7.5; 4.8–14.9 7.7; 5.6–11.2

D-dimer levels were progressively increased during normal pregnancy. F. XII activity was elevated significantly in every following gestational period. XIIa-dependent fibrinolysis was significantly heightened only in III trimester. High level of f. XII is possible player in regulation of balance between coagulation and fibrinolysis. Conclusion: D-dimer level and factor XII activity were significantly increased during normal pregnancy. XIIa-dependent fibrinolysis had statistically prolongation only in III trimester. Disclosure of Interest: None declared. Keywords: D-dimer level factor XII activity XIIa-dependent fibrinolysis normal pregnancy

FVB07 Technical and clinical validation of a modified thromboelastometry approach on a rotem device for the identification of patients at risk for hypo- and hyperfibrinolysis Kuiper GJAJM1,2,*, Van Oerle R2,3, Kleinegris M-CF2, Spronk HMH2, Lance MD1, ten Cate H2 and Henskens YMC3 1 Department of Anaesthesiology and Pain Treatment, Maastricht University Medical Center, Maastricht; 2Laboratory for Clinical Thrombosis and Haemostasis, Department of Internal Medicine, CARIM, Maastricht University Medical Center, Maastricht; 3 Haematological Laboratory, Maastricht University Medical Center, Maastricht, The Netherlands Objectives: Diagnosing patients at risk for hypo- or hyperfibrinolysis is challenging. We report the validation and applicability of a modified thromboelastometry (ROTEM) method to detect hypo- or hyperfibrinolysis in four patient groups. Methods: Venous citrated (3.2% w/v) blood was drawn from healthy volunteers and patients (sepsis, post cardiothoracic surgery, pregnancy, and liver disease). Recalcified (10 mM CaCl2) blood was assessed for fibrinolytic potential using either 35 pM tissue factor (TF), 35 pM TF and 125 ng/mL tissue plasminogen activator (tPA), or 35 pM TF and 175 ng/mL tPA for 2 h using the ROTEM method. Healthy volunteers were used to determine reproducibility (n = 1, measurements = 16) and reference ranges (n = 24). Results: At a final CaCl2 concentration of 10 mM optimal TF concentration was 35 pM (chosen to obtain clotting times (CT) < 1 min). For healthy volunteers, the coefficient of variation (CV) for CT was 9.5% and for MCF (maximum clot firmness) 1.8%. In the presence of 125 or 175 ng/mL tPA lysis time (LT) was achieved around 65 and 45 min respectively, with CV’s < 20% for CT, MCF, LOT (lysis onset time) and LT. Reference ranges for above parameters and fibrinolysis speed (FS = 75/[LT-LOT]) are shown in the figure. Compared to healthy volunteers (Mann–Whitney test P < 0.05) the MCF without tPA was increased in patients with sepsis (74 mm, IQR 68–78) and during pregnancy (68 mm, IQR 68–69), while post cardiothoracic surgery and liver disease patients had a decreased clot firmness (59 mm, IQR 50– 62 and 56 mm, IQR 49–61, respectively). During sepsis (2.3%/min, IQR 1.3–4.6) and post cardiothoracic surgery (2.6%/min, IQR 1.9– 4.0) fibrinolysis speed was reduced at 175 ng/mL tPA. Conclusion: Whole blood tPA induced fibrinolysis based ROTEM analysis was reproducible with CV’s less than 20% for all parameters. In patients with sepsis or post cardiothoracic surgery we observed a hypofibrinolytic potential, suggesting applicability of this method to assess alterations in fibrinolysis. Disclosure of Interest: None declared. Keywords: Diagnosis, Hyperfibrinolysis, Hypofibrinolysis, Rotation Thromboelastometry, Tissue Plasminogen Activator, Validation

9.6**; 6.1–24.3

*PI-II < 0.05; **PII-III < 0.05. © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

ABSTRACTS Image/Graph:

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sis. Recent studies have shown the importance of VEGF-A in enhancing the growth of lymphatic endothelial cells in lymph nodes (LNs) and the migration of dendritic cells into LNs. VEGF-A is produced in inflamed tissues and/or in draining LNs, where B cells are a possible source of this growth factor. We investigated the effect of B cellderived VEGFA in vivo using CD19Cre/hVEGF-Afl mice that express human VEGF-A (hVEGF-A) specifically in B cells. Methods: We created transgenic mice (CD19Cre/hVEGF-Afl) that express human VEGF-A specifically in B cells. Histopathological examination and immunohistochemical staining were used for the macroscopic analysis of tissues of the mice. Immunological functions of the mice were analyzed by cytokines measurement after lipopolysaccharide (LPS) challenge and ovalbumin (OVA) sensitization and challenge followed by the determination of serum Ab concentration. Results: We found that the hVEGF-A produced by B cells not only induced lymphangiogenesis in LNs, but also induced the expansion of LNs and the development of high endothelial venules (HEVs). Contrary to our expectation, we observed a significant decrease in the antigen-specific antibody production after immunization with OVA and in the proinflammatory cytokine production after inoculation with LPS in these mice. Conclusion: Our findings suggest immunomodulatory effects of VEGF-A: B cell-derived VEGF-A promotes both lymphangiogenesis and angiogenesis within LNs, but then suppresses certain aspects of the ensuing immune responses.In this study, we have shown that B cell-derived VEGF-A might play a role in maintaining the balanced immune responses, by orchestrating many aspects of the immune responses, including the expansion of lymphatic networks and the suppression of antibody production. Disclosure of Interest: None declared. Keywords: B cell, high endothelial venule, inflammation, lymph node, vascular endothelial growth factor

FVB09 Roles for transglutaminase 2 and blood clotting factor XIII-A in the maintenance of blood vessel structure Newell LM1,*, Standeven KF2, Grant PJ2, Pease RJ2 and Jackson CL1 1 Bristol Heart Institute, University of Bristol, Bristol; 2Division of Cardiovascular and Diabetes Research, Light Laboratories, Leeds, UK

FVB08 B cell-derived vascular endothelial growth factor a promotes lymphangiogenesis and high endothelial venule expansion in lymph nodes Hashiguchi T1,*, Ito T2, Shrestha B3, Miura N4, Nagasato T2, Takenouchi K1, Oyama Y1, Shimizu T1 and Maruyama I2 1 Laboratory and Vascular Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima; 2 Systems Biology in Thromboregulation, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan; 3Gandaki Medical College, Pokhara, Nepal; 4Department of Veterinary Medicine, Kagoshima University, Kagoshima, Japan Objectives: Vascular endothelial growth factor A (VEGF-A) is a prominent growth factor for both angiogenesis and lymphangiogene-


Objectives: Transglutaminase 2 (TG2) and Factor XIII-A (FXIII-A) are homologous enzymes that have been proposed to fulfil numerous overlapping essential functions, including the remodelling of blood vessels. However, we have observed that TG2 / .FXIII-A / mice are born at Mendelian frequency and are free from obvious skeletal defects. Both enzymes have been proposed to contribute to the stability of atherosclerotic plaques. We further bred these mice to generate apoE / .TG2 / .FXIII-A / triple knockout mice and also littermate control groups. We examined whether arterial repair was compromised in these mice. Methods: Male apoE / , apoE / .TG2 / apoE / FXIII-A / and triple knockout mice, (n = 30), were maintained from 6 weeks of age on a high fat diet for 3 months. At termination, mice were perfusionfixed. Buried fibrous caps in plaques in the brachiocephalic artery were counted, to determine previous plaque ruptures followed by overgrowth of new caps. Fibrotic regions in cardiac ventricles were detected with Masson’s trichrome and haemosiderin with Perl’s stain. In addition, female mice of each genotype were subjected to ligation of the left carotid artery or to sham ligation. Results: Changes in plaque area, composition and rupture frequency in the brachiocephalic artery were minimal between the genotypes. However marked cardiac ventricular fibrosis was apparent triple knockout mice (12.3% area involved, 2.1% SEM), while limited fibrosis was apparent in apoE / FXIII-A / mice (0.4% area involved,

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ABSTRACTS

0.24% SEM) and fibrosis was absent from other genotypes (P < 0.001). Haemosiderin was detected in fibrotic areas, suggesting that bleeding preceded fibrosis. Following carotid ligation, arterial rupture at 7–10 days was frequent in the triple knockout mice (6/12), but not observed in the other genotypes (0/26, P < 0.001). Conclusion: Neither FXIII-A nor TG2 is essential for atherosclerotic plaque stability and repair. However, combined deficiency leads to a marked reduction in blood vessel stability. Disclosure of Interest: None declared. Keywords: fibrosis, mouse, plaque rupture

FVB10 Relationship of platelet microparticles to complications and outcome of acute liver injury/acute liver failure (ALI/ALF) Gabriel DA1,2,*, Glover S1, Bowling R1 and Stravitz RT3 1 Invitrox, Inc., RTP; 2Division Hem/Onc, UNC School of Medicine, Chapel Hill; 3Gastoenterology, Medical College of Virginia, Richmond, VA, USA Objectives: Patients with ALI/ALF have increased circulating MP’s in proportion to the severity of the SIRS, systemic complications, and adverse outcome. Methods: Fifty patients with severe ALI, 78% with hepatic encephalopathy (HE), were recruited consecutively from a single center. The etiology of ALF was acetaminophen overdose in 50%. Platelet-poor plasma (PPP) and other laboratory parameters were collected on admission, and at day 3/4. MP’s were detected by ISADE and flow cytometry. ISADE (Invitrox, Inc) is a light scattering device that can accurately count and size MPs as small as 0.15 l. Results: 16/50 (32%) patients died and 27/50 (54%) recovered without LT. Decrease platelet count after admission correlated with poor outcome: higher venous ammonia (P < 0.001), lower arterial pH and bicarbonate P < 0.001), higher lactate (P < 0.01), and higher phosphate (P < 0.05). Decrease platelet count after admission was associated with SIRS (P < 0.01), progression to higher grade HE (P < 0.001), and death or LT (P < 0.01). MP’s of sizes 0.15–1.0 lm were detected by ISADE in all patients. CD41+ MP’s were detected in 27/31 (87%) of patients, indicating that the preponderance of MPs were platelet-derived. Admission MPs were proportional to platelet count reduction after admission (P = 0.07). Concentrations of 0.36–0.64 lm MPs correlated with laboratory predictors of poor outcome: lower admission bicarbonate (P = 0.001), higher phosphate (P = 0.0001), and higher creatinine (P = 0.03). Finally, MPs were directly proportional to the number of positive SIRS components (P < 0.001) and HE grade on admission (P < 0.001), and were significantly lower in spontaneous recovery vs. those who underwent LT or died (4.5E7 vs. 2.1E8/ mL, respectively; P = 0.0001), and lower in overall survivors than those who died (5.9E7 vs. 2.5E8/mL, respectively; P = 0.003). Conclusion: Platelet count reduction after admission for ALI/ALF connotes platelet fragmentation into MPs, which is associated with poor outcome. The exact role of MPs in ALI/ALF remains to be determined. Disclosure of Interest: D. Gabriel Shareholder of: Invitrox, Inc., S. Glover Employee of: Invitrox, Inc., R. Bowling Employee of: Invitrox, Inc., R. Stravitz: None declared. Keywords: None

FVB11 Activated protein C (APC) and thrombin modulate cardiomyocytes contractility via a protease activated receptor -1 (PAR1)- cardiac troponin I (CTI) pathway Alhamdi Y1,*, Wang G1 and Toh C-H2 1 Clinical Infection, Microbiology and Immunology, University of Liverpool, Liverpool, UK; 2Roald Dahl Haemostasis & Thrombosis Centre, Institute of Infection and Global Health, University of Liverpool, Liverpool, UK Objectives: Myocardial dysfunction is a major cause of morbidity and mortality. APC is an endogenous anticoagulant with anti-inflammatory properties which has been shown to improve cardiovascular function but direct effects on cardiomyocytes (CMs) contractility are unknown. In this study, we investigated the effects of APC & thrombin on left ventricular (LV) CMs and explored the underlying molecular mechanisms. Methods: LV CMs were isolated from adult rats using the Langendorff method. CM contractility (shortening) and intracellular calcium [Ca2+]i fluxes in response to doses of APC and thrombin were assessed using an edge-recognition system. Western blotting technique was used to study the relevant signalling pathways. Results: Of 5 nM APC doubles the contraction force of CMs. This positive inotropic effect was not associated with a rise in [Ca2+]i suggesting increased sensitivity of cardiac myofilaments to Ca2+ in response to low doses of APC. Blocking protein kinase C a (PKCa) with Go6976 (5 nM) augmented the APC-induced positive inotropic effect suggesting that PKCa is involved in this mechanism. Western blotting experiments showed that APC (5 nM) suppresses the activation of PKCa in CMs. However, at higher doses (≥ 20 nM), there is more activation of PKCa. Downstream of PKCa, APC (5 nM) induced de-phosphorylation of cTI which enhances its sensitivity to Ca2+. In contrast, thrombin produced a dose-dependent negative inotropic effect with clear toxicity at doses ≥ 2 U/mL. Both thrombin (2 U/mL) and APC (≥ 20 nM), induced a significant dose-dependent phosphorylation of cTI which was abrogated by blocking PKCa. The effects of both APC and thrombin were neutralized by blocking PAR-1. Conclusion: Paradoxical effects of APC and thrombin on CMs are mediated by PKCa & cTI downstream of PAR-1. This uncovers a novel pathway linking PAR-1 to cardiac myofilaments (troponins) which can be further studied to understand the pathophysiology of cardiac dysfunction. Disclosure of Interest: None declared. Keywords: APC, calcium, cardiomyocyte, thrombin

FVB12 Nitric oxide from enos reduces abdominal aortic aneurysms development in a mouse model with human-like hypercholesterolemia Momi S1,* and Gresele P1 1 Internal Medicine, University of Perugia, Perugia, Italy Objectives: To study the contribution of eNOS to atherosclerotic lesion formation, we generated a new strain of mice in which we combined a genetic model of eNOS deficiency with a mouse model of hyperlipidemia, the hyperlipidemic low density lipoprotein (LDLR / ) mouse, that develop atherosclerosis in a distribution that closely resembling human disease. Methods: After 16 weeks of Western-type diet cholesterol levels (by colorimetric assay), blood pressure (by tail cuff method), atherosclerotic lesions (by en face analysis) and the expression of matrix metalloproteinase (MMP)-2 and 9 (by zimography) in lesions extracts were evaluated.


ABSTRACTS Results: Mean arterial blood pressure was increased in LDLR/eNOS / mice and was comparable in degree to eNOS / animals (139 4.2 vs. 136 2.8 Hg, respectively); Serum cholesterol was enhanced by HFD, similarly in LDLR/eNOS / mice and in LDLR / mice (25.3 0.8 and 24.4 0.3 mM). The surface of aorta covered by lipidic plaques was 55.4 4% in LDLR/eNOS / mice and 45.4 12% in LDLR / mice. Interstingly enough, about 50% of LDLR/eNOS / animals kept on HFD for 16 weeks developed atherosclerotic abdominal aneurysms while they were not detected in LDLR / mice. The expression of MMP-9 and MMP-2 in extracts of AAA of LDLR/ eNOS / animals were enhanced as compared to LDLR / mice. Conclusion: Our data showed that eNOS deficiency induced a mild hypertension and increased atherosclerosis in HFD-LDLR / mice and promoted the development of spontaneous aortic aneurysms. Disclosure of Interest: None declared. Keywords: eNOS, LDLR, abdominal aortic aneurysm

FVB13 Cilostazol improves an accumulation of Ab by LRP-1 expression on endothelial cells Yasui H1,*, Kanno Y1, Kawashita E1, Nakagawa H1, Yamanaka M1 and Matsuno H1 1 DWCLA, Clin Pathol Biol, Kyo-Tanabe, Japan Objectives: The antiplatelet agent, cilostazol, works to restore and improve vascular endothelial cells in addition to controlling the platelet activating function, while contributing to the improvement of various pathologies. Cerebrovascular dysfunction is suspected as the cause for accumulation of bamyloid (Ab) inside the brain in Alzheimer-type dementia. In this study, Tg2576mice were used to investigate the effects of cilostazol on cognitive decline. Methods: The cognitive function of the mice was evaluated using a Moris Water Maze (MWM)test. bamyloid and LRP-1 in tissue were detected using both immunohistochemistry and western blotting method. These mRNA were also detected by PCR methods. Results: Significant hypoactivity was observed from 48 weeks old in the control group, continuing until 60 weeks old. When brain Abwas observed upon immunostaining, a clear accumulation of Abwas observed in the brain at 60 weeks old. The accumulated Abexhibited a tendency of local existence in vessels of the brain. A significant improvement in proximate memory was observed upon MWMtesting in groups administered cilostazol. The Abaccumulation inside the brain was observed along with a declining tendency in the cilostazol administration group compared to the control group, and in particular, a noticeable tendency was observed in the cerebral cortex. Regarding Tg2576mice, LRP-1clearly declined compared to the WTmice. The expression of LRP-1was maintained in the cilostazol administration group. These were observed by the expression of proteins and the expression level of mRNAupon Western blotting and the PCRmethod. Conclusion: Cilostazol improves the declined Abexcretion function of the brain vessels, significantly reduces the accumulation of Abinside the brain, and prevents the decline in cognitive function due to Alzheimer-type dementia. Disclosure of Interest: None declared. Keywords: None

FVB14 Procoagulant phospholipids activity in cerebrospinal fluid from patients with intracerebral haemorrhage Van Dreden P1,*, Woodhams B1, Rousseau A1, Hue G2, Lavoinne A2 and Vasse M3 1 STAGO, Gennevilliers; 2Institut de biologie clinique, CHU de Rouen, Rouen; 3Biologie Clinique, Hopital Foch, Suresnes, France © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

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Objectives: Brain haemostasis is very important as bleeding into neural parenchyma can result in paralysis, coma, and death. After brain injury or intra-cerebral haemorrhage (ICH) apoptosis plays an important role and procoagulant microparticles are shed from the membrane of apoptotic cells. The aim of this study was to investigate the generation of procoagulant phospholipids (PPL), D-Dimers (DDi) and tissue factor pathway inihibitor (TFPI) in ICH in different brain conditions. These parameters, measured in cerebrospinal fluid (CSF), during the acute phase of ICH could act as prognostic markers and help in the prediction of outcome. Methods: Fresh CSF (n = 100 patients) was collected by lumbar puncture between days 0 and 2 after hospital admission. In 31 patients no neurological or biochemical abnormalities were seen, and these samples were used as controls. Of the remaining patients: 12 had hydrocephalus, 9 had viral meningitis and 7 bacterial meningitis and 11 had a neurodegenerative disease. PPL, DDi and free TFPI levels were measured in all samples (reagents from Diagnostica Stago, Asnieres, France). Results: PPL and D-Di were significantly higher in ICH patients than in controls (P < 0.001). Significantly higher levels of PPL were observed in patients with ICH who died compared with survivors (P < 0.05). No significant difference in D-Di levels seen between survivors and non-survivors. PPL was significantly higher in ICH than in bacterial meningitis, viral meningitis, hydrocephalus and neurodegenerative diseases (P < 0.05). The levels of free TFPI in all CSF samples was below the detection limit (0.5 ng/mL). Conclusion: In this study high levels of PPL were detected in the CSF at early stage of ICH testifying the brain injury, and the extent of the vascular damage. The levels of PPL after spontaneous onset of ICH seem to correlate with clinical outcome in these patients. Changes in this parameter may provide additional information about the damage that occurs to the brain following ICH. Disclosure of Interest: None declared. Keywords: Procoagulant phospholipids, cerebrospinal fluid, intracerebral haemorrhage.

FVB15 Cleaved high molecular weight kininogen stimulates JNK/FOXO/MNSOD pathway for induction of endothelial progenitor cell senescence Zhu X1,2,*, Yoder MC3, Colman RW1 and YWu1 1 Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, USA; 2Cryus Tang Hematology Center, Soochow University, Suzhou, China; 3 Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA Objectives: High molecular weight kininogen (HK) is a major component of plasma kallikrein-kinin system (KKS), and is cleaved to its active form (HKa) upon the activation of this system. Although the KKS activation is widely involved in a variety of pathological settings, the activities of its activation product HKa remain largely unknown. Recently we have reported that HKa accelerates the onset of endothelial progenitor cell (EPC) senescence by induction of reactive oxygen species (Arterioscler Thromb Vasc Biol 2011;31:883), whereas the mechanism is not clear. In this study, we investigated whether HKa induces EPC senescence via stimulation of c-Jun N-terminal kinases (JNK)-related pathway. Methods: Human EPCs were treated with HKa in different concentrations for 72 h, which were followed by the measurement of phospharylation of JNK at Thr183/tyr185 and transcription factor FOXO4 at Thr451, as well as expression of Mn-superoxide dismutase (MnSOD) at protein and mRNA level. To narrow down the functional domain of HKa, recombinant proteins of human HK heavy chain (HC, 19– 380 aa) and light chain (LC, 390–644 aa) were generated for determining which domain(s) mediates the effect of HKa.

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ABSTRACTS

Results: In a concentration-dependent manner, HKa treatment induced phosphorylation of JNK at Thr183/tyr185, and its downstream phosphorylation of transcription factor FOXO4 at Thr451. Concomitantly, HKa upregulated the expression of MnSOD at protein and mRNA levels as measured by Western blot and real time PCR. Similar to HKa, the heavy chain (HC), but not the light chain, increased the percentage of senescent EPCs (63% 6.6 for HC v.s. 77.5% 6.02 for HKa). Moreover, HC at 100 nM increased FOXO4 phosphorylation at Thr451 and the expression of MnSOD in EPCs. Besides, both of HKa and its HC stimulated the production of intracellular H2O2. Conclusion: These above results demonstrate that HKa accelerates the onset of EPC senescence by stimulating JNK/FOXO4 /MnSOD pathway, and its effect is mediated by the HC. Disclosure of Interest: None declared. Keywords: kininogen, senescence, endothelial progenitor cells, reactive oxygen species

FVB16 Is prothrombin time a predictor of new-onset diabetes in hypertensive and coronary heart disease patients? Jong G-P1, Ma T2,* and Tien L3 1 Division of Cardiology, Armed Forces Taichung General Hospital; 2Department of the Health Service Management, China Medical University; 3Central Region Branch, Bureau of National Health Insurance, Taichung, Taiwan, China

thelial cells (ECs), an important component of the vessel wall, can alter the levels of thrombin required for sufficient coagulation. The aim of this study was to optimize the calibrated automated thrombogram (CAT) method to evaluate thrombin generation (TG) on ECs, with special regard to contribution of the intrinsic and extrinsic pathways. Methods: Human coronary arterial ECs (HCAECs) were cultured until confluency and starved overnight. Cells were incubated with normalpooled (NP) and factor XII-deficient (FXII def) platelet-poor plasma, together with a fluorogenic substrate for thrombin and calcium buffer to continuously monitor thrombin activity. Active site inhibited factor VIIa (ASIS) was used to block the extrinsic pathway and corn trypsin inhibitor (CTI) for blocking intrinsic activation. EA.hy926 cells, an EC-line with a stable phenotype, were assayed in the same way. The influence of culture conditions with low (1%) fetal bovine serum (FBS), a widely used component of cell culture medium, was evaluated as well. Results: HCAECs induced TG by means of both extrinsic and intrinsic activation as shown by TG in the absence of active extrinsic coagulation (addition of ASIS). Using EA.hy926 cells, which were cultured with a higher FBS concentration, intrinsic activation was still present in FXII def plasma with ASIS, which turned out to be related to the Image/Graph:

Objectives: Hypertensive and coronary heart disease (CHD) patients have an increased riskof developingdiabetes. Accumulating evidence suggests a close relation between metabolic disturbance and increased arterial thrombosis.However, data on the effect of arterial thrombosis on the development of new-onset diabetes (NOD) in hypertensive and CHD patients has not been well determined. We aimed to investigate the association between prothrombin time and NOD. Methods: This was a retrospective cohort study performed using data from claim forms provided to the Armed Forces Taichung General Hospital in Taiwan from January 2002 to December 2009. We estimated the odds ratios (ORs) of NOD associated with prothrombin time (INR: ≤ 1.5); nondiabetic subjects served as the reference group. Results: A total of 328 NOD cases were identified in 1680 hypertensive and CHD patients during the study period. In multiple Cox regression analysis, prothrombin time (INR: ≤ 1.5) was an independent predictor for NOD (OR, 1.12; 95% confidence interval (CI), 1.01–1.24), as were male sex, age, BMI, and additional use of diuretics, beta-blockerswhereas patients who take aspirin, angiotensin converting enzyme inhibitorsand angiotensin receptor blockerswere not. Conclusion: The results of this study suggest that prothrombin time (INR: ≤ 1.5) is an independent predictor of NOD in patients with hypertension and CHD. Increased arterial thrombosis may be involved in the development of diabetes in patients with hypertension and CHD. Disclosure of Interest: None declared. Keywords: arterial thrombosis, Coronary Heart Disease, Hypertension, New-onset diabetes, Prothrombin Time

FVB17 Thrombin generation on endothelial cells is initiated by both intrinsic and extrinsic activation and should be carried out in low serum conditions Geenen I1,2,*, Post M1, Molin D1, Van Oerle R3, ten Cate H3 and Spronk H3 1 Physiology, MUMC+, Maastricht; 2Surgery, MUMC+, Maastricht; 3Internal Medicine, MUMC+, Maastricht, The Netherlands Objectives: The process of thrombin generation involves numerous plasma proteases and cofactors. Interaction of these factors with endo© 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

ABSTRACTS presence of bovine FXII in FBS (Fig. 1). At low serum conditions, the contribution of bovine FXII was eliminated and ECs activated both the extrinsic and intrinsic pathway of coagulation. TG was inhibited in the presence of CTI and was higher compared to activation of the TF pathway in FXII def plasma (Fig. 2). Conclusion: When evaluating TG on ECs, low serum conditions should be used. Endothelial-related TG is characterized by both intrinsic and extrinsic activation. Disclosure of Interest: None declared. Keywords: coagulation assays, endothelial cells, extrinsic pathway, intrinsic pathway

FVB18 The research of inter-relationship between ADAMTS13 and human microvascular endothelial cells Wang A1,2,*, Yu Z2, Ma Z2, Ding K1, Liu X1, Wu J1, Sun Z1 and Ruan C2 1 Department of Hematology, Anhui Provincial Hospital, Hefei; 2 Key Laboratory of Thrombosis and Hemostasis of Ministry of Health, Jiangsu Institute of Hematology, suzhou, China Objectives: Like hepatic stellate cells, vascular endothelial cells are also a major source of plasma ADAMTS13, which has been confirmed in recent studies. However, these studies focus on some large vascular endothelial cells, and as to the research about ADAMTS13 expression in human microvascular endothelial cells or interaction on the surface of these cells is still unknown. Considering that ADAMTS13 regulates VWF-mediated platelet thrombus formation mainly on the surface of microvascular endothelial cells, so, our study is aim to investigate inter-relationship between ADAMTS13 and human microvascular endothelial cells. Methods: We used real-time quantitative PCR, Western blot technology and immunofluorescence microscopy to directly detect and observe the expression of ADAMTS13 in human microvascular endothelial cells. We also observed the expression and distribution of VWF in these cells by immunofluorescence microscopy. In addition, we also used flow cytometry to detect interaction between recombinant fulllength or C-terminal domains deleted ADAMTS13 protein and the membrane surface of these cells. Results: We found that ADAMTS13 expression did exist in microvascular endothelial cells and its supernatant, and there was some same distribution of stacking area between ADAMTS13 and VWF. Both full-length and C-terminal domain deleted ADAMTS13 protein could bind to the membrane surface of these cells, the average binding rate were 49.1%, 45.9% respectively. While, the binding of full-length ADAMTS13 protein to the membrane surface of these cells would reduce about 46% after 200 nM thrombospondin-1 blocking. Conclusion: The study suggests that human microvascular endothelial cells could also express and secret ADAMTS13, the balance between ADAMTS13 and VWF in these cells need to do a further study. ADAMTS13 could bind to the membrane surface of these cells, and this binding was not dependent on the C-terminal domain of ADAMTS13, and could be blocked by thrombospondin-1 protein. Disclosure of Interest: None declared. Keywords: ADAMTS13, microvascular endothelial cells, thrombosis, VWF

FVB19 Analysis of synthetic and biological microparticles on several flow cytometric platforms Mooberry M1,*, Fisher N2, Carraway MS3, Smith CB3, Kasthuri R4, Udis B2, Zucker R3 and Key N4 1 Medicine and Pediatrics; 2Microbiology and Immunology, University of North Carolina at Chapel Hill; 3NHEERL EPHD, US © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

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EPA, ORD, Chapel Hill, USA; 4Medicine, University of North Carolina, Chapel Hill Objectives: Microparticles (MPs) are membrane vesicles (0.1–1 lm) released from cells upon activation. The limit of detection of most standard flow cytometers is just below 1 lm; however, recent advances enable detection of particles < 0.5 lm. Synthetic beads are used to define size ranges and to standardize the MP gate, although beads differ in refractive index (RI) from biological MPs. Thus, the scatter of MPs in relation to beads is not equivalent and can vary between cytometers depending on the particular method used to detect and amplify scatter signals. Methods: MPs in plasma and MPs from platelets activated with ionophore (PMPs) were characterized and enumerated using counting beads, standard reference beads (MegamixTM, Biocytex), and a mixture of 1000, 800, 500, and 300 nm unstained NIST beads (BD) spiked with 200 nm FluoSpheres (Invitrogen). We compared flow cytometers with forward scatter (FSC) photomultiplier tube (PMT) amplification of scatter (BD FACS Canto SORP), FSC diode (Stratedigm S1000), and wide-angle diode (Beckman Gallios). Results: The primary scatter signal that detected differences in size of polystyrene beads < 300 nm was 90-degree side scatter (SSC), although not all cytometers had this capability. This ability to discriminate beads 300 nm and smaller, however, did not necessarily translate into improved detection of biological MPs. Minimization of background ‘noise’ and optimized electronics were also important factors for improved small particle detection. Our data illustrate the differences in light scatter characteristics between beads and biological MPs, which is influenced by both the RI and the method used to detect scatter. Conclusion: The field of MP analysis has great potential to improve our understanding of pathologic processes and aid in discovery of novel biomarkers in disease. The particular application of MP detection is an important consideration when interpreting results and selecting an instrument for MP work. Disclosure of Interest: None declared. Keywords: Flow cytometry, Microparticles

FVB20 Standardization of flow cytometry-based microparticle analysis for cytometers using side scatter for size measurements Poncelet P1,*, Robert S2, Faussat A-M3, Gameiro C4 and Mullier F5 1 R & T, BioCytex; 2Immuno-Hematology, UFR Pharmacy, INSERM-UMR-S608, Aix Marseille II University, Marseille; 3IFR 65, UPMC, Paris; 4BD Bioscience – Immunocytometry Systems, Rungis, France; 5Hematology Laboratory-NARILIS, UCL MontGodinne, Yvoir, Belgium Objectives: Reliable measurement of cell-derived microparticles (MP) is needed but their low size range (0.1–1 lm) creates a difficult challenge for detectionand counting by flow cytometry (FCM). Extending a 1st standardized protocolfor MP counts in the limited size range of 0.5–1 lm bead-eq.2, we expanded this range down-to 0.3 lm on Beckman-Coulter (BC) Gallios, thus detecting previously invisible ‘small’ MP3. VB SSC studies4 showed that this strategy, optimized on BC FCMrs measuring Forward Scatter (FSC) at high solid angle (1–19°), did not totally fit with FCMrs using low angle (1–8°) collection, including Becton-Dickinson (BD) FACS and LSRs. Side Scatter (SSC) was a more robust size-related triggering signal on such FCMrs3 but different bead-based references were needed since beads provide more intense SSC signals than biological particles of equal size. The aim is to provide scatter-based reference levels for threshold permitting general standardization of MP counts across platforms. Methods: Frozen PFP aliquots were used as common sources of platelet MP (PMP), defined via dual staining with CD41-PE and Annexin

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V-FITC. BD FCMrs: 2 FACS-Canto II, 1 FACS-Verse, 1 LSRII and 2 LSR-Fortessa equipped with a PMT for FSC. BC FCMrs: up-to 6 Gallios/Navios. The position of both ‘small’ and ‘large’ PMP subsets in dual-scatter plots was compared on each FCMr to that of calibrated beads covering a size range of 0.1–1 lm (Megamix-Plus). Results: Delineation of small PMP was made in FSC on BC Gallios using 0.3 lm and 0.5 lm beads; large PMP were found between 0.5 lm and 0.9 lm beads. The boundaries of the same two subsets were found on the SSC scale of BD FCMrs between 0.17 and 0.22 lm bead-eq (small PMP) and between 0.22 and over 0.5 lm bead-eq (large PMP). Conclusion: Similar counts were obtained on these size-defined PMP subsets using different platforms, demonstrating inter-instrument reproducibility. (i) Lacroix R, Semin Thromb Hemost 2010, (ii) Robert S., JTH 2009, (iii) Poncelet P, ISTH 2011, (iv) Lacroix R, JTH 2010. Disclosure of Interest: P. Poncelet Employee of: Full-time employee BioCytex, S. Robert: None Declared, A.-M. Faussat: None Declared, C. Gameiro: None Declared, F. Mullier: None Declared. Keywords: Biomarkers, Flow cytometry, microparticles, negative controls, phosphatidylserine expression

FVB21 The angiogenesis and anti-fibrosis effects of human adipose-derived stem cells overexpressing human HGF on acute myocardial infarction Zhu X1,*, Wu J1, Sun Z1, Cai X1, Zhu W1 and Liu X1 1 Hematology, Anhui provincial hospital, Hefei, China Objectives: The delivery of adipose-derived stem cells (ADSCs) for promoting repair of tissues has become a potential new therapy. Hepatocyte growth factor (HGF) is an important growth factor with the angiogenic, anti-fibrotic, and anti-inflammatory benefits. Therefore the transplantation of ADSCs into myocardial infarction may improve cardiac function through angiogenesis and anti-fibrosis, and combination of HGF may enhance these effects. Methods: ADSCs were isolated from human subcutaneous adipose tissue. Lentivirus vector with human HGF was constructed and then infected ADSCs. AMI rats were created by ligating the left coronary artery, and 24 h later, they were transfused via tail venous with PBS, GFP-labeled ADSCs, GFP-labeled ADSCs overexpressing HGF. The heart function was detected by echocardiogram.The GFP+ cells were detected directly under fluorescent microscopy and identified the endothelial cell differentiation in the heart. Immunohistochemistry and masson staining were made to observe the capillary density and the fibrotic area. The mRNA levels of collagen I and III, TNF-a and TGF-b1 were dectected by RT-PCR and real-time PCR. Results: ADSCs were identified by flow cytometry and being induced differentiation to adipocyte and bone.At 4 weeks after transplantation, GFP+ cells were detected in host myocardium in both AMI/ ADSC and AMI/ADSChHGF groups, but not in the AMI/PBS group. Some GFP+ cells expressed CD31. HGF level in myocardium was significantly increased in both transplantation groups, associated with a significant increase in capillary density. In contrast, a significant decrease in collagen I, III, TNF-a and TGF-b were observed in both transplantation groups, accompanied by a significant decrease in fibrotic area as compared with the AMI/PBS group. Consistently, LV function was significantly improved in both transplantation groups. Conclusion: This suggests that ADSCs combined with HGF gene transfer may be a useful strategy for the treatment of patients with ischemic heart disease. Disclosure of Interest: None declared. Keywords: acute myocardial infarction, adipose-derived stem cells, angiogenesis, hepatocyte growth factor

Haemorrhagic Disorders (Clinical) HDC01 Factor XIII in haemophilia a – effect on clot structure and permeability Okaisabor OO1,2,*, Foley J1, Sorenson B1,2 and Rea C1,2 1 Haemostasis Research Unit, Guy’s and St Thomas’ NHS Trust; 2 King’s College London, London, UK Objectives: Factor VIII (FVIII) deficiency in haemophilia A results in impaired clot-stabilising mechanisms, such as the activation of factor XIII (FXIII). Laboratory studies indicate clot stability in FVIII deficient plasma improves with addition of FXIII(1) and is a potential new therapeutic option. This study aimed to investigate clot structure and permeability following addition of supra-physiological levels of FXIII to haemophilia A plasma. Methods: To examine the effect of FXIII on clot pore size, clot permeability was assessed in FVIII deficient plasma, spiked with FVIII (0, 0.025, 0.1, 1 IU/mL) plus or minus FXIII 1 IU/mL. Clot formation was triggered with tissue factor (Innovin, final dilution 1:30000), phospholipid (4 lm) and calcium. Clots formed within a plastic tube were challenged with a pressure column of buffer. Pore size was calculated based on volume of fluid collected over time. Electron Microscopy images of clots were obtained by fixing in 2% glutaraldehyde, mounting and examining using an FEI Quanta 200F field emission scanning electron microscope (at 25 kV in high vacuum mode). Results: Addition of FXIII resulted in a 3-fold reduction in clot permeability and pore size in plasma containing 0 or 0.025 IU/mL FVIII (at 0.025 IU/mL the effect was statistically significant P = 0.027, Friedman Test). The benefit of adding FXIII was reduced as concentrations of FVIII increased (Figure 1). EM images supported this finding. Addition of FXIII alone to FVIII deficient plasma resulted in clots formed of closely packed fibres resembling normal clot structure (Figure 2). Image/Graph: Conclusion: The addition of FXIII to FVIII-deficient plasma results in decreased permeability and markedly improved clot structure. Its effect appears most remarkable at low concentrations of FVIII. Findings support the use of FXIII as a novel, factor-sparing agent in treating Haemophilia A. Reference: Rea CJ, Foley JH, Sorensen B. Factor XIII in the treatment of hemophilia A. N Engl J Med. 2012 Jan 19;366(3):281–-3. Disclosure of Interest: None declared. Keywords: None


ABSTRACTS A

B

C

HDC02 Factor c A1 domain substitution HIS99ARG results in super-instability of factor c and discrepancy between one-stage and two-stage Fc:C assay in a mild hemophilia A patient You G1,* and Wang X1 1 Ruijin Hospital of Shanghai Jiaotong University School of Medicine, Shanghai, China Objectives: To investigate the molecular mechanisms of super-instability of Fc and discrepancy of one stage and two stage Fc:C assay in a mild hemophilia A patient with His99Arg mutation. Methods: Phenotype diagnosis was performed.Fc:C was detected by one stage and two stage method.Patient’s plasma was incubated at different temperature (25, 4, 37, 56 °C) to assess the Fc:C at the designated time.ELISA was applied to measure the intact Fc remaining when the plasma was incubated at 56 °C at different time.All the exons of F8 gene and its flanking sequences were sequenced. 3D structure analysis was performed.His99Arg mutant expression plasmid was constructed and transfected into HEK293T, Fc:C and Fc:Ag of the stable expression products were assayed. Results: Fc:C of the proband was 14.7% detected by one stage assay but 1.2% by two stage assay. Fc:C dropped to < 1% from its initial value (14.7%) in 2 h when incubated at 25 °C or 4 °C. Fc:C dropped to 1.6% when incubated at 37 °C in 10 mins, which is corresponding with the result of two stage assay(1.2%). Fc:C was less stable compared with that of Fc wildtype following heat denaturation analysis, this reduced stability appeared to result from an about 200% increase in the dissociation rate judged by ELISA. Gene analysis revealed a A30716G substitution resulted in a His99Arg mutation. 3D structural analysis showed His99 residue closed to His1957(3.49 A)and Ser1959 (3.42 A)of A3 subunit,When His99 mutated into Arg99,the distance and 2.39 A,respectively.The changed into 4.19 A Fc stable expression products showed similar results in accordance with that of patient’s Fc. Conclusion: His99Arg mutation probably caused changes in the spatial structure of Fc, resulting in the decline of Fc:C. The increased dissociation rate of the heavy chain and light chain probably due to the weakened binding capacity of Cu2+ and A1 subunit that caused the instability and rapid inactivation of Fc. Interdomain instability also contributed to the discrepancy between one stage and two stage Fc:C assay. Disclosure of Interest: None Declared. Keywords: Gene mutation, Hemophilia A, Molecular mechanism, Phenotype diagnosis


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HDC03 Use of next-generation DNA sequencing for gene discovery in bleeding syndromes with defects of platelet function: a pilot study Lotta LA1,*, Tuana G1, Maino A1, Artoni A1, Lecchi A1 and Peyvandi F1 1 Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, U.O.S. Dipartimentale per la Diagnosi e la Terapia delle Coagulopatie, Fondazione IRCCS C a Granda - Ospedale Maggiore Policlinico, Universit a di Milano, Milano, Italy Objectives: Defects in platelet number, structure and function may result in bleeding tendency. Heterogeneous alterations have been described, including acquired and genetic forms with different pattern of inheritance. Platelet secretion defects (PSD) are heterogeneous platelet functional defects characterized by reduced granule secretion upon stimulation by different platelet aggregation agonists. PSD may be transmitted with autosomal dominant inheritance. The use of nextgeneration DNA sequencing may therefore help identifying genetic defects underlying inherited forms of PSD. Methods: We selected 2 Italian patients with PSD, clinical history of bleeding (bleeding severity score above 6) and at least one relative with a history of bleeding for exome (i.e. the protein coding area of the genome) sequencing. Also 7 healthy controls were chosen for sequencing. Sequencing was carried out by Illumina HiSeq after enrichment of target DNA sequences by solution-based capture. Annotation on databases of common (and likely neutral) genetic variation and exclusion of variants found in healthy controls were used as criteria to identify potentially disease-related genes and variants. Results: Sequencing was successfully performed with 39,545,400 (89.6%) of the target-sequence nucleotides covered by at least 20 reads. 25,300 single nucleotide variants (SNVs) and 205 indels were found on average in the 9 exomes. After removal of variants found in dbSNP and 1000Genomes database and of variants found in controls, 562 and 580 genes had at least one ‘novel’ potentially-associated variant in the two PSD patients. Intersection of the two gene-lists yielded 51 candidate genes. Conclusion: Next-generation DNA sequencing can be used to isolate candidate causal-genes for bleeding syndromes with defects of platelet function. Disclosure of Interest: None Declared. Keywords: Bleeding Tendency, Next Generation DNA Sequencing, Platelet Secretion Defect

HDC04 Head-to-head comparison of the optimul assay (96well plate aggregometry) with gold standard light transmission aggregometry for detection of acquired and inherited platelet defects Lordkipanidze M1,*, Chan MV2, Lowe GC1, Dawood BB1, Warner TD2, Watson SP1 and on behalf of UK GAPP study group 1 Centre for Cardiovascular Sciences, University of Birmingham, Birmingham; 2The William Harvey Research Institute, Barts & the London School of Medicine and Dentistry, London, UK Objectives: Platelet function tests are used to assess patients presenting with bleeding disorders and to monitor antiplatelet drugs. The 96-well plate assay (Optimul) was developed to reduce the time, blood volume and labour requirements of light transmission aggregometry (LTA) while maintaining the ability to study multiple platelet activation pathways simultaneously. Methods: Platelet function was assessed by LTA and Optimul in response to arachidonic acid (AA), ADP, collagen, epinephrine, TRAP, a thromboxane mimetic (U46619), and ristocetin. Doseresponse curves were generated and directly compared.

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Results: Platelet function was assessed in 20 healthy volunteers (nine male and 11 female, mean age 33, range 22–50), in 10 of which in vitro addition of aspirin 100 lM or cangrelor 1 lM was also tested, and in 15 patients (four male and 11 female, mean age 41, range 19–81) with bleeding suggestive of a platelet defect. Dose-response curves were readily assessable for both LTA and Optimul. For all agonists except collagen, the results were highly concordant in healthy volunteers. Both assays were robust and highly reproducible, with Optimul being completed in a much shorter time (10 min vs. 4 h) and requiring significantly less blood (6 ml vs 50 mL). Both assays detected platelet inhibition induced by aspirin or cangrelor (Figure). A platelet defect was detected by LTA in 60% (9/15) of participants with bleeding symptoms. Of these, 8 were also detected by Optimul. Optimul detected a platelet defect in three participants (most commonly in response to AA) that were classified as normal by LTA, with an overall detection rate for Optimul of 73% (11/15). Conclusion: By testing both drug-induced and genetic platelet defects, this exploratory study shows that the Optimul assay is sensitive to detect mild platelet defects, and could be used as an initial screening assay in patients presenting with bleeding or those on antiplatelet therapy. Funding: British Heart Foundation, Wellcome Trust. Disclosure of Interest: None Declared. Keywords: 96-well plate aggregometry, Acquired platelet disorder, Antiplatelet drugs, BLEEDING, Inherited platelet disorder, Light transmission aggregometry, Platelet function testing. Image/Graph:

HDC05 Hemostatic properties of red cell derived microparticles (RMP): promise of universal hemostatic agent Ahn YS1,*, Jy W1, Johansen ME1, Horstman LL1 and Bidot CJ1 1 Division of Hematology/Oncology, University of Miami, Miami, USA Objectives: Our understanding of RMP in hemostasis is limited. We reported that RMP are hemostatically active and well suited for hemostatic agent [JTH (Suppl 2). 269, 2011]. We futher investigated hemostatic properties of RMP as hemostatic agent. Methods: RMP were produced by high-pressure extrusion and hemostatic indicators evaluated by (i) annexin V binding, tissue factor (TF) on RMP by flowcytomety, and thrombin generation by CAT assay. (ii) RMP (1 9 108/mL) were added to factor-deficient plasmas and effect of RMP on clotting time (R), time to reach 20 mm (K), rate of of clotting (A), maximum amplitude (MA) and coagulation index (CI) on TEG were measured. (iii) Hemodilution was evaluated in normal whole blood diluted by saline. (iv) Effect of RMP on platelet adhesion and aggregation was assessed by IMPACT-R and aggregometry. (v) Blood samples of platelet and coagulation disorders were studied by TEG in the presence vs absence of RMP. Results: (i) RMP showed 20–30% binding to annexin V. No TF was detected on RMP but they generated thrombin robustly after a delay. (ii) In factor deficient plasma, RMP reduced R and K time and increased A, MA and CI variably. This effect was most pronounced in FVIII, FIX deficiencies. (iii) In hemodilution, R and K initially reduced unexpectedly upon dilution but this trend reversed > 50%


ABSTRACTS dilution: RMP reduced R, K and increased A, MA and CI. (iv) RMP enhanced platelet adhesion and aggregation at low concentrations of ADP, AA. (v) RMP corrected abnormality on TEG in blood of both platelet and coagulation disorders ex vivo. They included thrombocytopenias, thrombocytopathy and coagulopathy by anticoagulants and hemophilia with low inhibitor. Conclusion: (i) RMP had dual action, i.e. both primary and secondary hemostasis. (ii) Correction of TEG abnormality by RMP in both platelet and coagulation disorders further supports dual action of RMP. RMP appear well suited as universal hemostatic agent to treat bleeding disorders of both primary and secondary hemostasis. Disclosure of Interest: None Declared. Keywords: red cell microparticles, hemostatic properties, bleeding disorders.

HDC06 FVIII:C assay discrepancy and genotype-phenotype correlation in a group of mild haemophilia a patients Silva Pinto C1,*, Saturnino H1, Fidalgo T1, Marques D1, Goncßalves E1, Martinho P1, Oliveira AC1, Salvado R1, Martins N1 and Ribeiro L1 1 Departamento de Hematologia, Centro Hospitalar e Universitario de Coimbra, E.P.E., Coimbra, Portugal Objectives: Assuming that discrepancies have been reported in association with different genetic backgrounds and that this phenomenon is also explained by FVIII activation and incubation time variations between the different types of assays used to determine it, this study pretends to access a more accurate diagnosis in mild haemophilia A (MHA) in a group of patients routinely followed at our Haemophilia Centre. Methods: Forty patients with MHA from 25 families in whom F8 mutations had been identified. Patients’ hemorrhagic phenotype was evaluated. FVIII:C determined by one-stage (FVIII:C1 – FVIII deficient plasma, Siemens) and 2-stage chromogenic assays (FVIII:C2c -Factor VIII Chromogenic- Siemens, IL). FVIII:Ag determined by ELISA (Diagnostica Stago). Image/Graph:

Results: FVIII:C2c was lower than FVIII:C1: 24.8 11.2 UI/mL (8– 48 UI/mL) and 29.1 12.4 UI/mL (8–56 UI/mL), respectively. A discrepancy (FVIII:C2c/FVIII:C1 ≤ 0.6) was found in seven unrelated families (28%). FVIII:Ag levels (25 15.8 UI/mL) correlate better with FVIII:C2c. In five patients missense mutations were located in the A2 and A3 domains, the remaining in C1 and C2. All patients with discrepancy reflect CRM reduced phenotype. Our results are in trend with published reports: (i) FVIII:C1 revealed higher levels than the FVIII:C2c assay; (ii) 28% discrepancies identified had mutations mostly located in A2 and A3 domains (Table1). Clinically, 6 patients with discrepancy presented traumatic bleeding events and 2 had no bleeding history. © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

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Conclusion: No linear association between low levels of FVIII:C and bleeding tendency was found in patients with discrepancy. Which assay best reflects the coagulation potential and clinical phenotype remains unclear. MHA with FVIII:C1 within the normal range may be misdiagnosed, if FVIII:C levels would only be determined by FVIII: C1. This study helps clarifying the discrepancy phenomenon once other mutations no previously reported in association with FVIII:C assay discrepancy were identified. Disclosure of Interest: None Declared. Keywords: Assay discrepancy, FVIII:C, Mild haemophilia A

HDC07 Development of an alternative assay for the assessment of factor VIII inhibitor Bethesda titres Daniels S1,*, Heath A2, McMullan N3 and Raut S1 1 Biotherapeutics; 2Biostatistics, NIBSC, Potters Bar; 3University of Hertfordshire, Hatfield, UK Objectives: The Bethesda/Nijmegen modification is currently the standard method for detecting anti-FVIII antibodies (inhibitors) in patients. However, the variability associated with these inhibitor assays is high with inter-laboratory CVs of 22–128% and inter-assay CVs of 0.5–36% indicating the need for standardisation and possible development of new assays to measure inhibitors accurately and reproducibly. The aim of this study was to assess if an inhibitor reference standard could be used, in a dilution series with a sigmoid parallel line based bioassay analysis, to accurately determine Bethesda titres. Methods: Different inhibitor samples, diluted in FVIII deficient plasma over a wide dilution range, were used to assess the inhibitory effects on FVIII activity profiles of a Normal Pooled Plasma. For this bioassay equal volumes of the inhibitor dilutions and imidazole buffered normal plasma (0.1 M, pH7.4) were mixed and incubated for 2 h at 37 °C. Chromogenic assays were then carried out on these samples to determine the residual FVIII activity profiles. A sigmoid parallel line based bioassay programme was used to determine the Bethesda titres of both high and low titre inhibitors by analysis of the FVIII activity profiles, relative to an inhibitor reference standard with an assigned titre of 9.2 BU/mL. Results: Initial results suggest that a polyclonal anti-FVIII antibody reference standard can be used to determine the Bethesda titre of inhibitors, whose titres differ by < 5% from those determined by the Bethesda/Nijmegen method and with inter-assay variation of 2–16%. Conclusion: The data provides proof of principle for the assessment of FVIII inhibitor titres which can be achieved both accurately and reproducibly, by the analysis of FVIII activity profiles over a wide antibody dilution range using a sigmoid parallel line bioassay, relative to an anti-FVIII antibody reference standard. Disclosure of Interest: None Declared. Keywords: Bethesda/Nijmegen, Factor VIII, Inhibitors

HDC08 Genetic analysis of twenty Egyptian patients with congenital afibrinogenemia Abdelwahab M1,*, Neerman-Arbez M2, Koegel J2 and Shaker O3 1 Pediatric Hematology, Cairo University Pediatric Hospital, Cairo, Egypt; 2Genetic Medicine and Development, University of Geneva Medical Center, Geneva, Switzerland, 3Biochemistry, Kasrelainy Hospital, Cairo University, Cairo, Egypt Objectives: To assess genotype of Egyptian patients with congenital afibrinogenemia and see if there is phenotype genotype correlation in our population with historic multiethnic variation. Methods: All patients were subjected to thorough history taking and clinical evaluation.Afibrinogenemia was diagnosed when plasma

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immunoreactive fibrinogen levels were undetectable .Samples were taken for genetic testing after a consent. Genomic DNA was isolated using standard protocols, exons and intron-exon junctions of the fibrinogen genes were amplified by polymerase chain reaction (PCR) and sequenced. Results: We report the genetic analysis of 20 afibrinogenemic patients (age 3.5–25 years) from consanguineous marriages with wide phenotypic variability. Umbilical bleeding occurred in 70% of patients, epistaxis in 60%, oral bleeding in 40%, muscloskeletal bleeds in 35%, genitourinary in 15%, intracranial haemorrhage in 15%, gastrointestinal bleeding in 5%. To date, more than sixty distinct mutations have been identified in patients with afibrinogenemia, in homozygosity or compound heterozygosity. We identified three different mutations in our patient group, all in homozygosity as expected given the consanguinity. These were a nonsense mutation in FGA exon 5: c.718C > T; p.Q240X (Q221X without the signal peptide) that we previously described (Abdel Wahab et al., 2010), a novel missense mutation in FGB exon 6: c.853C > T; p.R285C (R255C without the signal peptide) and a novel frameshift mutation in FGB exon 8: c.1305delTinsCA. Conclusion: Interestingly, as in other studies, patients having the same mutation varied considerably in onset and severity of bleeding even among siblings. In conclusion, although all afibrinogenemic patients have unmeasurable functional fibrinogen, genotype-phenotype correlations are not easy to establish, both for bleeding and thrombosis risk. Disclosure of Interest: None Declared. Keywords: afibrinogenemia, Congenital, Egyptian

HDC09 Mutations causing severe factor XIII deficiency in Pakistan Borhany M1,*, Shamsi T1, Cairo A2 and Peyvandi F2 1 Haematology, Haemostasis & Thrombosis, National Institute of Blood Disease & Bone Marrow Tranpsplantation (NIBD), Karachi, Pakistan; 2A. Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione I.R.C.C.S. C a Granda Ospedale Maggiore Policlinico, Universit a degli Studi di Milano and Luigi Villa Foundation, Milan, Italy Objectives: Factor VII deficiency is the most common among Rare bleeding disorders(RBDs) but in Pakistan we have seen a high number of Factor XIII (FXIII) deficiency in comparison to other RBDs. Here, we describe two novel mutations in the F13A affected with severe FXIII deficiency. Methods: In our centre 14 unrelated patients affected with FXIII deficiency with repeated bleeding symptoms are followed. All laboratory coagulation tests showed normal results except FXIII activity, which was undetectable in plasma using 5M urea clot solubility test and Photometric assay (Berichrom). FXIIIA antigen levels were then determined.The molecular analysis was performed by direct sequencing of the coding regions, intron/exon boundaries and 5′ and 3′ untranslated regions of the F13A. Results: A novel splice site mutation, c.1460 + 1G>A (IVS11 + 1G > A) in the core domain was identified in 6 patients (five homozygote and one heterozygote) belonging to three different families. The use of the Berkely Drosophilia Genome Project human database predicted that this mutation would probably result in the complete abolition of the donor splicing site. The other novel mutation, c.1126 C > T, p. Trp376Arg (Exon 9), was also identified in the core domain, present in one patient in homozygous state. The use of Poly Phen-2 predicted that this mutation could be damaging for the protein with a score of 1.0. Both mutations are associated with undetectable FXIII activity and 2% of FXIIIA antigen level in homozygous patients. The one in heterozygous state has 60% of antigen and 53% of activity. Conclusion: We have identified two novel mutations leading to congenital FXIII deficiency. We will characterize all other severe affected

patients of our centre to verify if these variants could be recurrent mutations in our specific geographic area. This molecular analysis might help for prevention of these disorders in our region through prenatal diagnosis in families with already one severe affected child. Disclosure of Interest: None Declared. Keywords: Rare bleeding disorders,Factor XIII deficiency, Pakistan

HDC10 VWF exerts a protective effect on FVIII from inhibitor inactivation both in vitro and in vivo Shi Q1,*, Kuether EL1, Schroeder JA1, Perry CL1, Fahs SA1, Gill JC1 and Montgomery RR1 1 Medical College of Wisconsin, Blood Research Institute, Children’s Research Institute, Milwaukee, WI, USA Objectives: The important association between VWF and FVIII has been investigated for decades, but the influence of VWF on FVIII inhibitors is still controversial. In this study, we explored the effect of VWF on the reactivity of FVIII inhibitors. Methods: The plasma from immunized VWFnullFVIIInullmice, purified plasma IgG from inhibitor patients, or antibody (hMoAb) from inhibitor patients’B-cell clones were used for in vitro studies. Inhibitors were incubated with rhFVIII either with or without VWF. The remaining FVIII:C after inactivation was determined by chromogenic assay and the inhibitor titers were calculated. For in vivo study, FVIIInull or VWFnullFVIIInullmice were infused with inhibitory plasma and rhFVIII followed by a tail clip survival test. Results: VWF doesn’t affect the FVIII:C measured in the chromogenic assay. VWF has a dose-dependent protective effect on FVIII from inhibitor inactivation in both mouse and human samples. The ratio of inhibitor titers in the absence of VWF to its presence were 6.82 5.80 (n = 27) in mouse inhibitory plasma, 5.00 3.36 (n = 4) in human purify IgG, and 6.06 1.18 (n = 3) in hMoAb, respectively. We found that a pre-formed complex of VWF with FVIII provides more effective protection from inhibitors versus competitive binding of VWF and antibodies to FVIII. This protective effect was confirmed by in vivo studies. When rhFVIII was infused into FVIIInull mice to 2 U/ dL followed by inhibitor infusion, all mice with inhibitor titer of 2.5 BU/mL (n = 4) survived tail clipping, and 2/4 survived with either 25 or 250 BU/mL. If inhibitory plasma was infused first followed by rhVIII infusion, then only 2/6 mice with 2.5 BU/mL inhibitors survived tail clipping and none survived with 25 BU/mL or higher. In contrast, when rhFVIII was infused into VWFnullFVIIInullmice followed by inhibitor infusion, no animals survived tail clipping. Conclusion: Our studies demonstrate that VWF exerts a protective effect, reducing inhibitor inactivation of FVIII, both in vitro and in vivo. Disclosure of Interest: None Declared. Keywords: FVIII, Inhibitor, VWF

HDC11 von Willebrand Factor Propeptide (VWFPP) to von Willebrand factor antigen (VWF:AG) ratio in different subtypes of congenital and acquired von Willebrand disease (VWD) Stufano F1,*, Canciani MT1, Baronciani L1, Cozzi G1, La Marca S1, Pagliari MT1, Garcia Oya I1, Siboni SM1 and Peyvandi F2 1 U.O.S. Dipartimentale per la Diagnosi e la Terapia delle Coagulopatie, A. Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione I.R.C.C.S. Ca’ Granda Ospedale Maggiore Policlinico; 2U.O.S. Dipartimentale per la Diagnosi e la Terapia delle Coagulopatie, A. Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione I.R.C.C.S. Ca’ Granda Ospedale © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

ABSTRACTS Maggiore Policlinico, Universit a degli Studi di Milano and Luigi Villa Foundation, Milan, Italy Objectives: Some previous reports evaluated the utility of the VWFpp/ VWF:Ag ratio in identify VWD subtypes characterized by a reduced VWF survival (type 1, type Vicenza, type 2B). The role of this marker merits further study especially that it may carry implications for management. The aim of the study was to assess the VWFpp/VWF:Ag ratio in a large sample of patients with VWD followed at the Haemophilia and Thrombosis Centre in Milan. Methods: In 149 patients with a diagnosis of VWD according to the ISTH-SSC guidelines, VWFpp was measured by a commercial enzyme-linked immunoabsorbent assay (ELISA) (Sanquin) and VWF: Ag was measured by an in house ELISA using polyclonal antibodies (DAKO). Eighty healthy volunteers were also evaluated to establish the normal range ( 2 SD from the mean). Results were expressed as VWFpp/VWF:Ag ratio. Results: As shown in the Table, VWD type 2 patients had a reduced survival of VWF (elevated VWFpp/VWF:Ag ratio) except for 2A/IIC and 2A/IIH VWD subjects. According to VWFpp/VWF:Ag ratio and VWF intra-platelet levels, we were able to characterize three groups of VWD type 1 patients associated with different pathogenic mechanisms. The Vicenza type VWD had the highest VWFpp/VWF:Ag ratio value among congenital cases. In patients with acquired von Willebrand syndrome, the VWFpp/VWF:Ag ratio was considerably high except in cases with Essential Thrombocythemia. Conclusion: Due to comparable results for most VWD type 2 patients, the VWFpp/VWF:Ag ratio cannot be used as a tool to discriminate VWD type 2 subtypes. However, it can be used to delineate patients that could benefit from 1-desamino-8-D-arginine vasopressin (DDAVP) infusion compared with VWF replacement therapy. The VWFpp/VWF:Ag ratio may be useful to distinguish patients affected with VWD type 1 and borderline VWF levels from normal individuals. Disclosure of Interest: None Declared. Keywords: von Willebrand factor propeptide, VWD, VWF, VWFpp/ VWF:Ag ratio Image/Graph:

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HDC12 Combined APTT and waveform analysis increases the diagnostic capability of standard APTT assays Leong L1,*, Ramsey P1, Tang L1, Sim D1, Murphy JE1, Laux V1 and Myles T1 1 U.S. Innovation Center, Bayer HealthCare, San Francisco, CA, USA Objectives: Accurate diagnosis of patients with factor deficiencies may require extensive testing. Waveform analysis assesses the kinetics of coagulation by monitoring the increase in turbidity (optical change) over time. Methods: To assess the utility of waveform analysis in the diagnosis of patients with FVIII deficiencies, 17 hemophilia A donor plasma were subjected to aPTT on the ACL TOP, followed by waveform analysis of the raw data. The plasma was obtained from donors who had been subjected to factor-specific and Bethesda assays. One donor was crossreactive material (antigen)-negative, and two had FVIII inhibitors, at approximately 2 and approximately 110 BU. With the exception of one donor who had slightly higher FVIII activity levels (1–2%), the bulk of the hemophilia A plasma contained < 1% FVIII levels. The plasma were tested by standard aPTT, and re-tested as necessary. The aPTT results were initially assessed without the benefit of waveform analysis. Results: The aPTT failed to yield a clot time in some of the samples. Application of waveform analysis, which allowed visualization of coagulation kinetics, resolved all the aPTT issues. Waveform analysis indicated that aPTT failures can be attributable to prolongation of the clot time beyond the assay duration or to altered coagulation kinetics and consequent clot time determination errors. Clot time determination errors could be corrected manually using the on-board ACL TOP software or using waveform analysis to determine the time needed to achieve maximal acceleration of coagulation. Conclusion: These results indicated that application of waveform analysis to monitor the kinetics of coagulation can enhance the accuracy of standard aPTT assays. The improved accuracy will positively impact laboratory throughput by decreasing the number of re-tests. Disclosure of Interest: L. Leong Employee of: Bayer HealthCare, P. Ramsey Employee of: Bayer HealthCare, L. Tang Employee of: Bayer HealthCare, D. Sim Employee of: Bayer HealthCare, J. Murphy Employee of: Bayer HealthCare, V. Laux Employee of: Bayer HealthCare, T. Myles Employee of: Bayer HealthCare. Keywords: None.

HDC13 Presentation and management of acute coronary syndromes (ACS) among adult persons with hemophilia (PWH): initial results of an international, retrospective, ten-year survey Fogarty PF1,*, Mancuso ME2, Kasthuri RS3 and GEHEP Global Emerging Hemophilia Experts Panel 1 Penn Comprehensive Hemophilia and Thrombosis Program, University of Pennsylvania, Philadelphia, PA, USA; 2Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Ospedale Maggiore Policlinico, Milan, Italy; 3Division of Hematology and Oncology, University of North Carolina, Chapel Hill, Chapel Hill, NC, USA Objectives: Little data is available on acute coronary syndromes (ACS) comprising unstable angina (UA), non-ST elevation MI (NSTEMI), and ST-elevation MI (STEMI) among PWH. Methods: Members of an international consortium representing over 2000 PWHretrospectively completed case-report forms of episodes of ACS in adult PWH from 2002–present. Demographics, type/severity of HA/HB, cardiovascular risk factors (CRFs), interventions, hemo© 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

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static protocol, revascularization outcomes, and complications were recorded. Results: Fifteen episodes of ACS (7 UA, 5 NSTEMI, 3 STEMI) occurred among 14 PWH. The median age was 56 years (range, 31– 72 years), although 5/14 patients were younger than 50 years. 5/14 patients had mild HA/HB. 3/14 patients (including 1 who suffered two episodes of ACS) had HIV. 10/15 episodes were associated with ≥ 3 CRFs. Only 1/14 patients (38 years. with severe HA and STEMI) was receiving prophylaxis (ppx) at baseline. In 5/15 episodes, the initial ACS protocol was altered due to the underlying HA/HB, mostly by holding/delaying aspirin (ASA). Five episodes each were managed medically, led to percutaneous coronary intervention (PCI), or coronary artery bypass grafting (CABG). 10/10 PCI or CABG, supported by factor infusion in 9/10 cases, resulted in successful revascularization. Among five patients undergoing PCI, all received stents (BMS or DES) and ≥1 month of dual antiplatelet therapy followed by ASA. Only one of these patients (mild HA, no secondary ppx given during chronic management) experienced bleeding (GI) requiring an interruption of antiplatelet agents, whereas 4 patients with non-mild HA/HB on secondary ppx did not bleed. No ACS-related deaths occurred during initial/definitive management. Conclusion: ACS occurs among PWH (including young PWH) typically in association with multiple CRFs. Modification of the standard initial ACS protocol in some cases did not lead to worse revascularization outcomes. Secondary ppx may be necessary to protect from bleeding due to chronic antiplatelet agents even among mild HA/HB. Disclosure of Interest: P. Fogarty Grant / Research support from: Baxter Healthcare, Biogen IDEC, CSL Behring, Consultant for: Bayer Healthcare, Baxter Healthcare, Biogen IDEC, M. Mancuso Grant / Research support from: Bayer Healthcare, R. Kasthuri Grant / Research support from: Bayer Healthcare. Keywords: aging, cardiovascular, coronary, Haemophilia.

HDC14 Fluorescent quantitation PCR method applied to do carrier and prenatal diagnosis in haemophilia A and B families with large deletion mutations Lu Y Department of Laboratory Medicine, Ruijin Hospital, Shanghai, China Objectives: Large deletions are found in about 5% of haemophilia patients. Though the patient with large deletion can be confirmed, carrier identification in affected families is indefinable by direct sequencing. The aim of this study was to do the carrier and prenatal diagnosis in haemophilia families with large deletions. Methods: LD-PCR and PCR were adopted for the screening of the INV22 and INV1 respectively. The F8/F9 gene coding and boundary sequences were analyzed by direct sequencing. Seven STR sites related to F8/F9 gene were combined together to do the linkage analysis. Fluorescent quantitation PCR of the exon 15 in F8 gene, exon 2 and 4 in F9 gene, ALB gene as the internal control, was used to quantitate the relative copy numbers of the related segments. Results: The HA patient was found to have the deletion of exon 15 for unsuccessful amplification of it. The fluorescent quantitation PCR of the exon 15 showed that the index’s relative copy number of this fragment was zero. While, to his mother, grandmother and sister, the relative copy number of exon 15 was one copy respectively, which indicated that all of the three relative females were carriers with the causative deletion mutations. Luckily, the prenatal diagnosis showed the carrier sister had a health female fetus with the relative copy number of two. For the HB family, it was failed to amplify the exon 2–4 segments in the index. The results of the fluorescent quantitation PCR manifested that the copy numbers of the exon 2 and 4 were zero in the patient and one copy in his mother who was a female haemophila carrier. The female who requested for the carrier detection was detected

to have two copy numbers of the exon 2 and 4 respectively. Furthermore, the linkage analysis results were coincident with the genetic analysis. Conclusion: This report illustrated that fluorescent quantitation PCR method represented an efficient method to determine the carrier status and do the prenatal diagnosis in haemophilia families with large deletion mutations. Disclosure of Interest: None Declared. Keywords: deletion, fluorescent quantitation PCR, haemophila, mutation.

HDC15 Thrombin generation, thrombodynamics and thromboelastography in the patients with different bleeding phenotype of severe hemophilia A Tarandovskiy I1,*, Balandina A2, Panteleev M3, Kumskova M2, Kopylov K2 and Ataullahkanov F3 1 The Laboratory of Molecular Mechanisms of Hemostasis, Center for Theoretical Problems of Physicochemical Pharmacology; 2 National Research Center for Hematology MHSD; 3Center for Theoretical Problems of Physicochemical Pharmacology, Moscow, Russian Federation Objectives: To investigate the possible mechanisms of different bleeding hemophilia A (FVIII 97.5th percentile (> 200 IU/dL; n = 234). Using a factor VIII level below the 25th percentile (≤ 86 IU/dL) as the reference category, relative risks of recurrence increased with higher percentiles of factor VIII levels: hazard ratios were 1.4 (95% CI, 0.8–2.4) for p25– p50, 1.8 (95% CI, 1.1–3.0) for p50–p75, 2.3 (95% CI, 1.4–3.8) for p75– p90, 2.6 (95% CI, 1.5–4.3) for p90–p97.5, and 3.5 (95% CI, 2.1–6.0) for levels above p97.5. These relative risk estimates did not materially change after adjustment for sex, age and body mass index. Conclusion: High levels of factor VIII are a predictor for recurrent venous thrombosis. Disclosure of Interest: None declared. Keywords: Factor VIII, prediction, Recurrent Venous Thrombosis.

LSPE05 Acustar HIT-IGG and heparin-induced multiple electrode aggregometry: a useful combination for rapid diagnosis of type-II HIT Minet V1, Douxfils J2,3,*, Bailly N1, Osselaer J-C4, Chatelain C3,5, Elalamy I6, Chatelain B1,3, Dogn e J-M2,3 and Mullier F1,2,3 1 Hematology Laboratory, CHU Mont-Godinne, Yvoir; 2 Pharmacy, University of Namur; 3Namur Thrombosis and Hemostasis Center (NTHC), Namur; 4Blood Transfusion Center; 5 Hematology, CHU Mont-Godinne, Yvoir, Belgium; 6 ^pital Tenon, Paris, France Hematology, Ho Objectives: Early type II heparin-induced thrombocytopenia (HIT) diagnosis is essential to improve clinical outcomes of this potentially lethal condition but remains challenging. HemosILâ AcuStar HIT and heparin-induced multiple electrode aggregometry (HIMEA) were recently proposed as new rapid methods for diagnosis of type-II HIT. The primary objective of this study was to study performances of AcuStar HIT-IgG(PF4-H), AcuStar HIT-Ab(PF4-H) and HIMEA. The secondary objective was to compare these assays with PF4 Enhancedâ, Light Transmission Aggregometry (LTA), 14C-Serotonin Release Assay (SRA) and clinical outcomes. Methods: Sera HIT-suspected patients (n = 104) were studied retrospectively by AcuStar HIT-IgG (PF4-H), AcuStar HIT-Ab(PF4-H). HIMEA was performed on 81 patients. These tests were compared with ELISA (PF4 Enhancedâ), LTA, SRA and clinical outcomes data by Chi-Square tests and ROC Curves. Clinical outcomes were available for each patient (including nine positive type-II HIT). Results: The cut-off recommended by the manufacturer for AcuStar HIT-IgG and AcuStar HIT-Ab(PF4-H) (i.e. 1 AU) showed positive predictive value (PPV) of 64.3% and 45.0%, respectively. When clinical outcome was considered as the reference, negative predictive values of AcuStar HIT-IgG(PF4-H), AcuStar HIT-Ab(PF4-H) and HIMEA were 100%. The PPV reached 75.0%, 81.8% and 80.0%, respectively. The cut-offs were 2.89 AU, 9.41 AU and 276 AU respectively. Seventy-nine patients presented a medium-high pretest probability requiring biological testing. AcuStar HIT-IgG(PF4-H) allowed to exclude © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

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the diagnosis of HIT in 65 of these patients. Among the 12 positive AcuStar HIT-IgG(PF4-H), 9 patients were confirmed HIT and HIMEA allowed to exclude the diagnosis of HIT in 2 out of 3 non-HIT patients. Conclusion: The combination of AcuStar HIT-IgG and HIMEA with optimized cut-offs is useful for rapid and accurate diagnosis of type-II HIT. Disclosure of Interest: None declared. Keywords: Acustar, Diagnostic Tools, Electrode aggregometry, TypeII HIT.

LSPE06 Determination of dabigatran in plasma, serum, and urine samples from patient with atrial fibrillation Harenberg J*, Marx S and Kraemer R 1 Clnical Pharmacology, Ruprecht-Karls University Heidelberg, Mannheim, Germany Objectives: Dabigatran at doses of 110 mg bid and 150 mg bid showed equivalence or superior efficacy and safety compared to warfarin in patients with non-valvular atrial fibrillation. Impairment of renal function decreases elimination from blood followed by an increase of plasma concentration of dabigatran. We analysed dabigatran in plasma, serum and urine from patients during steady state conditions of therapy in these patients. Methods: Twenty eight outpatients and 50 healthy volunteers were included into the study while on dabigatran treatment for a minimum of 3 months. Controls of the general health status and of concentrations of dabigatran in plasma, serum, and urine were performed over several months. Chromogenic S2238 substrate assay was performed from plasma and from serum and urine samples as described (patent application GB1110502.0). Concentrations of dabigatran were determined by appropriate standard curves from pooled normal plasma, serum and urine of healthy controls. Data were analysed using mircrosoft excel 2003 and SAS software release 9.2. Results: Concentrations of dabigatran in controls were 0.01 0.01 lg/mL (plasma, n = 50), 0.05 0.05 lg/mL (serum, n = 50), and 0.02 0.01 lg/mL (urine, n = 50). During therapy the concentrations of dabigatran were: 0.11 0.09 lg/mL (plasma, n = 164), 0.09 0.08 lg/mL (serum, n = 41), and 6.19 5.70 lg/mL (urine, n = 82) Differences between the samples without and during intake of dabigatran were significant for plasma (P < 0.001), serum (P < 0.005), and urine (P < 0.001). The concentration of dabigatran in plasma and serum samples correlated significantly (r = 0.69338, P < 0.01). The correlation of plasma and serum samples with urine samples was low due to the time dependence of drug intake. Conclusion: Determination of dabigatran in serum and urine samples may offer an alternative approach of determination compared to plasma samples. Urine sampling is easy, not invasive and can be performed repetively. A point of care method is currently developed for urine samples. Disclosure of Interest: None declared. Keywords: anticoagulant assay, dabigatran etexilate, new anticoagulants, standardization.

LSPE07 Determination of rivaroxaban in plasma, serum, and urine from patients undergoing total hip and knee replacement surgery €mer R3 and Weiss C4 Harenberg J1,*, Schulze A2, Marx S1, Kra 1 2 Clnical Pharmacology; Orthop€ adisch-Unfallchirurgisches Zentrum, Ruprecht-Karls University Heidelberg, Mannheim; 3 Institute of inorganic chemistry, Ruprecht-Karls University Heidelberg, Heidelberg; 4Institute of Biometry and Biostatistics, Ruprecht-Karls University Heidelberg, Mannheim, Germany

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ABSTRACTS

Objectives: Rivaroxaban is determined in plasma samples by coagulation or chromogenic anti-factor Xa assays. Rapid and easy to perform assays to measure rivaroxaban in serum have not been described. Due to glomerular filtration of about 30% rivaroxaban can be detected in urine. We have developed methods to determine rivaroxaban in serum and urine in patients undergoing THR and TKR. Methods: One fifty patients were included into a study undergoing elective THR and TKR surgery. Plasma, serum, and urine samples were obtained before and 4–6 days while on therapy with rivaroxaban 10 mg od. Samples were taken 12 h after intake of rivaroxaban. Chromogenic S2222 substrate assay was performed from plasma samples and from serum and urine samples (international patent application PCT/EP2011/005586). Creatinine clearance (CrCl) was calculated from serum creatinine values (Cockroft-Gault equation). Results: Concentrations of rivaroxaban before treatment were 14.3 12.3 ng/mL (plasma), 17.7 17.4 (serum), and 18.6 18.0 ng/mL (urine). During therapy the concentrations were: 66 41 ng/mL (plasma), 81 40 ng/mL (serum), and 1524 1346 ng/mL (urine). Differences between the samples before and during intake of rivaroxaban were all significant (P < 0.0001). At day 4–6 of treatment patients < 70 years (n = 74) had higher CrCl (84 37 mL/min) and lower plasma (58 30 ng/mL) and serum concentrations of rivaroxaban (73 29 ng/mL)compared to those ≥ 70 years who had a lower CrCl (60 25 mL/min, P < 0.001) and a higher plasma (73 48 ng/ mL, P = 0.0294) and serum concentrations of rivaroxaban (89 47 ng/mL, P = 0.0161). Concentrations of rivaroxaban in urine were 2485 2170 ng/mL for younger and 1877 1641 ng/mL for olders (P = 0.5460). Conclusion: Determination of rivaroxaban in serum samples may offer an alternative compared to plasma samples. Urine sampling is easy and not invasive. A point of care method is currently developed for urine samples. Higher plasma and serum concentrations of rivaroxaban are reflected by a not significant lower excretion. Disclosure of Interest: None declared. Keywords: anticoagulant assay, new oral anticoagulants, rivaroxaban, standardization.

LSPE08 Prevalence of factor V Leiden in 318 iranian individual referred cases and the diagnostic power of two different APC-R screening tests Ahmadinejad M1,*, Ahmadzadeh N1, Farhadi E1, Atarodi K1, Balooch S1, Seyed Mortaz L1, Negari S2, Maghsudlu M3, Amirizadeh N4 and Ahmadinejad Z5 1 Reference Coagulation Lab, Iranian Blood Transfusion Research Center; 2Molecular Lab, Pathobiology Lab Center; 3Donor Selection Department; 4Research Center, Iranian Blood Transfusion Research Center; 5General Lab, NIOC Hospital, Tehran, Iran, Islamic Objectives: Factor V leiden mutation (FVL) is a common cause of inherited thrombophilia. In clinical laboratories, the diagnosis is primarily based on activated protein C resistance screening test (APC-R) followed by a genetic assay to identify the genotype (heterozygous and homozygous). Due to high cost of genetic assays in Iran, we decided to evaluate the sensitivity and specificity of a new APC-R screening test (Pentapharm APC-R Pefakit) in comparison with our current method (Stago APC-R) and further to assess the diagnostic power of the new method for discrimination between heterozygotes and homozygotes. Methods: In a 6- month period (April to September 2009), 318 patients who referred to Iranian Blood Transfusion Organization Reference Coagulation Laboratory for thrombophilia screening tests were evaluated both for FVL genotype and for two different APC-R assays. Results: Among 318 cases, 34 (10.6%) had mutations for FVL, which 29 (9.1%) were heterozygote and 5 (1.5%) homozygote. Using a cut off ratio of 2.3 for Pefakit, the sensitivity and specificity values of

100% and 99.3% for detection of FVL mutation were found. Using a predefined cut off of 120 s for Stago APC-R kit, the values were 97% and 95.6%, respectively. A range between 1.1–2.1 for FVL heterozygotes and < 1.1 for homozygotes could completely discriminate between heterozygote and homozygote cases, except for a heterozygote patient who was on warfarin therapy and diagnosed as a homozygote. No interference was seen either by lupus anticoagulant or any other evaluated parameters. Conclusion: It appears that the prevalence of FVL is slightly lower (about 10%) in our thrombophilic cases, in comparison with European countries (about 21%). The new APC-R screening test (APC-R Pefakit) has a high sensitivity and specificity as well as a high power in discrimination between heterozygotes and homozygotes. Disclosure of Interest: None declared. Keywords: APC-R, factor V Leiden.

LSPE09 Heparin-induced multiple electrode aggregometry is a promising and useful functional tool for heparininduced thrombocytopenia diagnosis: confirmation in a prospective study Gerotziafas G*, Galea V, Robert F, Elalamy I and Elalamy I Service d’Hematologie biologique, Tenon University Hospital, Paris, France Objectives and Background: Heparin-Induced Thrombocytopenia (HIT) is a potentially lethal adverse effect of heparin therapy. Accurate and rapid HIT laboratory diagnosis when HIT is suspected is crucial. The combination of an immunological assay with a functional test improves the accuracy of HIT but functional assays are currently limited to a few laboratories. Multiplateâanalyzer (Dynabyte, Munich, Germany) is a practical, semi-automated and easy-to-perform platelet aggregation assay. Aim: To explore whether heparin-induced platelet aggregation in whole blood assessed by Multiplateâ (Heparin-Induced Multiple Electrode Aggregometry, HIMEA) can replace platelet aggregation test (PAT) in platelet rich plasma. For this purpose HIMEA performance in HIT diagnosis was prospectively evaluated. Methods: HIMEA and PAT were compared to serotonin release assay (SRA) in 200 well characterized consecutive patients suspected for HIT. Results: HIMEA was more sensitive than PAT (72.7 vs. 68.2%) but had more or less the same NPV (96.7% vs. 96.1%, respectively) compared to SRA. Additionally, HIMEA was found to slightly more specific than PAT (98.3 vs. 97.2%). A 100% PPV was found for both HIMEA and PAT. Conclusion: HIMEA is at least as sensitive and as specific test as PAT for detection of HIT platelet-activating antibodies. The combination of an immunological assay with HIMEA could be a feasible option in non specialized laboratories for HIT diagnosis optimization. Disclosure of Interest: None declared. Keywords: Heparin Induced Thrombocytopenia, Multiplate.

LSPE10 Preanalytical variables of the calibrated automated thrombogram: towards a standardized and validated procedure Loeffen R*, Kleinegris M-C, Loubele ST, Pluijmen P, Fens D, van Oerle R, ten Cate H and Spronk HM Biochemistry, Maastricht University Medical Center, Maastricht, The Netherlands Objectives: Thrombin generation (TG) assays are sensitive methods to assess the overall clotting potential of plasma and despite their commonly use in thrombosis research standardization of preanalytical © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

ABSTRACTS conditions is missing. In order to setup a standardized protocol, we determined preanalytical variables of and validated the Calibrated Automated Thrombogram (CAT) method. Methods: TG was assessed by means of the CAT using MP- (0 pM tissue factor, 4 lM phospholipids) and PPLow-reagent (1 pM tissue factor, 4 lM phospholipids). Results: Blood drawn into 3.2%(w/v) citrated tubes through intravenous catheters or butterfly needles showed significantly more hemolysis compared to venipuncture using conventional needles, whereas the needle system had no influence on plasma thrombin generation. The brand and material of the collection tube (seven sources), however, strongly influenced thrombin generation (ETP ranging from 0 to 620 nM min). This was mainly due to variations in contact activation, as addition of corn trypsin inhibitor attenuated the differences. TG in the first tube was significantly higher compared to the second drawn tube, with an average 20% increase in peak height. Storage of whole blood for 6 h at 4 °C, RT or 37 °C prior to centrifugation altered TG significantly. However, TG after storage at 37 °C was most comparable to direct processing (< 10% difference in peak height). Plasma is best analyzed immediately after thawing, since storage at 4 °C, RT or 37 °C for up to 60 min decreased or increased TG significantly. When not possible, thawed plasma remains most stable when stored at RT. Based on these results, we setup an in house standardized protocol, which was used for validation, resulting in coefficients of variations below 15% for all CAT derived parameters at both the MP- and PPLow trigger. Conclusion: Thrombin generation was largely influenced by pre-analytical conditions, demonstrating the need for an international standardized protocol. Disclosure of Interest: None declared. Keywords: pre analytical variables, standardization, thrombin generation test.

LSPE11 Laboratory monitoring techniques for dabigatran and rivaroxaban – method suitability and variability between European laboratories Helin T1,*, Pakkanen A2, Lassila R3 and Joutsi-Korhonen L1 1 Coagulation Disorders Unit, Clinical Chemistry, HUSLAB, Helsinki University Central Hospital; 2Labquality Ltd; 3 Coagulation Disorders Unit, Internal Medicine and Clinical Chemistry, HUSLAB, Helsinki University Central Hospital, Helsinki, Finland Objectives: Laboratory assays to measure plasma concentration of novel oral anticoagulants (NOACs) are under development. Routine monitoring is unnecessary. Under special circumstances, eg. bleeding, thrombosis or surgery, bioactivity assessment is vital. Methods: NOACs dabigatran (Dabi) and rivaroxaban (Riva) were examined. Plasma spiked with Dabi (120 and 300 lg/L) or Riva (60, 146 and 305 lg/L) was sent to European laboratories (115 labs for Dabi, 38 for Riva). Samples were donated by Boehringer Ingelheim and Bayer, respectively, and delivered by the external quality assessment organisation Labquality. International normalized ratio (INR) and activated partial thromboplastin time (APTT) were analyzed for all samples, whereas thrombin time (TT) only for Dabi and a specially calibrated anti-factor Xa activity (anti-Xa) for Riva. Results were reported by 73 labs with Dabi (15 different INR and APTT reagents) and 22 labs with Riva samples (5 INR and 4 APTT reagents). Analysis of variance (ANOVA) was used for comparison. Results: Both NOACs increased INR: the increase was modest, albeit larger for Dabi with higher coefficient of variance (CV) (Table). Both NOACs dose-dependently prolonged APTT. Again, the increase and CV were larger for Dabi. The INR and APTT results varied reagentdependently (ANOVA P < 0.005). Dabi significantly prolonged TT in


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all 12 labs reporting results. Anti-Xa provided good estimates for lower Riva concentrations. INR (Dabi n = 73, Riva n = 22)

Dabi 120 lg/L Dabi 300 lg/L Riva 60 lg/L Riva 146 lg/L Riva 305 lg/L

APTT (Dabi n = 72, Riva n = 19)

Anti-Xa (n = 8)

Mean Mean CV (%) (s)

Mean CV (%) (lg/L) CV (%)

1.30 1.54 1.07 1.13 1.33

21.6 17.8 14.3 14.9 15.5

24.1 25.9 6.7 10.7 14.6

68.3 91.1 34.7 40.2 43.3

– – 67 134 207

– – 8.1 9.0 33.8

Conclusion: INR and APTT are unsuitable for NOAC monitoring due to variabilities between reagents and laboratories. TT is overly sensitive, but suitable as a screening tool to detect Dabi. Calibrated anti-Xa quantifies Riva. The variability seen with spiked samples is likely to be even larger in clinical samples. Disclosure of Interest: T. Helin: None Declared, A. Pakkanen: None Declared, R. Lassila Paid Instructor for: Novo Nordisk, Baxter, Bayer, Boehringer Ingelheim, Octapharma, Pfizer, Speakers Bureau: Swedish Orphan Biovitrum, L. Joutsi-Korhonen Paid Instructor for: Labquality, Novo Nordisk. Keywords: anti-Xa assay, APTT, dabigatran, INR, laboratory monitoring, NOAC, rivaroxaban, thrombin time.

LSPE12 In vitro assessment of the applicability of prothrombin complex concentrate as antidote for the direct factor XA inhibitor rivaroxaban Leyte A*, Dinkelaar J and Molenaar PJ Hematology and Clinical Chemistry Laboratory, Onze Lieve Vrouwe Gasthuis, Amsterdam, The Netherlands Objectives: Rivaroxaban has been approved as antithrombotic agent for prevention of venous thrombo-embolism for specific indications. At present no antidote is appointed. In this in-vitro study, using ProThrombin Time (PT) and Endogenous Thrombin Potential (ETP) assays, we evaluated the influence of Prothrombin Complex Concentrate (PCC: Cofact) on the anticoagulant effects of Rivaroxaban. Methods: Blood samples from 9 healthy volunteers were spiked with Rivaroxaban up to profylactic levels (200–800 lg/L). PCC was added to these samples in concentration ranges as used clinically to reverse anticoagulant effects of vitamin K antagonists (VKA), and PT and ETP were measured (Innovinâ and ETP reagent on a BCS-XP analyzer, Siemens). Results: Addition of Rivaroxaban to the donor plasmas resulted in dose-dependent elongation of PT and ETP lagtime, and in decrease of ETP area under curve (AUC, thrombin generated). PT and lagtime could not be normalized by addition of PCC irrespective of the Rivaroxaban concentration, whereas AUC could be normalized in some combinations. Conclusion: In vitro, PCC in amounts regular for VKA reversal did not neutralize the Rivaroxaban effect on PT or ETP lagtime. Relatively high doses of PCC were needed to reverse the effect on total thrombin formation, which in vivo may translate to unfortunate overshoot of prothrombotic forces depending on respective clearance profiles of Rivaroxaban and the components of the PCC. Disclosure of Interest: None declared. Keywords: antidote, Prothrombin Complex Concentrate, rivaroxaban.

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ABSTRACTS

LSPE13 Validation of two age adapted d-dimer cut-off values for exclusion of deep venous thrombosis in suspected elderly primary care patients Schouten HJ1,2,*, Koek HL2, Oudega R1, Geersing GJ1, Janssen KJ1, van Delden JJ1 and Moons KG1 1 Julius Center for Health Sciences and Primary Care; 2 Department of Geriatrics, University Medical Center Utrecht, Utrecht, The Netherlands Objectives: With increasing age the D-dimer concentration increases and its specificity decreases. Age adapted D-Dimer cut-off values were proposed and validated in secondary care patients. Their use increases the number patients in whom venous thromboembolism could be safely excluded. We analysed whether the use of age adapted D-Dimer cut-off values can be translated to primary care patients suspected of deep venous thrombosis (DVT). Methods and Design: Cross sectional. Setting: One hundred and ten general practitioner practices in the Netherlands. Patient: Of 1374 consecutive patients with suspected DVT. Main Outcome Measures: The proportion of patients with D-Dimer values below two proposed age adapted cut-off levels (i.e. age 9 10 lg/L in patients aged > 50 years, or 750 lg/L in patients aged > 60 years) in whom DVT could be excluded, and the number of false negative results. Results: Using the Wells score, 647 patients had an unlikely clinical probability of DVT. In these patients, DVT could be excluded in 47.8% by using the age dependent cut-off value (age 9 10 lg/L in patients aged > 50 years) versus in 42.0% by using the conventional cut-off value of 500 lg/L (increase of 5.7%, 95% CI 4.1–7.8%). This resulted in respectively 0.5% and 0.3% false negative cases (increase of 0.2%, 95% CI 0.04–8.6%).The increase in exclusion rate by using the age dependent cut-off value was highest in the oldest patients. In patients aged >80 years, deep venous thrombosis could be safely excluded in 35.5% using the age dependent cut-off value compared to 21.0% using the conventional cut-off value (increase of 14.5%, 95% CI 6.8–25.8%). Compared to the age dependent cut-off value, the cutoff value of 750 lg/L had a comparable exclusion rate (47.4%, all ages) and false negative rate (0.3%). Conclusion: Combined with a low clinical probability, the use of age adapted D-Dimer cut-off levels resulted in a considerable increase in proportion of elderly patients in primary care in whom DVT could be safely excluded compared to the conventional cut-off value. Disclosure of Interest: None declared. Keywords: Age dependent D-dimer cut-off value, D-dimer, Deep venous thrombosis, Elderly.

LSPE14 The cost-effectiveness of ‘point of care’ D-dimer tests to rule out deep venous thrombosis in primary care Hendriksen J1,*, Geersing GJ1, van Voorthuizen S1, ten CateHoek A2, Joore M3, Moons K1 and Koffijberg E1 1 Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht; 2Departments of Clinical Epidemiology and Medical Technology Assessment and Internal Medicine; 3Departments of Epidemiology and General Practice, School of Public Health and Primary care (CAPHRI), Maastricht University Medical Center, Maastricht, The Netherlands Objectives: During recent years, several quantitative and qualitative point-of-care (POC) D-dimer tests have become available. These tests provide results within minutes and can help primary care physicians (PCPs) exclude deep venous thrombosis (DVT) during the consultation of the patient. Nevertheless, PCPs are hesitant to refrain from the

conventional laboratory tests as it is unclear which D-dimer test is best, that is, has the most favourable cost-effectiveness in ruling out DVT. The objective of this analysis is to identify the most cost-effective diagnostic work-up strategy for DVT in primary care using a POC or hospital based laboratory D-dimer test. Methods: We extended the previously developed Markov simulation model of the AMUSE study for the current analysis and used a 10-year time-horizon. Primary care diagnostic strategies to rule out or confirm DVT using a Simplify, Cardiac, Triage or Nycocard POC D-dimer test and hospital based strategies using a latex assay were compared. Results: The Simplify D-dimer resulted in 6.9661 QALYs at the cost of €8167 per patient. All other tests resulted in additional health benefits at additional costs. At the expense of an extra €72 (95%CI €-2 to €166), 0.0046 QALY (95% CI 0.0013 to 0.0122) can be gained by using the Cardiac strategy (ICER €15,500/QALY). Although the Triage strategy would result in even more health benefits than the Cardiac strategy (+0.0014 QALY [95% CI 0.0036 to 0.009]) the additional cost of € 99 per patient (95% CI €33–€193) results in an unfavourable ICER of €71,226/QALY. Conclusion: Applying a POC D-dimer test in primary care (most notably the Cardiac test) is a cost-effective strategy compared to regular laboratory test strategies. Therefore, PCPs should be encouraged to use these POC tests as an attractive and cost-effective alternative to labour-intensive and time-consuming referral of the patient for a hospital based laboratory test. Disclosure of Interest: None declared. Keywords: Cost-effectiveness, D-dimer, Point-of-care devices, primary care.

LSPE15 Fibrin-related marker comparison in sepsis: correlation to global and molecular parameters of haemostatic dysfunction Toh JMH1,*, Ken-Dror G2 and Downey C3 1 Liverpool College; 2Biostatistics, University of Liverpool; 3 Haematology, Royal Liverpool University Hospital, Liverpool, UK Objectives: Sepsis is associated with an increase in intravascular fibrin formation. Its detection is through fibrin-related markers (FRM) of which D-Dimer (DDI) and fibrin degradation products (FDP) are the most commonly used. However, soluble fibrin monomer (FM) has been considered a better FRM as it is an early precursor of fibrin. Methods: With the aim of examining this hypothesis, FDP, DDI and FM (Stago, France) assays were used on 105 plasma samples from patients with sepsis, systemic inflammatory response syndrome (SIRS) and healthy individuals. They were correlated with each other and also with antithrombin (ATIII, Stago) as a molecular indicator of thrombin activity and fibrinogen (FIB), prothrombin time (PT) and platelets (PLT) as indicators and global parameters used in identifying disseminated intravascular coagulation (DIC). Results: Pearson correlation coefficient showed strong correlation between the 3 FRM (r = 0.59–0.98) and that increases in all three FRM correlated significantly to reduced AT levels (P < 0.005). The only FRM to correlate significantly with PT, FIB and PLT was FM. DDI and FDP correlated to PT only. In clinical terms, all three FRM significantly discriminated patients with sepsis (Mean in lg/mL: 26.05 for FDP, 16.59 for FM, 6.80 for DD) from normals (1.42, 4.54 and 0.29, respectively) but not from SIRS. In 27 patients with sepsis who had serial samples from three consecutive days, all three FRM values showed reducing or increasing trends in terms of survival or death, respectively but neither analysis of variance for continuous variables (ANOVA) nor non-parametric tests, due to small sample size, showed statistical significance. Conclusion: In summary, this study in evaluating three FRM showed general equivalence as markers of intravascular fibrin formation with FM performing better in relation to the global haemostatic tests used


ABSTRACTS in DIC scoring. FM could be more appropriate than D-dimer as the FRM to measure in the DIC scoring system. Disclosure of Interest: None declared. Keywords: Antithrombin, D-dimer, DIC, fdp, fibrinogen, fibrinolytic parameters, Sepsis.

LSPE16 Large inter-individual variation of the pharmacodynamic effect of anticoagulant drugs on thrombin generation Bloemen S*, Hemker CH and Al Dieri R Synapse BV, CARIM, Maastricht, The Netherlands Objectives: Anticoagulation by standard dosage of an inhibitor of thrombin generation presupposes predictable pharmacokinetics and pharmacodynamics of the anticoagulant. We tested the latter by adding a fixed concentration, around IC50, to a series of 44 plasmas from apparently healthy donors and determining their effect in vitro on thrombin generation. Methods: Thrombin generation was determined by the calibrated automated thrombinography (CAT) in platelet poor plasma of 44 apparently healthy subjects spiked with around IC50 of otamixaban, melagatran, unfractionated heparin, dermatan sulfate and pentasaccharide. Results: The inter-individual coefficient of variation (CV) of the endogenous thrombin potential (ETP) and peak height before inhibition were 18% and 16%, respectively and became 20–24% and 24– 43% after inhibition. Conclusion: For any type of anticoagulant tested, the anticoagulant response to a fixed concentration of antithrombotic is highly variable from one individual to another. Direct inhibitors of thrombin and factor Xa are not different from heparin (-likes) in this respect. Dose adaptation, therefore, is likely to improve the treatment with any of these anticoagulants. Disclosure of Interest: None declared. Keywords: anticoagulants, antithrombotics, inter-individual variation, thrombin generation.

LSPE17 Laboratory recommendations for the monitoring of rivaroxaban Douxfils J1,2,*, Mullier F1,2,3, Chatelain C2,4, Robert S1, e J-M1,2 Chatelain B2,3 and Dogn 1 Pharmacy, University of Namur; 2Namur Thrombosis and Hemostasis Center (NTHC), Namur; 3Hematology Laboratory, 4 Hematology, CHU Mont-Godinne, Yvoir, Belgium Objectives: Rivaroxaban does theoretically not require monitoring but it may be useful to prevent thrombosis or bleedings in some specific clinical settings. The aim of the study was to determine which coagulation assay could be used to assess the impact of rivaroxaban on secondary haemostasis. Methods: Rivaroxaban was spiked at concentration ranging from 11 to 1090 ng/mL in platelet-poor plasma. The following routinely used or more specific coagulation assays were performed: activated partial thromboplastin time (aPTT); prothrombin time (PT); dilute prothombin time (dPT); prothrombinase-induced clotting time (PiCT); ecarin clotting time (ECT); reptilase time (RT); Biophen direct Xa inhibitor (Biophen DiXaI); Liquid anti-Xa (LAX) and thrombin generation assay (TGA). Results: A concentration dependent prolongation of aPTT, PT, dPT, PiCT was observed. PT and dPT were the most sensitive chronometric assays but results varied depending on the reagent. dPT slightly reduce the sensitivity but it also depends on the reagent. For PT, Triniclot PT


55

Excel S was the most sensitive reagent while for dPT it was Recombiplastin diluted 1/64. Biophen DiXaI and LAX showed a concentration dependent decrease in OD/min and the highest sensitivity but are not available 24 h a day. Both test showed a linear correlation but Biophen DiXaI may be used on a wider range of concentration. Moreover, Biophen DiXaI is insensitive to heparins and fondaparinux. In TGA, Cmax was the most sensitive parameter with the tissue factor induced pathway. Conclusion: PT can be used as screening test to assess the risk of bleedings. Nevertheless, a more specific and sensitive assay such as Biophen DiXaI using calibrators should be used to determine correctly the concentration of rivaroxaban in plasma. In addition, cut-offs associated with the risk of bleedings should be defined. Standardization of the time between the last intake of dabigatran and the time of blood collection is mandatory. Disclosure of Interest: None declared. Keywords: anti-Xa assay, bleeding, monitoring, prothrombin time, rivaroxaban, thrombosis.

LSPE18 Laboratory recommendations for the monitoring of dabigatran Douxfils J1,2,*, Mullier F1,2,3, Chatelain C2,4, Robert S1, e J-M1,2 Chatelain B2,3 and Dogn 1 Pharmacy, University of Namur; 2Namur Thrombosis and Hemostasis Center (NTHC), Namur; 3Hematology Laboratory; 4 Hematology, CHU Mont-Godinne, Yvoir, Belgium Objectives: Due to low bioavailability and inter-individual variability, monitoring of dabigatran may be required in specific situations to prevent the risk of bleeding or thrombosis. The aim of the study was to determine which coagulation assay(s) could be used to assess the impact of dabigatran on secondary haemostasis. Methods: Dabigatran was spiked at concentrations ranging from 5 to 943 ng/mL in pooled citrated human platelet-poor plasma. The following clotting assays were performed: Prothrombin Time (PT); activated Partial Thromboplastin Time (aPTT); Thrombin Time (TT); Ecarin Clotting Time (ECT); Ecarin Chromogenic Assay (ECA); Prothrombinase-induced Clotting Time (PiCT); Activated Clotting Time (ACT); Hemoclot Thrombin Inhibitor (HTI) and Thrombin Generation assay (TGA). Results: A concentration-dependent prolongation of PT, dPT, and aPTT was observed while aPTT was the most sensitive. The results varied mostly due to the clotting reagent. HTI, ECT and TGA were the most sensitive tests but are not available 24 h a day..Dabigatran induced a concentration-dependent delay and inhibition of tissue factor-induced TGA. Cut-offs related with higher risk of bleeding were defined for aPTT and HTI. For aPTT, cut-off values for each reagent were proposed taking into account the inter-reagent variability. Nevertheless, for one particular reagent, aPTT varies according to the lot number. Consequently, each lab should calibrate each lot of aPTT reagent on each coagulometer. Conclusion: aPTT could be used for the monitoring of dabigatran after calibration and as screening test for the risk of overdose but specific cut-off for each reagent must be used. However, because of its higher sensitivity, good reproducibility, excellent linear correlation, its simplicity of use and possibilities of automation, HTI should be considered as the gold-standard. Standardization of the time between the last intake of dabigatran and the time of blood collection is mandatory. Disclosure of Interest: None declared. Keywords: bleeding, dabigatran, hemoclot thrombin inhibitors, monitoring, thrombosis.

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ABSTRACTS

LSPE19 Comparison of prothrombinase-based apc resistance assay and FV Leiden genotyping Horvat I*, Herak DC and Zadro R Department of Laboratory Diagnostics, University Hospital Centre Zagreb, Zagreb, Croatia Objectives: Factor V Leiden (FV Leiden) is one of the most common thrombofilic risk factors in general population. Nowdays, when laboratory management is under strong pressure to reduce the number and costs of analysis, the goal is to find out a reliable functional assay that can serve as first-line screening for FV Leiden. The Pefakit APC-R Factor V Leiden (Pentapharm Ltd, Basel, Switzerland) is a functional assay designed to detect activated protein C resistance phenotype (APCR) mostly due to FV Leiden. In contrast to more commonly used APTT-based assays, this is a prothrombinase-based assay, exclusively measuring the susceptibility of plasma FVa to APC-mediated inactivation. The aim of this study was to evaluate the analytical performance of the Pefakit APC-R Factor V Leiden assay in order to find out the sensitivity and specificity of the assay in comparison to DNA analysis. Methods: We analyzed 158 patient plasma samples reffered to our laboratory for thrombophilia work-up. By DNA analysis we identified 100 non-carriers, 54 heterozygous and 4 homozygous patients for FV Leiden. The original manufacturer APCR assay was adapted for the Behring Coagulation System analyzer (Siemens Medical Solutions Diagnostics, Germany) and results were expressed as APC ratios. Results: The obtained mean APCR ratios were: 4.04 (range 2.19–5.70) for FV Leiden non-carriers and 1.59 (range 1.30–2.00) for heterozygotes. The same APC ratio of 1.00 was obtained for all four homozygotes. The analyzer and assay specific cut-off value of 2.00, obtained by ROC curve analysis, provided 100% sensitivity and 100% specificity, with clear discrimination between non-carriers, heterozygotes and homozygotes. Conclusion: In conclusion, Pefakit APC-R Factor V Leiden assay showed an excellent performance in reliable and accurate distinguishing between FV Leiden carriers and non-carriers. Disclosure of Interest: None declared. Keywords: Factor V Leiden, APC resistance, coagulation assay.

LSPE20 Evaluation of Pro C global assay as a screening test for thrombophilia in women with adverse pregnancy outcome Naz A1,*, Hossain N2, Shamsi TS3 and Khan N4 1 Thrombosis and hemostasis, National Institute of Blood Diseases; 2Gynae and Obstetric, Dow University of Health Sciences; 3Haematology, National Institute of Blood Diseases; 4 Statistcs, Dow University of Health Sciences, Karachi, Pakistan Objectives: To determine if Pro C Global assay can be used as a screening test for Thrombophilia. Methods: Case Control Study. It was conducted from June 2008 to August 2011 at Department of Obstetrics & Gynecology Unit 3 Dow University of Health Sciences and National Institute of Blood Disease. Cases were women with adverse pregnancy outcome. Adverse pregnancy outcome included women with intrauterine demise, or pregnancy losses. Controls were women without any adverse pregnancy outcome. Pro C Global assay, Free Protien S, Protien C, Antithrombin III, Factor V lieden and PTTLA were performed on both groups. If result of PTTLA were > 37 s, lupus anticoagulant and anticardiolipin antibodies were carried out. Simple descriptive, chi square test, Univariate Odds ratio and Kappa statistics were performed for data analysis by using SPSS version 17.0. Results: A total of 372 tests were done out of 384 women. This included 269 controls and 113 cases. The interrater reliability for the

raters (Pro C Global &Protein S) was found to be j = 0.048 (P < 0.073) The interrater reliability for the raters (Pro C Global & Protein C) was found to be j = 0.019 (P < 0.619) The interrater reliability for the raters (Pro C Global & FV Leiden) was found to be j = 0.242 (P < 0.001) The interrater reliability for the raters (Pro C Global & Antithrombin III) was found to be j = 0.019 (P < 0.619). Conclusion: Pro C Global assay is not a good screening test for thrombophilia. Disclosure of Interest: A. Naz Grant / Research support from: Higher Education Commission, N. Hossain: None Declared, T. Shamsi: None Declared, N. Khan: None Declared. Keywords: Thrombophilia, Pro C Global, adverse pregnancy outcome, Pakistan.

LSPE21 Thrombodynamics assay in healthy and diseased subjects Balandina AN1,2,*, Lipets EN3, Soshitova NP3, Polokhov DM4, Vasil’ev SA3,5,6,7, Panteleev MA1,3,4,7 and Ataullakhanov FI1,3,4,7,8 1 Molecular Hemostasis, Center for Theoretical Problems of Physicochemical Pharmacology; 2Biochemistry, Centre of Pediatric Hematology, Oncology and Immunology; 3Research and Development, HemaCore LL; 4Physical Biochemistry; 5 Theoretical Coagulologic; 6Molecular Hemostasis, National Research Center for Hematology; 7Physics, Moscow State Univeristy; 8Biochemistry, Centre of Pediatric Hematology, Oncology and Immunology, Moscow, Russian Federation Objectives: We developed a novel in vitro assay for coagulation diagnostics, called thrombodynamics, that takes into account spatially heterogeneity of blood clotting. Here, we attempted to evaluate effects of normal-, hypo- and hypercoagulation plasma samples on thrombodynamics. Methods: In thrombodynamics fibrin clot formation in a thin layer of nonstirred platelet free plasma where clotting was initiated by a surface with immobilized tissue factor and monitored using videomicroscopy. 37 healthy subjects, 10 patients with coronary heart disease undergoing the heparin therapy and 48 patients with Hodgkin’s disease undergoing the polychemotherapy were included in the study. Patients represented the models of hypo- and hypercoagulation conditions, respectively, which was conformed by activated partial thromboplastin time (2–2.5 and 0.7 from norm respectively). Results: We monitored the following characteristics: clot initiation (Tlag, time of clot formation near the TF surface), propagation (Vst, rate of fibrin clot propagation) and plasma procoagulant potential (SC, spontaneous clotting outside the clot growth region, and Vst). Tlag does not differ (P = 0.05) between these groups of subjects: means SD were 0.8 0.2 min (in healthy subjects), 1.0 0.3 min (in hypocoagulation) and 0.8 0.4 min (in hypercoagulation). The Vst differed between the groups (P < 0.05): 24 3 lm/min (in healthy subjects), 5.5 1.8 lm/min min (in hypocoagulation case) and 30 9 lm/min (in hypercoagulation case). No SC was observed in normal and hypocoagulation cases, but SC was present in 40% of subjects with hypercoagulation. Conclusion: The most sensitive parameters of thrombodynamics to hypo- and hypercoagulation conditions are Vst and SC. The data indicate that this spatially heterogeneous experimental approach has a potential for coagulation diagnostics. Support: RFBR grants 10-01-91055, 11-04-00303, 11-04-12080. Disclosure of Interest: A. Balandina Grant / Research support from: HemaCore LLC, E. Lipets Employee of: HemaCore LLC, N. Soshitova Employee of: HemaCore LLC, D. Polokhov Grant / Research support from: HemaCore LLC, S. Vasil’ev Employee of: HemaCore LLC, M. Panteleev: None Declared, F. Ataullakhanov Employee of: HemaCore LLC. Keywords: in vitro diagnostics, thrombodynamics. © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

ABSTRACTS LSPE22 Characterization of the role of cancer cells in the promotion of thrombus formation under shear Baker SM*, McCarty OJ, Berny-Lang MA and Tormoen GW Biomedical Engineering, Oregon Health & Science University, Portland, OR, USA Objectives: The routine observation of tumor emboli in the peripheral blood of patients with carcinomas raises questions about the clinical relevance of these circulating tumor cells. Thrombosis is a common clinical manifestation of cancer and circulating tumor cells may play a pathogenetic role in this process. The presence of coagulation-associated molecules on cancer cells has been described, but the mechanisms by which circulating tumor cells augment or alter coagulation remains unclear. In this study we utilized suspensions of a metastatic adenocarcinoma cell line, MDA-MB-231, as a models of circulating tumor cells to determine the thromobogenic activity of these blood-foreign cells. Methods: Experiments were performed to characterize the procoagulant phenotype of MDA-MB-231 cells and the effects of MDA-MB231 cells on the kinetics of clot formation in both open and closed systems. Results: In human plasma, MDA-MB-231 cells significantly enhanced clotting kinetics. The effect of MDA-MB-231 cells on clotting times was cell number-dependent and inhibited by a neutralizing antibody to tissue factor (TF) as well as inhibitors of activated factor X and thrombin. Using fluorescence microscopy, we found that MDA-MB-231 cells supported the binding of fluorescently-labeled thrombin. MDAMB-231 cells were shown to interact with platelets and fibrin-rich clots in a shear dependent manner. In a model of thrombus formation under pressure-driven flow, MDA-MB-231 cells significantly decreased the time to occlusion. The procoagulant activity of MDA-MB-231 cells was found to correlate with spatial separation in both closed, wellmixed systems and open, flowing systems. Conclusion: Our findings indicate that the presence of breast cancer cells in blood can stimulate coagulation in a TF-dependent manner, suggesting that tumor cells that enter the circulation may promote the formation of occlusive thrombi under shear flow conditions. Disclosure of Interest: None declared. Keywords: cancer cells, coagulation, shear, thrombus formation.

LSPE23 Prolonged clot lysis time is a risk factor for venous thrombosis Karasu A1,*, van Hylckama Vlieg A1, Luddington R2, Baglin CA2, Rosendaal FR1,3 and Baglin TP2 1 Clinical Epidemiology, Leiden University Medical Center, Leiden, The Netherlands; 2Haematology, Addenbrooke’s Hospital, Cambridge, UK; 3Thrombosis and Haemostasis Research Center, Leiden University Medical Center, Leiden, The Netherlands Objectives: The risk of venous thrombosis is determined by one’s ability to form a clot, inhibit clot formation, and dissolve clots, i.e., by procoagulant, anticoagulant and fibrinolytic factors. Hypofibrinolysis is a decreased capacity to dissolve blood clots and can be identified by a prolonged clot lysis time (CLT). Our aim was to study the association between reduced fibrinolysis, as measured by a prolonged CLT, and the risk of a first venous thrombosis in a case-control study, i.e. THE-VTE study. Furthermore, we wanted to determine whether the association between CLT and venous thrombosis is explained by the endogenous thrombin potential (ETP). Methods: Between March 2004 and December 2008, consecutive patients with a first episode of deep venous thrombosis of the leg (DVT) or a pulmonary embolism (PE) from the anticoagulant service at Addenbrooke’s Hospital, Cambridge and the anticoagulation clinic in Leiden were invited to participate. All participants provided a blood © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

57

sample 2–3 months after discontinuation of anticoagulation. CLT and ETP were measured in 584 patients and 334 controls. The risk of venous thrombosis was assessed by calculating odds ratios (OR) with 95% confidence intervals after adjustment for age, sex, and study centre. Results: Hypofibrinolysis, i.e., CLT above the 90th percentile in controls (> 122 min), was associated with a 1.8-fold increased risk of a first VT (OR = 1.8, 95% CI: 1.2–2.7). The risk of venous thrombosis was positively associated with CLT, i.e., when analyzed in quartiles of CLT as measured in controls, those with a CLT in the highest quartile (> 105 min) compared with the lowest quartile (< 76 min) had the highest risk of VT (OR = 2.7, 95% CI: 1.8–4.0). After adjustment for the ETP, the risk estimates only marginally changed (OR > P90 vs. ≤ P90 = 1.6, 95% CI: 1.0–2.5). Conclusion: These results indicate that a prolonged CLT is a risk factor for venous thrombosis, which is not explained by endogenous thrombin generation. Disclosure of Interest: None declared. Keywords: clot lysis time, endogenous thrombin potential, Risk factor, Venous thromboembolism.

LSPE24 Lyophilized normal and abnormal lupus control plasma can be used for assay control in DRVV and APTT based lupus anticoagulant tests Wagner L*, Kaufmann V, Mager J, Geiter S and Leitner M Technoclone GmbH, Vienna, Austria Objectives: To rule out a lupus anticoagulant antibody according to actual guidelines two assays that are sensitive to these antibodies, a DRVV based and a PTT based one, must be negative. Lyophilized plasmas with assigned values for both DRVV and APTT based lupus tests are available to be used as positive and negative controls. The aim of this study was to evaluate the performance of these plasmas for use in DRVV and APTT based Lupus anticoagulant assays (LA). Methods: Technoclotâ LA Screen and Technoclotâ LA Confirm are simplified DRVV reagents for detection of LA in one-stage clotting tests. Lupus Anticoagulant Test is a modified aPTT. In the test an activator of SiO2/AI2O3 suspension and two phospholipid concentrations is used. Lupus Inhibitor Plasma and Lupus Inhibitor Plasma Low are lyophilized plasma are positive controls, prepared from selected lupus inhibitor plasma strongly and low positive in most Lupus Inhibitor screening and confirmatory assays. The Platelet Poor Plasma is prepared from normal plasma, with platelet content reduced to a level where it does not influence the Lupus tests. The assays were performed automated using the Ceveronâ alpha, BCS and AMAX coagulation analyzer. Results: Evaluated parameters were LA screen and LA confirm clotting times, ratio LA screen/LA confirm, normalized ratio LA screen/ LA confirm and LCA index. Recovery of target values for all controls, tests and analyzers were in the range 100 10%. Inter-assay CVs were between 1.59 and 5.32%. Conclusion: This study shows that with all control plasma and the tested reagents reliable and reproducible results were obtained. Results between used analyzers do not differ more than 5.32%. Normal and abnormal controls are recommended for a complete quality control program when patient samples are tested for lupus anticoagulant. The same controls can be used for DRVV and APTT based tests. Normal control can be used for normalization of ratio results. Disclosure of Interest: L. Wagner: None Declared, V. Kaufmann Employee of: Technoclone GmbH, J. Mager Employee of: Technoclone GmbH, S. Geiter Employee of: Technoclone GmbH, M. Leitner Employee of: Technoclone GmbH. Keywords: APTT silica based lupus anticoagulant assay, DRVVT assay, LCA index, Lupus anticoagulant negative control, Lupus anticoagulant positive control, lupus anticoagulant test.

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ABSTRACTS

LSPE25 von Willebrand factor level in newly diagnosed patient with multiple myeloma after the first cycles of induction therapy Hait E1,*, Abdulkadyrov K2, Papayan L1 and Golovina O1 1 Laboratory of Blood Coagulation; 2Haematology Department, Russian Research Institute of Haematology and Transfusiology, Saint-Petersburg, Russian Federation Objectives: Patients with multiple myeloma (MM) have an increased risk of thromboembolic complications. The special therapy causes the activation of endothelium in myeloma patients. Von Willebrand factor (VWF) is a marker of endothelial activation and it’s increased level may be a sign of hypercoagulation and risk factor of cardiovascular disease. Aim: To investigate VWF plasma level (VWF:Ag) and ristocetin- cofactor activity (VWF: RCo) before and after first cycles therapy in patients with newly diagnosed MM. Methods: We investigated platelet–poor plasma from 25 patients with newly diagnosed MM at the age 51 till 78 years; 13 (52%) persons had the primary thrombotic history and 12(48%) didn’t have thrombotic anamnesis. Control group comprised 82 age-, sex-matched healthy individuals. VWF:Ag were measured by ELISA (Technozym, VWF: Ag, ELISA, Austria). Measurements were made before and after first cycles of therapy. Differences were evaluated with non-parametric statistical methods (Wilcoxon test, Statistica 6.0) and significance assumed for P < 0.05. Results: VWF: Ag and VWF: RCo plasma levels before and after start of therapy did not significantly differ (154% vs. 181% and 200% vs. 210%, P < 0.05, respectively) in the total group of patients. However, the measured values were distinct in subgroups of patients with and without thrombotic history (Table)

Patients

Without thrombotic history (n = 13)

With thrombotic history (n = 12)

Laboratory parameters

VWF:Ag (%) Me

VWF:RCo (%) Me

VWF:Ag (%) Me

VWF:RCo (%) Me

Before therapy After therapy

145 210*,**

136 190*,**

300* 210*,**

230* 120**

*P < 0.05 for difference with controls; **P < 0.05 for difference with values before therapy. Conclusion: VWF plasma level is markedly alter after the first cycle of induction therapy. VWF:Ag and VWF:RCo plasma levels are bring down and heighten in patients with primary thrombotic history or without it, respectively. High levels of VWF:Ag give evidence of endothelial activation and may be one of the reasons of thrombotic complication in MM patients. Disclosure of Interest: None declared. Keywords: multiple myeloma, von willebrand factor.

LSPE26 Unfractionated heparin (UFH) and low molecular weight heparins (LMWH) counteract thrombin generation (TG) and proangiogenic properties of MDA.MB.231 breast cancer cells Diani E*, Marchetti M, Vignoli A and Falanga A Division of Immunohematology and Transfusion Medicine, Department of Oncology-Hematology, Ospedali Riuniti di Bergamo, Bergamo, Italy Objectives: Tumor cell procoagulant activity is involved in both thrombotic diathesis and tumor progression in cancer. Therefore, inhibiting this target might improve the malignant disease prognosis.

A beneficial effect of LMWH on both thrombosis rate and survival in cancer patients has been shown. We evaluated whether the procoagulant and proangiogenic potential of MDA.MB.231 cells may be affected by two LMWH (dalteparin and bemiparin) or UFH. Methods: The capacity of heparins (from 0.01 to 1 IU/mL) to inhibit the TG induced by breast cancer cells was assessed by using the Calibrated Automated Thrombogram (CAT) assay. In addition, the antiangiogenic activity was evaluated as the capacity of heparins to inhibit endothelial capillary tube formation induced by MDA.MB.231 conditioned medium in a Matrigel-based assay. Results: The results show that heparins significantly counteracted breast cancer cell-induced TG in a dose-dependent manner, being the endogenous thrombin potential (ETP) and peak height (Peak) the most affected parameters. At 0.4 IU/mL concentration, UFH completely inhibited TG, while dalteparin reduced by 70% and 80%, and bemiparin by 40% and 60%, ETP and Peak, respectively (P < 0.01). Capillary-tube formation induced by MDA.MB.231 was also dosedependently counteracted by heparins. At the concentration of 0.1 IU/ mL bemiparin determined a 100% inhibition (P < 0.05) of angiogenesis, followed by dalteparin (75%, P < 0.05) and UFH (35%, P = n.s.). Therefore, differently from what observed with the TG, the capacity to inhibit angiogenesis was greater for LMWH compared to UFH. Conclusion: This study demonstrates that, in the same experimental system, heparins, toghether with the antiangiogenic capacity, can effectively counteract the prothrombotic stimulus of tumor cells. Our data further support the possible use of LMWH as adjuvant therapy in cancer. Disclosure of Interest: None declared. Keywords: Angiogenesis, breast cancer, heparins, LMWH, procoagulant activity, thrombin generation.

LSPE27 The European action on anticoagulation (EAA) prothrombin time /international normalised ratio (PT/ INR) line – a rapid method of deriving local international sensitivity index (ISI) and mean normal PT (MNPT) for use with coagulometers Poller L1,*, Ibrahim S1 and Jespersen J2 1 Central Facility, European Action on Anticoagulation, Faculty of Life Sciences,University of Manchester, Manchester, UK; 2 Hospital of South West Denmark, University of Southern Denmark, Esbjerg, Denmark Objectives: The PT/INR Line provides local INR without ISI or MNPT (Clin Chem 2010;56:1608–17, J Thromb Haemost 2011;9:140– 8). ISI are required for INR using automated systems. The PT/INR Line is obtained using a set of 5 ECAA lyophilised plasmas, ISI and MNPT determined and incorporated into automated test systems allowing easier INR derivation. Methods: PT/INR Line ISI and MNPT were determined from 56 calibrations at 28 centres using 42 human, 8 bovine and 6 rabbit reagents, with certified INR of the 5 ECAA plasmas plotted against local PT. ISI and MNPT from slope and intercept estimates [ISI = 1/slope, MNPT = e(intercept)]. ISI were compared with local calibrations (ECAA FDA-approved method). Local INR were also obtained for a validation plasma from ISI calibrations and the PT/INR Line. Results: Mean INR for the 5 ECAA plasmas was for human 2.41, bovine 2.04 and rabbit thromboplastins 2.80. Table shows ISI and MNPT results with three different reagent species, with human, mean local ISI of 0.93 was significantly different compared to the mean PT/ INR Line derived ISI of 0.99 [P < 0.001]. Differences between local manual calibration ISI and PT/INR derived ISI with rabbit and bovine reagents was not significant. Table ISI/MNPT with mean INR of the validation plasma (vp) with the 3 reagent species before and after with local ISI calibrations and the PT/INR Line © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

ABSTRACTS Manufacturer’s values

Local ISI calibrations

PT/INR Line

Difference between local and PT/INR Line - ISI

59

Reagent species

N

ISI/MNPT

VP INR (SD)

ISI/MNPT

VP INR

ISI/MNPT

VP INR

Paired t-test

Human Rabbit Bovine

42 6 8

0.94/10.9 1.22/12.8 1.02/37.7

2.74 (0.33) 2.85 (0.23) 2.30 (0.13)

0.93/11.8 1.44/13.2 1.11/37.9

2.47 (0.13) 2.83 (0.19) 2.10 (0.08)

0.99/12.7 1.40/13.0 1.19/39.0

2.45 (0.12) 2.80 (0.23) 2.13 (0.08)

P < 0.001 P = 0.46 P = 0.16

Conclusion: The PT/INR Line gave small differences in ISI compared with human reagent’s local calibrations. The PT/INR Line ISI combined with its MNPT gave good overall INR agreement with conventional ISI calibration over the range of coagulometers and reagents. Disclosure of Interest: None declared. Keywords: PT/INR Line, coagulometers, thromboplastins, ISI, INR deviation.

LSPE28 European action on anticoagulation (EAA). local INR derivation simplified by the PT/INR line and a spreadsheet from the world wide web Poller L1,*, Ibrahim S1 and Jespersen J2 1 Central Facility, European Action on Anticoagulation, Faculty of Life Sciences,University of Manchester, Manchester, UK; 2 Hospital of South West Denmark, University of Southern Denmark, Esbjerg, Denmark Objectives: WHO Prothrombin Time (PT) standardisation based on INR and WHO reference thromboplastins provides a good basis for ensuring the safety of vitamin K-antagonists but the complexity of its demands aggravated by the universal replacement of the manual PT technique by coagulometers has made implementation difficult. Individual coagulometers require International Sensitivity Index (ISI) calibration as they have considerable effects on INR even between instruments of the same manufacture. Image/Graph:

Line) based on a set of only 5 certified European Concerted Action on Anticoagulation (ECAA) plasmas (Clin Chem 2010;56:1608–17, J Thromb Haemost 2011;9:140–8, Am J Clin Pathol 2011;135:732–40). To obtain the PT/INR Line, certified INRs are plotted against the local PT results of the set of 5 ECAA calibrant plasmas. Orthogonal regression analysis is required for conventional WHO INR derivation, the PT/INR Line using simpler linear regression analysis gives reliable INR (see figure). Results: INR derivation is now made simpler using a spreadsheet available to download online from the EAA Website (www.anticoagulants.co.uk) (J Clin Pathol 2011;64:930–2) without requiring (i) local ISI, (ii) MNPT, (iii) thromboplastin IRP, (iv) manual PT testing, (v) sets of 60 anticoagulated patients and 20 normal subjects tested at 3 different centres, (vi) orthogonal regression analysis and (vii) any mathematical calculation. Conclusion: The PT/INR Line is a simple method using only 5 certified ECAA plasmas to derive INR. It does not require manual PT testing, an international sensitivity index (ISI) or a mean normal prothrombin time (MNPT). It can be used in place of local ISI calibration and has been shown to give reliable INR (Clin Chem 2010;56:1608–17). Application is easier with the assistance of the spreadsheet. Disclosure of Interest: None declared. Keywords: PT/INR Line, coagulometers, thromboplastins, ISI, INR deviation.

LSPE29 Characterization of thrombin generation (TG) by the calibrated automated thrombography (CAT) in atrial fibrillation (AF) patients on permanent oral anticoagulation therapy (OAT) with warfarin Marchetti M*, Giaccherini C, Lerede T, Maggioni A, Cefis M and Falanga A Immunohematology and Transfusion Medicine, Ospedali Riuniti Di Bergamo, Bergamo, Italy

Methods: In a series of recent reports the EAA has demonstrated the reliability of a simplified INR derivation method using the newly introduced prothrombin time/international normalised ratio (PT/INR © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

Objectives: In this work we utilized the CAT assay to characterize the TG potential in patients with AF. The correlation between TG and PT-INR was investigated to understand whether TG can be used to identify those subjects at higher risk of thrombo-hemorrhagic events. Methods: Ninety consecutive AF patients (34F/56M; mean age: 73 9 years) were recruited at the Bergamo Hospital OAT center. Seventy-seven patients had a target PT-INR within 2–3 range, and 13 within 3–4. CAT assay was performed in platelet-poor plasma at 5 pM TF and 4 lM phospholipids in absence and presence of activated protein C (APC); the normalized APC sensitivity ratio (nAPCsr) was calculated. Patients were followed-up for an average time of 2 years. Results: Compared to controls, AF patients have significant (P < 0.01) decreased ETP (1229 114 vs. 435 190 nM*min) and peak (256 44 vs. 109 51 nM), and prolonged lag-time (2.0 0.3 vs. 4.1 1.5 min) and ttPeak (4.6 0.6 vs. 6.2 1.7 min). INR significantly correlates with all TG parameters (r2 = 0.6), also after correction for sex and age. Interestingly, patients with similar INR showed significant variability in TG, particularly those in 2–3 INR range. APC significantly inhibits ETP in patients and controls, however the

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ABSTRACTS

nAPCsr of patients was significantly lower (0.6 0.3 vs. 1.05 0.63).During follow-up, 2 bleeding events (gastric and cerebral hemorrhage) occurred in 2 patients, despite being in INR range. Interestingly, one of them (INR = 2.54) presented with a very low ETP value (184 nM*min) and nAPCr. No thrombotic events were registered. Conclusion: Patients on warfarin have a significant decrease in TG, which partially correlates with INR. The wide variability of TG in subjects with similar INR values suggests a higher sensitivity of CAT in detecting hemostatic abnormalities. Finally, the low nAPCsr indicates that these patients are also more sensitive to APC anticoagulant activity. Studies are necessary to investigate the ability of TG to predict complications in patients on OAT. Disclosure of Interest: None declared. Keywords: atrial fibrillation, Oral Anticoagulants, prothrombin time, thrombin generation.

LSPE30 The effect of preanalytical and analytical variables on spatial fibrin clot formation (the thrombodynamics assay) in healthy subjects Balandina AN1,2,*, Lipets EN2,3, Soshitova NP3, Tarandovskiy ID1, Shcherbina IA2,4, Polokhov DM4, Poletaev AV4, Panteleev MA1,4,5,6 and Ataullakhanov FI1,2,4,5,6 1 Molecular Hemostasis, Center for Theoretical Problems of Physicochemical Pharmacology; 2Biochemistry, Centre of Pediatric Hematology, Oncology and Immunology; 3Molecular Hemostasis, HemaCore LLC; 4Physical Biochemistry, National Research Center for Hematology; 5Research and development, HemaCore LLC; 6Physics, Moscow State Univeristy, Moscow, Russian Federation Objectives: Blood coagulation in vivo is a spatially heterogeneous process, where clot grows from the site of activation towards the bulk of intact blood. Methods: Thrombodynamics is based on fibrin clot formation in a thin layer of nonstirred plasma where clotting is initiated by a surface with immobilized tissue factor (TF) and monitored using videomicroscopy. We monitored the following characteristics: clot initiation (Tlag, time of clot formation near the TF surface), propagation (Vst, rate of fibrin clot propagation) and plasma procoagulant potential (SC, spontaneous clotting outside the clot growth region, and Vst). Results: A total of 37 healthy subjects (15 male, 22 female, aged 33 12 [19–60]) were examined. All parameters followed the Gaussian distribution and the means SD were: 0.8 0.2 min (Tlag), 24 3 lm/min (Vst) and no SC was observed (for the further characterization only Vst was presented). There was no sex difference (24 2 lm/min for male vs. 25 3 lm/min for female, P = 0.05) or age difference (24 3 lm/min for 20–30 aged vs. 24 3 lm/min for 40–60 aged, P = 0.05). There was difference between platelet poor plasma (PPP, one centrifugation at 1600 g for 15 min) or platelet free plasma (PFP, PPP centrifugation at 10,000 g for 5 min): 23 4 lm/ min and 28 5 lm/min respectively (P < 0.05, n = 8). No difference in blood sampling type was registered: 25 5 lm/min for vacuum tube with citrate and 26 4 lm/min for self-flowing to the citratecontaining tube (P = 0.05, n = 5). The parameter reproducibility (3 independent sampling) was 23 2 lm/min (n = 3). Analytical error was 10% (n = 10). The time of parameters stability for fresh samples was 24 h (at 20 °C). No statistically valid seasonal variations were registered (21 2 lm/min in spring/summer vs. 22 3 lm/min in autumn/winter, P < 0.05, n = 5). Conclusion: Thrombodynamics should be used with PFP within 24 h after withdraw of blood, normal values are applicable for male and female of 20–60 years old. Support: RFBR grants 10-01-91055, 11-04-00303, 11-04-12080.

Disclosure of Interest: A. Balandina Grant / Research support from: HemaCore LLC, E. Lipets Employee of: HemaCore LLC, N. Soshitova Employee of: HemaCore LLC, I. Tarandovskiy Grant / Research support from: HemaCore LLC, I. Shcherbina Grant / Research support from: HemaCore LLC, D. Polokhov Grant / Research support from: HemaCore LLC, A. Poletaev Grant / Research support from: HemaCore LLC, M. Panteleev: None Declared, F. Ataullakhanov: None Declared. Keywords: spatial clot growth assay, thrombodynamics.

LSPE31 Assessment of haemostasis in the primary multiple myeloma patients using different laboratory methods Gracheva M1,2,*, Urnova ES3, Mendeleeva LP3, Sinauridze EI3, Balandina AN1,2, Lipets EN2,4, Gribkova IV3, Vasiliev SA3, Parovichnikova EN3, Savchenko VG3 and Ataullakhanov FI1,2,3,4,5 1 Center for Theoretical Problems of Physicochemical Pharmacology RAS; 2Center of Pediatric Hematology, Oncology and Immunology; 3National Research Center for Hematology; 4 HemaCore LLC; 5Moscow State University, Faculty of Physics, Moscow, Russian Federation Objectives: Patients with Multiple Myeloma (MM) have an increased risk of blood clotting disorders. The aim of this study was to compare different tests in the assessment of haemostasis. Methods: The study included 18 primary patients observed in the Department of Chemotherapy of Hemoblastoses and Bone Marrow Transplantation, Research Center for Hematology. The following methods were performed to assess the clotting parameters: (i) Activated Partial Thromboplastin Time (APTT), (ii) Prothrombin Index (PI), (iii) Endogenous Thrombin Potential (ETP), (iv) Thrombodynamics (a new method based on a spatial fibrin clot growth registration). Thrombodynamics is characterized by steady-state growth rate of clot formation (Vst) and spontaneous clots formation - the time of half-clotting of the whole experimental plasma volume (St), that normally absent. Results: PI was normal for all patients (97 17%). Thrombodynamics demonstrated hypercoagulation state in 10 patients (56%), Vst was 47 15 lm/min (the normal value is 24 3 lm/min), St was 22 8 min and only one patient had no spontaneous clots. Among this group ETP test showed hypercoagulation state in half of the cases – nine patients (area under the curve was 1500 200 nM*min whereas in norm 1000 200 nM*min), while APTT was shortened for three patients – 15% (27 2 s vs. 33 3 s in norm). Patients with normal and hypocoagulation in Thrombodynamics test had also the same indications for ETP test (ETP = 730 240 nM*min), but APTT had indicated hypercoagulation in two cases (11%) – 26 and 28 s. Conclusion: More than a third of patients with primary MM present hypercoagulation disorders in Thrombodynamics and endogenous thrombin potential tests. Standard APTT and PI tests registered hypercoagulation substantially in fewer cases. Support: RFBR grants 10-01-91055, 11-04-00303, 11-04-12080. Disclosure of Interest: M. Gracheva Grant / Research support from: HemaCore LLC, E. Urnova: None Declared, L. Mendeleeva: None Declared, E. Sinauridze: None Declared, A. Balandina Grant / Research support from: HemaCore LLC, E. Lipets Employee of: HemaCore LLC, I. Gribkova: None Declared, S. Vasiliev: None Declared, E. Parovichnikova: None Declared, V. Savchenko: None Declared, F. Ataullakhanov Employee of: HemaCore LLC. Keywords: Multiple Myeloma, Haemostasis, Thrombodynamics.


ABSTRACTS LSPE32 Inhibitory effects of bortezomib on platelet aggregation induced by ADP, collagen and ristocetin in patients with multiple myeloma ska E1, Zajdel K1, Nowicki A1, Rupa-Matysek J1,*, Gil L1, Wojtasin Dytfeld D1 and Komarnicki M1 1 Department of Haematology with Transplantation Unit, Poznan , Poland University of Medical Sciences, Poznan Objectives: Multiple myeloma (MM) therapy may affect prothrombotic and anticoagulant processes. Patients (pts) receiving thalidomidebased regimens are at increased risk of venous thromboembolism (VTE), while incidence of VTE with bortezomib-based regimens is very low. In vitro study indicates the reduction of adenosine diphosphate (ADP)-induced platelet aggregation by bortezomib. Aim: The aim of the study was analysis of the effect of bortezomib on platelet aggregation induced by agonists in pts with MM. Methods: A total of 26 pts with relapsed/refractory MM with median 57.5 (range 44–71) years, receiving bortezomib-based regimens were included in the study. Optical platelet aggregometry was performed in PRP (Chrono Log Corp) with agonists (2 lg/mL collagen, 5 and 3.5 lM ADP, 1.25 and 0.5 mg/mL ristocetin). Platelet aggregation was measured during two 21-day cycles on day 1 (pre-bortezomib), 4, 8 and 11. Results were analysed in two groups: treated with (n = 17) or without thalidomide (Thal, n = 9), using Friedman ANOVA with post-hoc testing. Results: Thrombotic complications were observed in 4 pts. In both groups, no statistically significant changes in mean platelet aggregation on day 1, 4, 8 and 11 were found during the 1st cycle, independent of Thal exposure. During the 2nd cycle in group without Thal, significant reduction in mean platelet aggregation were seen: 5 lM ADP (P = 0.0099, post-hoc day 1 vs. 8); 3.5 lM ADP (P = 0.0146, day 1 vs. 11), collagen (P = 0.0019, day 4 vs. 8), 1.25 mg/mL ristocetin (P = 0.0017, day 1 vs. 8 and 1 vs. 11), 0.5 mg/mL ristocetin (P = 0.0049, day 4 vs. 11). In group treated with Thal significant platelet aggregation inhibition by 0.5 mg/mL ristocetin (P = 0.002, day 4 vs. 8) and collagen (P = 0.033, day 1 vs. 4) were found. Transient reduction in platelet counts was observed in all pts, but statistically significant only in group without Thal. Conclusion: Low incidence of VTE in relapse/refractory MM pts may be explained by inhibitory effects of bortezomib prolonged exposure on platelet functions and counts. Disclosure of Interest: None declared. Keywords: platelet aggregation, bortezomib, multiple myeloma, thrombosis, ADP, collagen, ristocetin.

LSPE33 Comparison of the use of TFPI blockage and plasma dilution to increase the sensitivity of thrombin generation assay to measure the procoagulant activity of microvesicles Gheldof D*, Francßois M, Bernard C, Jean-Michel D and Christian C Hematology Laboratory-NARILIS CHU Mont-Godinne, Universit e Catholique de Louvain, Namur, Belgium Objectives: Patients with cancer have a 7–10- fold increased risk of developing venous thromboembolism (VTE). Circulating microvesicles (MVs) could be a predictive biomarker for VTE in cancer. Thrombin generation assay (TGA) is a useful technique to detect procoagulant activity (PCA) of MVs. However, TGA suffers from a lack of sensitivity due to the presence of Tissue Factor Pathway Inhibitor (TFPI) in


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plasma. The aim of this study is to improve the sensitivity of TGA to Tissue factor by limiting the interference of TFPI. Methods: Serial dilutions of MDA-MB231 cells were incubated for 45 min at 37 °C to generate MVs. Samples were then centrifuged or not and cells or the supernatants (Sup) were used for TGA assay. Normal pooled plasma was incubated with TFPI inhibitors or is diluted two times to decreased plasma level of TFPI. Lagtime is used as surrogate marker of TGA to detect PCA of MVs. Results: (i) Use of TFPI inhibitor decreased two times the cells concentration needed for a significant reduction of lagtime. Moreover, the use of TFPI inhibitor decreased 2.4-fold the intra-assay variability. (ii) The plasma dilution decreased two times cells concentration needed for a significant reduction of lagtime by cells. However, plasma dilution did not increase the sensitivity to MVs in the supernatant in comparison to undiluted plasma. Conclusion: TFPI inhibitor increased sensitivity to MVs PCA and decreased variability of TGA. Plasma dilution is less effective than the use of TFPI inhibitor to increase sensitivity to MVs TGA. Disclosure of Interest: None Declared. Keywords: cancer, microvesicles, Thrombosis.

LSPE34 Lupus anticoagulant screen and confirm reagents utilizing the FX activating venom from vipera lebetina Goldford MD* R2 Diagnostics, Inc., South Bend, USA Objectives: A number of snake venoms directly activate FX to FXa, independently of FVa, at the physiologic cleavage site Arg52-Ile53. When formulated in dilute venom, phospholipid-dependent reagents, FX activating venoms are the basis for the integrated LA screen and confirm assays. Although Russell’s Viper venom, in the form of the DRVVT, is the most well known venom assay for LA, availability of the venom can be limited. A FX activating venom from Vipera lebetina was used to create LA screen and confirm reagents. Methods: Detectin VL and Correctin VL are commercially available screen and confirm assays for LA in which Vipera lebetina venom provides the FX activating protease. During development and regulatory submissions these reagents were compared to commercially available Russell Viper venom reagents in studies that included method comparison studies and precision studies. Stago DRVV S and DRRV C were used as the comparator reagents, and data was collected on the STA Compact. Results: Excellent agreement of Detectin VL and Correctin VL was demonstrated with the Stago DRVV S and DRVV C screen and confirm assays. A comparison of the Normalized Ratio determined with both sets of reagents yielded a positive percent agreement of 98%, a negative percent agreement of 96%, and an overall percent agreement of 94%. Regression analysis of the raw clot times for Detectin VL compared to Stago DRVV S yielded a slope of 1.014, an intercept of 4.20, and an r2 of 0.923. For Correctin VL compared to Stago DRVV C the regression statistics were a slope of 1.002, an intercept of 5.23, and an r2 of 0.866. Precision estimates of the Detectin VL and Correctin VL clotting times were < 2% for repeatability and < 4% for within-device. Conclusion: Detectin VL and Correctin VL are FDA 510(k) cleared assays. Optimized as ‘drop-in’ replacements for Russell’s Viper reagents, Detectin VL and Correctin VL provide the clinical lab with an alternative source of reagents for its LA testing algorithm. Disclosure of Interest: M. Goldford Employee of: employee of r2 Diagnostics. Keywords: anti-phospholipid syndrome, LA, lupus, test, thrombosis.

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LSPE35 Correlation of peak thrombin results obtained by TGA measurement on Ceveronâ alpha tga using fast setting and standard setting Wagner L*, Kaufmann V, Mager J and Unzeitig G Technoclone GmbH, Vienna, Austria Objectives: In big studies it has been shown that measurement of thrombin generation expressing the results as Peak Thrombin may help identify patients with cancer at high risk of VTE1 as well as those at high risk of recurrence of VTE2. Manual measurement of thrombin generation is influenced by different factors, as the fluorescent reader used, the source of tissue factor and phospholipids in the trigger reagents and the technician performing the test. To enter in routine a standardized, quick and automated thrombin generation method is needed. Ceveronâ alpha allows the measurement of Peak Thrombin by thrombin generation (TGA) setting either stopping measurement after peak thrombin has been reached or registering the whole thrombin generation curve. Aim of the study was to evaluate the two settings for peak thrombin generation on Ceveronâalpha with the new fluorogenic module for determination of TGA in routine under standardized conditions. Methods: In the present study 2 settings of peak thrombin determination on Ceveron alpha for thrombin generation measurement were used. In routine setting (fast setting) the measurement stops after peak thrombin has been reached. In standard setting the whole thrombin generation curve is registered. The 2 settings were evaluated and performance characterized using Technothrombinâ TGA for Thrombophilia and the controls TGA Control High and TGA Control Low. Results: CVs of peak thrombin for TGA Control Low with Peak Thrombin in the low range (30–40 nM Thrombin) were found to be < 5%, with intra-assay CVs < 3% and interassay CV < 5%. CVs of peak thrombin for TGA Control High with Peak Thrombin in the range (200–300 nM Peak Thrombin) were found to be < 5% with intra-assay CV < 2.5% and interassay CV < 5%. Conclusion: Our results show that using the Ceveronâ alpha TGA peak thrombin measurement can be performed under standardized conditions, with very good precision and within 20 min using the routine settings. Disclosure of Interest: L. Wagner Employee of: Technoclone GmbH, V. Kaufmann Employee of: Technoclone GmbH, J. Mager Employee of: Technoclone GmbH, G. Unzeitig Employee of: Technoclone GmbH. Keywords: automated thrombin generation, routine thrombin generation, Standardized thrombin generation.

Methods: Blood samples were taken at different intervals (0.5; 1.0; 1.5; 2.0; 3.0; 4.0; 6.0 h) after a single dose at the instauration of the treatment and 6 days later. The following coagulation assays were performed: Prothrombin Time (PT) (Neoplastin CI+, Neoplastin R; Triniclot PT Excel S), Biophen Direct Xa Inhibitor and Liquid anti-Xa. Results: Results showed that the anticoagulant effect of rivaroxaban was not affected by a CYP3A4*1G polymorphism. Indeed, using calibrated PT or specific chromogenic assays, the rivaroxaban concentration was within the therapeutic range for AF (mean Cmax = 290 ng/ mL and mean Ctrough = 32 ng/mL). Conclusion: In conclusion, although rivaroxaban is metabolized by CYP3A4, CYP3A4*1G polymorphism is not associated with a decrease of plasmatic concentrations at normal therapeutic doses. Disclosure of Interest: None declared. Keywords: acenocoumarol, CYP3A4 polymorphism, monitoring, rivaroxaban, warfarin.

Platelet Basic Science PBS01 Discoid platelet aggregations without endothelium disruption visualized by in vivo molecular imaging, and contribution of inflammatory cytokines Nishimura S1,* and Nagasaki M1 1 Department of Cardiovascular Medicine, The University of Tokyo, Tokyo, Japan Objectives: To elucidate the underlying mechanisms of adult common diseases based on chronic inflammation, it is vital to examine the multi-cellular kinetics in living animals, and the thrombosis mechanism without endothelial cell (EC) disruption remains unclear. Methods: Therefore, we developed in vivo imaging technique based on single- and multi-photon microscopy, and we assessed dynamic cellular interplay in thrombosis models (Fig 1, 2008 JCI). We visualized that rapidly developing thrombi composed of discoid platelets without EC disruption was triggered by ROS (Fig 2). Image/Graph:

LSPE36 Impact of CYP3A4*1G Polymorphism on plasmatic concentration profile of rivaroxaban (Xareltoâ) assessed by coagulation assays Douxfils J1,2,*, Mullier F1,2,3, Chatelain C2,4, Chatelain B2,3 and Dogne J-M1,2 1 Pharmacy, University of Namur; 2Namur Thrombosis and Hemostasis Center NTHC, Namur; 3Hematology Laboratory; 4 Hematology, CHU Mont-Godinne, Yvoir, Belgium Objectives: A 52 years old woman suffering for atrial fibrillation (AF) was previously treated by acenocoumarol or warfarin to prevent stroke without success (INR < 2). A genetic analysis searching for polymorphism of CYP3A4, VKORC1, CYP2C9, F7, GGCX, CALU or EPHX1 was performed. The results showed a CYP3A4*1G polymorphism associated with a fast-metabolizer profile. Dabigatran was contraindicated due to a weight higher than 110 kg. Therefore, we proposed to switch the patient to rivaroxaban 20 mg od and monitor the plasmatic concentrations at different times by using different coagulation assays.

Results: Using this technique, we elucidated that Lnk (adapter protein) regulates integrin signaling leading to stabilization of developing thrombus (2010 JCI). In addition, we established the culture system of human iPS-derived platelets, and we confirmed their functional role in vivo (2010 JEM). In addition, we elucidated the contribution of inflammatory cytokines, ROS, and integrin signaling to our thrombosis models (2011 Blood). The inflammatory cytokines including TNF© 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

ABSTRACTS alpha and IL-1 could be key components of the EC response, acting through regulation of vWF mobilization to the cell surface (Fig 3). Thrombus formation was then initiated by the binding of platelet GPIb-alpha to endothelial vWF. Integrin activation was required for late phase thrombus stability. Conclusion: In sum, using our imaging system can be a powerful tool to analyze thrombus formation. We clarified the mechanism of discoid platelet aggregations on undisputed endothelium. The initial platelet aggregation subsequently leads to irreversible integrin- and actindependent thrombus development. Inflammatory cytokine signaling in ECs also played pivotal role. Disclosure of Interest: None declared. Keywords: In vivo imaging, Inflammatory Cytokines, Thrombosis.

PBS02 Rapid recruitment and microaggregate formation of human platelets by the colonizing bacterium streptococcus oralis under shear rates that mimic physiological conditions

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PBS03 Three dimensional reconstruction of intact human integrin alphaiibbeta3 in a phospholipid bilayer nanodisc Choi W-S1,*, Rice WJ2 and Coller BS1 1 Laboratory of Blood and Vascular Biology, The Rockefeller University; 2New York Structural Biology Center, New York, NY, USA Objectives: Integrin aIIbb3 is a hetrodimeric transmembrane signaling receptor that plays a central role in hemostasis and thrombosis. While an x-ray crystallographic structure of the ectodomain is available, there is no high resolution three-dimensional structure of full-length aIIbb3 in a lipid bilayer. Moreover, it is difficult to reconcile the existing crystal structure with the either x-ray and neutron density shapes obtained by small angle scattering techniques or the reconstructed electron cryomicroscopy image. Image/Graph:

Tilley D1,*, Douglas C2 and Kerrigan SW1 1 Molecular & Cellular Therapeutics and The School of Pharmacy, The Royal College of Surgeons in Ireland, Dublin, Ireland; 2 School of Dentistry, University of Sheffield, Sheffield, UK Objectives: Infective Endocarditis (IE) is considered to be the 4th leading cause of life threatening infectious disease and is associated with considerable morbidity and mortality. It is characterised by the formation of platelet-bacteria thrombi on heart valves which, if untreated, can lead to valve failure. The oral bacteria, Streptococcus oralis, is amongst the pathogens most commonly isolated from IE patients. Streptococcus oralis contribution to the growing platelet fibrin thrombus is critical for the development of IE; however, the mechanism of rapid platelet recruitment and activation is poorly understood. Methods: Whole blood was perfused over immobilized S. oralis at flow rates of 50 s (venous) and 800 s (arterial) in a parallel flow chamber. Experiments were recorded in real time and visualized using fluorescence microscopy. Results: The interaction between platelets and S. oralis led to the development of single platelet adhesion (50 s) or microaggregate formation (800 s). Depletion of plasma proteins failed to affect platelet adhesion at either flow rate (50 s control 46 13 platelets v plasma depleted 21 8 platelets, P = NS; 800 s control 254 57 vs. plasma depleted 299 29, P = NS). Preincubating platelets with abciximab, failed to have any effect on the interaction at either (50 s control 57 5 platelets v abciximab 53 42 platelets, P = NS; 800 s control 311 + 52 vs. abciximab 242 + 4, P = NS). Platelet binding to S. oralis led to the development of filopodial and lamellipodial extension with full platelet spreading. Preincubating platelets with abciximab prevented platelet spreading on S. oralis (control 30 11% platelets spread v abciximab 0.07 0.07% platelets spread, P < 0.05). Conclusion: These results suggest that platelets adhere directly to S. oralis under physiological conditions that mimic venous and arterial shear. This interaction results in the transmission of a signal that we propose strengthens the thrombus formation on heart valves of IE patients. Disclosure of Interest: None declared. Keywords: bacteria, infective endocarditis, platelet activation, platelet adhesion, spreading.


Methods: Intact aIIbb3 was purified from octyl glucoside-solubilized platelets and inserted into phospholipid nanodiscs. aIIbb3 nanodiscs were negatively stained and imaged by electron microscopy at 120 kV. The locations of the aIIb b-propeller and b3 PSI domain were identified using mAbs 10E5 and 7H2, respectively. Three-dimensional reconstruction of aIIbb3 in the nanodiscs was performed using the random conical tilt (RCT) method by collecting and correlating 12,504 pairs of images obtained without and with a 50° tilt. Results: The negative stain EM images we obtained differ from those of Ye et al. [J. Cell. Biol. 2010. 188(1)], who were the first to study aIIbb3 nanodiscs, in that we consistently find the aIIb b-propeller domain on the top of the image rather than pointed down toward the lipid bilayer. Three-dimensional reconstruction of the aIIbb3 random resolution shows both a less compact strucconical tilt images at 24 A

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ture than the crystal structure of the ectodomain and torsion of the leg domains. The RCT reconstruction fits the outlines of aIIbb3 from electron cryomicroscopy and both small angle x-ray and neutron scattering better than does the crystal structure. Conclusion: We provide a novel RCT-based 3-dimension reconstruction of aIIbb3 in a lipid bilayer in the absence of detergent. This reconstruction appears to be able to reconcile previous reconstructions of aIIbb3 using other techniques. Disclosure of Interest: None declared. Keywords: platelet, aIIbb3, nanodisc, electron microscopy.

PBS04 Platelet flow-induced protrusion (FLIPR) formation is a shear and calpain induced process leading to platelet disintegration and the formation of procoagulant microparticles Tersteeg C1,*, Heijnen H1, Pasterkamp G2, Floris C1, Urbanus RT1, Maas C1, Hoefer I2, Mak-Nienhuis EM2, De Angelis V1, De Groot PG1 and Roest M1 1 Clinical Chemistry and Hematology; 2Experimental Cardiology, UMC Utrecht, Utrecht, The Netherlands Objectives: Activated platelets are a major source of pro-coagulant and pro-inflammatory microparticles. Despite their importance in circulation, the formation of microparticles after platelet activation was only visualized under static conditions up till now. Methods: We studied the formation of microparticles under physiological conditions of flow in whole blood or platelet rich plasma from healthy donors. Perfusion chambers with stenosis-like obstructions were used to disturb the laminar flow profiles. Results: Long membrane strands of up to 250 mm were observed downstream of the stenosis, and these strands originate from spread platelets (further named “flow-induced protrusions” [FLIPR]). FLIPR formation was observed predominantly from disintegrating platelets that were spread on physiological surfaces including fibrinogen, fibronectin, vWF, collagen peptides and collagen. After 30 min of flow FLIPRs were formed from 3.1 1.1% of the platelets spread in areas with non-linear flow profiles. Similarly, aggregates formed on collagen at high shear show FLIPR formation directly downstream the aggregates, within 15 min of perfusion. Platelets that formed FLIPRs expressed anionic phospholipids, determined by Annexin-V and prothrombin binding. FLIPRs further disintegrate into procoagulant microparticles. Isolation of these disintegrated FLIPRs showed that they were able to accelerate thrombin generation in a thrombin generation assay. Destabilizing microtubules with nocodazole increased the FLIPR formation to 30.5 4.2%, while inhibiting actin polymerization using cytochalasin D showed no effect. FLIPR formation is depending on the presence of extracellular calcium and was completely blocked with the calpain inhibitor calpeptin. Conclusion: Our study introduces a novel mechanism of platelet microparticle formation from protruding platelets in a calpain and shear dependent process. Platelet disruption will lead to FLIPR formation and the formation of procoagulant microparticles. Disclosure of Interest: None declared. Keywords: microparticles, Platelets, shear

PBS05 Synergy of platelet receptors in thrombus formation: a microarray approach De Witt SM*, Lamers MM, Swieringa F, Kruchten R, Farndale RW, Clemetson KJ, Cosemans JMEM and Heemskerk JWM Biochemistry, Maastricht University, Maastricht, The Netherlands Objectives: The development of a high throughput perfusion assay, employing a microarray of platelet-adhesive matrix proteins, to deter-

mine which substrate-receptor interactions support the thrombusforming process. Methods: Arrays of matrix proteins and derived peptides were microspotted using a robot. Spots contained ligands for GPVI and/or a2b1, GPIb, CLEC-2, aIIbb3, CD36, avb3 or a6b1. Coating density of spots was evaluated by protein and immune staining. Whole blood was perfused over arrays at 1600/s. Phase contrast and multicolor confocal microscopic images were captured in real-time and after post-hoc staining. Using normalized scoring scales, fibrinogen-binding, P-selectin and PS expression, thrombus formation and size were assessed from recorded images. Results: No to single platelet adhesion occurred to surfaces containing ligands of CLEC-2, a2b1 CD36, aIIbb3 or avb3. Platelet adhesion,activation and thrombus size increased with surfaces of ligands of aIIbb3 < GPIb20 9 109/L. TEG showed shortened R and K times with an increased a angle when compared to normal whether or not the patient was septic. The MA was reduced in the majority but 29.2% had even normal MA despite PCs < 20 9 109/ L. 42.8% of these had a PC > 20 9 109/L by FC. Conclusion: Prophylactic platelet transfusions could potentially be reduced if one incorporates TEG and/or FC in more precisely assessing platelet counts and global coagulation even if current thresholds are kept the same. 1. British Journal of Haematology, 2003, 122, 10–23;2. AM J Clin Pathol Mar; 115(3): 460, 2001 Disclosure of Interest: None Declared. Keywords: platelet transfusion, TEG, flowcytometry.

PC15 Hemorrhagic risk due to platelet dysfunction in myelodysplatic patients. The correlations with anemia severity and iron overload Popov VM1,*, Vladareanu AM2, Bumbea H2, Begu M2, Iova AM2, Gaman M2, Popescu M3, Stocheci C4 and Kovacs E5 1 Hematology, County emergency Hospital, Pitesti; 2Hematology, Emergency University Hospital, Bucharest; 3Neurosurgery, County emergency Hospital, Pitesti; 4Emergecy Unit, County emergency Hospital, Pitesti; 5Byophisics, University of Medicine and Pharmacy, Bucharest, Romania Objectives: Patients with myelodysplastic syndrome (MDS) present hemorrhagic complications although they do not have severe thrombocytopenia. There are a few studies which have reported the decrease of platelet aggregation (Girtovitis FI et al, Acta Haematol. 2007; Mittelman M et al, Int J Hematol. 2000). Little is known about the anemia and effects of iron overload on platelet activation in these patients (Lynch S, Soslau G in Platelets 2011).

Aim: The evidence of any correlation between low platelet response and severity of anemia or iron overload. Methods: The retrospective study included 34 MDS patients classified according to FAB proposals and 29 healthy volunteers. All patients have hemorrhagic complications although their platelet count was above 100 000/mmc. Platelet function was analyzed by optical aggregometry using: ADP, collagen, ristocetin and epinephrine. The value of platelet aggregation variables (the curve amplitude, amplitude and duration of the ADP and collagen curve lag phase) were correlated with hemoglobin value (Hb) and the level of ferritin. Results: Platelet response for all reagents was reduced in MDS patients comparatively to volunteers. The platelet response induced by collagen and ristocetin was statistically different according to FAB category of risk. Anemia decreased the platelet aggregation amplitude: collagen r = 0.395, P = 0.0029; ristocetin r = 0.556, P = 0.002. Duration of the lag phase induced by collagen was negatively correlated with Hb level: r = 0.315, P = 0.019. We have obtained a correlation between high level of ferritin and variables of platelet aggregation curve induced by ADP: disaggregation incidence (r = 0.866, P = 0.01) and the absence of the second curve (r = - 0.717, P = 0.013). Conclusion: Dysfunctions of platelet in MDS patients have an important role in the increased risk of hemorrhage. The mechanism which was involved in decreased platelet response maybe the defective platelet production with more platelet abnormalities and the severity of anemia or iron overload. Disclosure of Interest: None Declared. Keywords: myelodysplastic syndrome, iron overload, platelet membrane, platelet aggregation, anemia, hemorrhage risk.

PC16 Preoperative bleeding risk factors in TAVI patients ska - Jelonkiewicz K1,*, Witkowski A2, Daz browski M2, Czerwin ska E2, Chmielak Z2, Banaszewski M3, Ksiez z_ ycka- Majczyn 4 1 ska J3 Kusmierski K , Hryniewiecki T , Demkow M5 and Stez pin 2 1 Department of Acquired Cardiac Defects; Department of Cardiology and Interventional Angiology; 3Intensive Cardiac Care Clinic; 4Department of Cardiac Surgery and Transplantology; 5 Department of Coronary Artery Disease and Structural Heart Disease, Institute of Cardiology, Warsaw, Poland Objectives: Bleeding complications worsens prognosis after interventions. The aim of the study is to estimate the risk of bleeding complications in TAVI pts. Methods: A single-center analysis of serious, in-hospital bleedings according to VARC scale. Prognosis value of known bleeding risk factors and used anticoagulant therapy was estimated. We enrolled 83 consecutive pts. 62–91 yrs (81.10 7.20) who underwent TAVI in 2009–2011. The uni- and multivariate analysis consisted of: v2 or Fisher exact test and logistic regression analysis. Antithrombotic therapy before TAVI in 83 pts. (bleedings no./pts. no.)

P value

ASA (25/42) Clopidogrel (22/22)*: Clopidogrel + ASA (10/15) Clopidogrel+ASA+VKA (4/6) VKA+ASA (2/5) Without treatment (6/8)†

0.006 0.0003 0.002 0.003 NS 0.009

Results: Characteristic of population according to the risk factors: hypertension 68(81.93%), abnormal renal function 49(59.04%), liver insufficiency 3(3.61%), stroke/TIA 13(15.66%), bleeding history 12 (14.46%), anaemia 43(51.81%), labile INR 5(6.02%), drugs (antiPLT/NSAIDs) 73(87.95%), alcohol 2(2,4%), female 54(65.06%), DM2 33(39.76%), mean BMI (25.54 4.0), transapical implantation


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16(19.275%). 51(61.44%) pts. had serious bleedings. The results of univariate analysis of pre-TAVI in comparision to VKA (6 pts. 0 bleedings) treatment are presented in Table.

PC18 Evaluation of platelet dysfunction in viral liver disease: relationship to disease severity

In multivariate analysis, the history of anaemia (P = 0.03);(3.76 OR: 95%CI: 1.273–11.114; P = 0.01), higher than mean age (P = 0.01); (2.712 OR: 95%CI: 1.101–6.678; P = 0.03), use of Clopidogrel before TAVI (P = 0.005);(4.48 OR: 95%CI: 1.119–17.996; P = 0.03), BMI 50 9 109/L. After 16 mo of treatment with romiplositm, pregnancy at 6 weeks’ gestation was discovered, and romiplostim was discontinued. She was treated with IVIG but to maintain the platelet level above 30 9 109/L, frequent infusions every 8–10 day were required, leading to severe side effects. IVIG was discontinued, and romiplostim was resumed at 10 weeks’ gestation, using doses up to 10 lg/kg/week (higher than the pre-pregnancy requirement), with good response. In late pregnancy four administrations of IVIG were required to keep the value close to 100 9 109/L. A healthy child was delivered with no bleeding complications, after which romiplostim was continued. Conclusion: Romiplostim is pregnancy category C based on animal studies showing potential harm to fetuses; it has not been studied systematically in pregnant women with ITP. Our case suggests its safety in selected patients, although doses higher than those that were effective pre-pregnancy may be required. Further studies, using data from national registries, are needed to evaluate use of romiplostim in this clinical scenario. Disclosure of Interest: A. Giermasz: None Declared, J. Knoche: None Declared, P. Fogarty Grant / Research support from: Amgen, Inc., Consultant for: Amgen, Inc. Keywords: immune thrombocytopenia, ITP, pregnancy, romiplostim.


Objectives: Liver cirrhosis is a major health problem in egypt, especially complicating viral hepatitis.The development of portal hypertension and oesophageal varices are the major complications of liver cirrhosis. This study aimed to assess the platelet function in patients with liver cirrhosis and to study the relationship between it and different stages of liver cirrhosis, presence of oesophageal varices as well as other liver function tests. Methods: Platelet function was tested in patients suffering from viral hepatic cirrhosis (no:50), using PFA-100. It is correlated by other liver function tests and disease severity assessed by child-pugh stage of cirrhosis, degree of ascites,oesophageal varices and splenomegally. Results ResultsAggregation studies demonstrated abnormal response in 70% of cases with a mean of 192.6 + 85.2 s. The prolongation in closure time was higher in child stage B, followed by stage A and C.Interstingly, it is more prolonged in mild ascites than moderate or resistant ascites. Closure time was more prolonged in grade I, II oesophageal varices than grade III and IV. Significant associations were established for platelet function with S.bilirubin and oesophageal varices. Significant associations were obtained between spleen size and albumin,child stage,ascites and grades of oesophageal varices. Conclusion: Platelet function by PFA-100 could be used to stratify cirrhotic patients and grades of oesophageal varices.However further studies should be continued to clarify the complex defect of platelet function in viral liver cirrhosis and to reveal its efficacy in predicting bleeding. Disclosure of Interest: None Declared. Keywords: cirrhosis,platelet dysfunction,PFA100,hepatitis C,varices, ascites.

PC19 Relationship between von Willebrand factor and platelet parameters in adult dengue infection patients Sukorini U*, Setyawati S and El Khair R Department of Clinical Pathology, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia Objectives: Dengue is the most important arthropod-borne viral disease and widespread as reemerging disease. In an attempt to reveal certain aspects of the pathogenesis of the bleeding disorder in dengue hemorrhagic fever especially related to vasculopathy and thrombopathy, there is need for understanding of the relationship between platelet parameters and plasma von Willebrand factor. Methods: This cross sectional study was conducted at Sardjito General Hospital, Yogyakarta, Indonesia. Adult hopitalized patients were included in the study based on the inclusion and exclusion criteria. Diagnosis of dengue infection was made according to serological status of IgG and IgM antidengue and WHO 2005 criteria. The Person correlation test was performed to know the relationship between von Willebrand factor with the platelet count and mean platelet volume (MPV), respectively. Results: Fifty two patients were included in the study consist of 19 males and 33 females. The mean level of von Willebrand factor is higher in non-O group vs. O-group population (240.5% vs. 258.8%, P = 0.551) but non significantly different. There is an inverse correlation between the von Willebrand factor and platelet count (r = - 0.368, P = 0.007), but a positive relationshion between von Willebrand factor

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ABSTRACTS

and the mean platelet volume (r = 0.393, P = 0.004) was found among these patients. Conclusion: There is a relationship beetween endothelial activation with amount of platelet and mean platelet volume. Our present data suggest that the von Willebrand factor involved in the mechanism of thrombocytopenia and affect the turn over of platelets. Disclosure of Interest: None Declared. Keywords: correlation, dengue, mean platelet volume, platelet count, von Willebrand factor.

PC20 Thrombin generation test in the monitoring of antiplatelet therapy effectiveness Smirnova O1, Namestnikov Y1, Matvienko O1, Berezovskaya G2, Golovina O1 and Papayan L1,* 1 Laboratory of Blood Coagulation, Russian Research Institute of Haematology and Transfusion; 2Cardiology department, Almazov Federal Heart, Blood and Endocrinology Centre, St.Petersburg, Russian Federation Objectives: Thrombin generation test (TGT) in platelet-rich plasma (PRP) is increasingly being employed as a global method to assess the procoagulant role of platelets and its modifications by antiplatelet agents. The aim of our study was to evaluate a possibility of using TGT for the assessment of efficacy of antiplatelet therapy. Methods: Venous blood was obtained from 17 patients after the successful coronary stent implantation. All of them were given clopidogrel and aspirin (75 and 150 mg per day, accordingly). The control group comprised 19 healthy volunteers. TGT was performed in fresh PRP with the platelet count adjusted to 150 9 109/l by dilution with autologous platelet poor plasma. Thrombin generation was measured in a Fluoroscan Ascentâ fluorometer at 1 pmol/l tissue factor concentration using dedicated software. The following parameters were analyzed: lag time of thrombin generation (LT), endogenous thrombin potential (ETP), maximum concentration of thrombin (Peak), time to reach the peak (TTP), the initial rate of thrombin generation (Rate = Peak / [TTP–LT]). Statistical analysis of the results was performed by non-parametric methods using the median (Me), 95% confidence interval (95% CI) and Mann-Whitney U test (Statistica 6.0). Results: Our data showed the decreasing of ETP, Peak and Rate and prolongation of LT and TTP in patients vs. controls. In the case of Peak, Rate and TTP the differences were statistically significant (Table). Table. Thrombogram parameters in the control group and group of patients (Me, 95% CI). Thrombogram parameters

Controls

Patients

ETP, nMmin Peak, nM Rate, nM/min LT, min TTP, min

2010 (1900.9–2113.7) 139.8 (124.2–149.4) 12.3 (10.6–14.4) 15.5 (14.4–18.5) 27.5 (25.7–29.9)

1830.5 (1736.6–2047.4) 123.8* (100.8–127.8) 7.8* (6.5–9.9) 18 (15.8–21.2) 34.3* (30.2–36.6)

Conclusion: Thrombin generation measurement in PRP can be useful in the monitoring of antiplatelet therapy effectiveness. Disclosure of Interest: None Declared. Keywords: anti-platelet therapy, thrombin generation test.

PC21 Thromboelastographic testing to determine individual response to clopidogrel Bryant BJ1,*, Treat J1,2, Scalia F3, McQueen I4 and Sharks P4 1 Blood Bank, University of Texas Medical Branch, Galveston, TX; 2 Blood Bank; 3Surgery; 4Pharmacy, Arkansas Heart Hospital, Little Rock, AR, USA Objectives: Activation of clopidogrel is decreased in patients with reduced CYP2C19 function. CYP2C19 has 30 variants with varying enzyme levels. Reduction in drug activation can be genetic or due to CYP inhibiting medications. Genetically, the presence of CYP2C19 reduced-function alleles has been reported as high as 30%. Thromboelastographic (TEG) testing was undertaken instead of expensive genetic testing to determine patients at risk for suboptimal therapy to clopidogrel. Methods: Patients on clopidogrel underwent TEG testing to determine the rate of inhibition of ADP-dependent platelet aggregation. Target inhibition goal for therapeutic efficacy was 40% inhibition from patient baseline level. Normal responders had therapeutic inhibition levels. Non-responders had little to no change from baseline. Slow responders had intermediate levels of inhibition. Slow responders on medications that were CYP substrates were analyzed. The inhibition of aggregation was plotted against the number of substrates each patient was taking. Results: Of the 49 patients tested, 35% (17/49) were normal responders, 55% (27/49) were slow responders, and 10% (5/49) were nonresponders. In the slow responder category, 13 (48%) were on medications that were CYP substrates. Sub-analysis of these slow responders demonstrated a linear decrease in inhibition of aggregation from 29% with 1 substrate to 14% with 3 substrates. Conclusion: TEG testing can be used to evaluate a patient’s response to therapy with clopidogrel. Many genetic variations or disease induced physiological changes in the CYP enzymes and/or concomitant dosing of CYP3A4/CYP2C19 active agents can result in resistance to anti-platelet therapy. Testing individual response to antiplatelet therapy is important in predicting a patient’s response to therapy and critical in modifying a patient’s drug regimen, ensuring patient safety, and achieving desirable outcomes. Disclosure of Interest: None Declared. Keywords: anti-platelet therapy, Clopidogrel, thromboelastographic testing.

PC22 Effect of cytochrome P450 polymorphism on platelet reactivity after treatment with clopidogrel in patients scheduled for percutaneous coronary intervention Saad AA1,*, Abd Elsalam AM2, Kamal GM3, Abou El-Ezz NF4 and El-Hagracy RS3 1 Clinical Pathology; 2Cardiology; 3Internal Medicine; 4 Community, Environmental & Occupational Medicine, Ain Shams University, Cairo, Egypt Objectives: Clopidogrel reduces cardiovascular events in patients with atherosclerotic disease. In order to acquire its antiplatelet effect, clopidogrel requires oxidation by the hepatic cytochrome P450 3A (CYP3A) to generate an active metabolite. The CYP3A subfamily consists of the 3A5 enzyme and three other isoforms.Gene variants of CYP3A5 are responsible for major individual differences in CYP3A enzymatic activity. The aim of the present work is to study the effect of CYP3A5 polymorphism on clopidogrel response and post-stent complications among Egyptian patients scheduled for percutaneouscoronary intervention(PCI). In addition, to assess post-clopidogrel platelet reactivity (PPR) as a predictor of post-stent complications.


ABSTRACTS Methods: This study included 45 patients scheduled for elective PCI at Ain Shams University hospitals. All the studied patients were subjected to detection of CYP3A5 A6986G polymorphism and assessment of clopidogrel response using adenosine diphosphate (ADP)-induced platelet aggregation. Results: Wild, heterozygous and homozygous genotypes were detected in 2.2%, 31.1% and 66.7% of the patients respectively. A trend for lower PPR in expressor genotype group was found (P = 0.08). No influence of the CYP3A5 genotypes on post-stent complications was found (P = 1.0). Clinical presentation, smoking, previous acute coronary syndrome (P < 0.05) were the main influencing factors of the PPR. Logistic regression analysis demonstrated that the stent length and PPR are the main predictors for post-stent complications (95% CI = 1.014–1.950 and 0.999–1.181; P = 0.04 and 0.05 respectively). Conclusion: CYP3A5 genetic polymorphism does not contribute to the observed variability in the inhibitory effect of clopidogrel. Low response to clopidogrel is a novel risk factor for ischemic events after PCI.The evaluation of low response to clopidogrel may help to identify patients at increased risk that may benefit from intensified antiplatelet strategy. Disclosure of Interest: None Declared. Keywords: CYP3A5 polymorphism, Clopidogrel, percutaneous coronary intervention.

Thromboemolic Disorders PE01 Markers of coagulation and inflammation in relation to the post thrombotic syndrome Bouman A*, Smits J, ten Cate H and ten Cate-Hoek A Maastricht University Medical Centre, Maastricht, The Netherlands Objectives: Post thrombotic syndrome (PTS) occurs in 20–50% of patients after deep venous thrombosis (DVT). Although several risk factors for PTS are known, it is difficult to accurately predict which patients will develop PTS. Biomarkers could be a valuable tool for PTS risk assessment. Objective: To investigate whether increased levels of factor VIII, Creactive protein (CRP) or D-dimer over time are associated with the development of PTS in patients after acute DVT. Methods: PTS status was assessed using the Villalta scale. Blood sampling was performed at three points during follow-up: one month after discontinuation of anticoagulant treatment (T0) and at 12 (T1) and 24 (T2) months after index DVT. Results: Data were derived from a prospective cohort study including 228 consecutive patients after acute DVT. At T1, both levels of Ddimer (median 725 ng/mL (interquartile range, IQR 400–1400) versus 378 ng/mL (251–652) P = 0.004) and CRP (median 3.9 mg/l (IQR 1.6–8.5) versus 2.4 mg/l (1.0–4.3) P = 0.018), were increased in patients with PTS, compared to patients without PTS, respectively. FVIII was not associated with PTS. Univariate analysis demonstrated associations between PTS and several clinical characteristics. However, in the multivariate logistic regression analysis only varicosities (OR 12.8 95% CI: 3.0–55.2 P = 0.001), previous ipsilateral DVT (OR 6.4 95% CI: 1.5–27.2 P = 0.011), and CRP > 5 mg/l on T1 (OR 7.8 95% CI: 2.4–25.6 P = 0.001) remained significantly associated with PTS. Conclusion: Besides previous ipsilateral DVT and varicosities, CRP > 5 mg/L at 1 year after the acute event of DVT was a strong and independent predictor for PTS. Persistent inflammation rather than hypercoagulability may be the most important etiological factor in PTS, and may be a target for future therapy. The development of a risk score for PTS, including both clinical risk factors and biomarker levels, such as


77

CRP, might be desirable for the identification of patients prone to PTS. Disclosure of Interest: None declared. Keywords: Biomarkers, CRP, D-dimer, Factor VIII, Post thrombotic syndrome.

PE02 Alternative diagnoses of pulmonary embolism in primary care Erkens PM1,*, Lucassen W2, Heugten MV3, Rietjens L3, Geersing G-J4, Prins M3, Cate HT3 and Stoffers J3 1 General Practice/Clinical Epidemiology, Maastricht University, Maastricht; 2Academic Medical Center, Amsterdam; 3Maastricht University, Maastricht; 4University Medical Center Utrecht, Utrecht, The Netherlands Objectives: A list of alternative diagnoses as seen in primary care might help the general practitioner (GP) in judging the probability of PE. This study investigated alternative diagnoses in primary care after excluding PE. Methods: This study is a sub-study of the Amsterdam Maastricht Utrecht Study on thromboembolism that validated the use of the Wells CDR for PE combined with point-of-care D-Dimer testing in 598 consecutive adult patients suspected of PE in primary care in the Netherlands (Amuse-2). After medical history and physical examination, all patients were referred to secondary care and diagnosed according to local protocols. PE was confirmed or refuted based on a composite reference standard, including, spiral CT and 3 months of follow-up. Results: In the study population (71% female; mean age 48 years) venous thromboembolism (VTE) was present in 73 patients (prevalence 12.2%). Odds Ratio of PE for whom the GP thought PE was most likely, was 4.7 (95%CI: 2.5–8.6). The most frequent alternative diagnoses after excluding PE were: thoracic pain/dyspnoea e.c.i. (41.5%%), pneumonia (12.4%), myalgia (11.6%), exacerbation COPD (4.4%), panic disorder/hyperventilation (3.8%) and upper respiratory tract infection (2.1%). Low risk patients with a Wells score ≤ 4 and a negative D-dimer test were significantly more diagnosed with a clinically non-relevant diagnosis such as thoracic pain/dyspnoea e.c.i., myalgia and panic disorder/hyperventilation (OR 0.4; 95% CI: 0.3–0.6; P = 0.001), while high risk patients with a Wells score > 4 or a positive D-dimer test were more often diagnosed with clinically relevant diagnoses (OR 2.0; 95% CI: 1.4 – 3.0; P = 0.001). Conclusion: The item ‘PE is more likely than an alternative diagnosis’ has a high predictive value in predicting PE. The most common alternative diagnoses of PE in primary care are thoracic pain/dyspnoea e.c.i, pneumonia and myalgia. High risk patients were more often diagnosed with clinically relevant diagnoses than low risk patients. Disclosure of Interest: None declared. Keywords: alternative diagnoses, primary care, pulmonary embolism.

PE04 Compliance of recommendations with french clinical practice in the management of thromboembolism in patients with cancer: the Carmen study Sevestre M1,*, Farge D2, Debourdeau P3 and Groupe Francophone Thrombose et Cancer 1 CHU Amiens, Amiens; 2Hopital Saint Louis, Paris; 3Hopital Desgenettes, Lyon, France Objectives: Long-term treatment with low-molecular-weight heparin (LMWH) is recommended for treatment of venous thromboembolism

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(VTE) in cancer patients. Few data are available on compliance in this population. Our study measured whether the management of VTE in patients with cancer was consistent with French recommendations. Methods: Compliance with recommendations (CR+) was analysed according to malignancy and VTE from a 500-patient cross-sectional observational study run between May and October 2010. CR+ was defined as the compliance to initial 10-day treatment followed by longterm LMWH for at least 3 months, avoiding LMWH in patients with renal insufficiency (SRI). All inpatients with a diagnosis of cancer and VTE of less than 6 months were included in the study. Results: Of 500 patients included in 47 centers, 242 (49%) were male, 81 (18%) had local (T+), 83 (18%) had loco-regional (N+) and 287 (64%) had metastatic malignancies. Malignancies were gastro-intestinal (25%), gynaecologic (23%), pulmonary (21%), haematological (14%), urologic (10%) or other (8%). Twelve patients had SRI. Overall, treatment was CR+ in 289/500 patients (58% [95% CI: 53% >62%]). Out of 12 patients with SRI only 3 (25%) were treated longterm with vitamin K antagonists (VKA), as usually recommended. Tumour site influenced CR+ (P = 0.02). Treatment for haematological malignancy was poorly compliant with recommendations (32%) while patients with lung malignancy had the best compliance (68%). TNM stage and VTE location had no influence on treatment compliance. Conclusion: In French practice, treatment of cancer-related VTE is CR+ in 58% of cases. TNM stage and VTE location do not influence compliance which remains insufficient, especially in patients with haematological malignancy. Disclosure of Interest: None declared. Keywords: recommendation, cancer, venous thrombosis, pulmonary embolism.

PE06 Unusual thromboses and thrombophilia: a case-control study

PE05 D-Dimer and ultrasound in combination Italian study (dulcis) to establish the optimal duration of anticoagulation for venous thromboembolism: preliminary results

Chapin J1,*, Christos P2 and DeSancho MT1 1 Division of Hematology-Oncology, New York Presbyterian Hospital-Weill Cornell Medical School; 2Clinical and Translational Science Center, Weill Cornell Medical College, New York, NY, USA

Cosmi B1,*, Legnani C2, Ghiraduzzi A3, Testa S4, Vittorio P5, Favaretto E2 and Palareti G1 1 1Department. Angiology & Blood Coagulation; 2Department Angiology & Blood Coagulation, S.Orsola-Malpighi University Hospital, Bologna; 3Department Internal Medicine I, Angiology, Arcispedale Santa Maria Nuova, Reggio Emilia; 4Haemostasis & Thrombosis Center, General Hospital, Cremona, Cremona; 5 Department Clinical and Experimental Medicine, Division of Clinical Cardiology, University Hospital, Padova, Italy Objectives: To evaluate the efficacy and safety of a procedure employing residual vein obstruction (RVO) and D-dimer to establish the individual risk of recurrence and thus the necessity to prolong or stop anticoagulation after idiopathic deep vein thrombosis (DVT) and/or pulmonary embolism (PE). The specific aims of the study are: i) to obtain a recurrence rate less than 5% per year in the first and second

N Age Thrombophilia Cancer JAK2 PGM FVL APLAs PC/PS/AT

DVT-PE 100 40 65 1 0 9 20 18 11

year after anticoagulation is suspended according to the procedure ii) to allow anticoagulation suspension in at least 40% of all subjects Methods: multi-centre management study in which D-dimer is measured after at least 3 months of anticoagulation in patients with RVO less than 4 mm in case of a previous DVT and/or normal pulmonary arterial pressure with echocardiography in case of previous PE and those who have undergone at least 12 months of therapy for previously altered RVO. If D-dimer is below age and gender specific cutoffs, anticoagulation is stopped and D-dimer is re-assessed after 15, 30, 60 and 90 days. If all the D-dimer measurements are below the cut-offs, anticoagulation is definitely stopped and patients are followed-up for two years. If one of these D-dimer measurement is above the cut-offs, anticoagulation is resumed for at least 6 months and patients are re-evaluated Results: As of 31th December 2011, 965 out of 2116 screened patients (46%) have been enrolled. Of these 397 (41%) stopped anticoagulation because of all D-dimer measurements below the cut-offs and 7 of these patients had a recurrent event (1.7% pt-y; 95% CI: 1–4%). In 463 subjects in whom anticoagulation was resumed, 9 major bleeding events were observed (2.4%; 95% CI: 1–5%). Conclusion: These preliminary results show that a management strategy based on RVO and D-dimer allows stopping anticoagulation in at least 40% of patients with a low risk of recurrence after a first episode of idiopathic DVT and/or PE. Final results of the study are expected at the end of 2012. Disclosure of Interest: None declared. Keywords: deep vein thrombosis, pulmonary embolism, D-dimer, vitamin K antagonists, hypercoagulability.

Objectives: Unusual venous thromboses (UVT) occur in the cerebral (CVT), splanchnic (SVT) and upper extremity veins (UEDVT). UVTs are distinct from deep vein thrombosis and pulmonary embolism (DVT/PE). Our objective was to evaluate thrombophilias between with UVTs and DVT-PE. Methods: Retrospective case-control study of patients referred to our hematology clinic from January 2006 to December 2011. Cases were UVT and controls were DVT/PE. Demographic data and risk factors collected included thrombophilia, pregnancy, puerperium, and use of female hormones. Results: There were 48 patients with UVT (14 CVT, 18 SVT, and 16 UEDVT) and 100 patients with DVT/PE (Table). The majority of patients were female. Thrombophilias were identified in 37% of UVT patients and in 65% of DVT/PE patients (P = 0.003). None of the patients with UVT had deficiencies of protein C, S or antithrombin while there were

UVT 48

P

CVT 14

P

UEDVT 16

P

SVT 18

P

37 18 3 4 7 6 3 0

0.14 0.003 0.1 0.01 0.4 0.4 0.08 0.02

33 5 1 0 4 2 1 0

0.06 0.04 0.2 – 0.05 1.0 0.5 0.4

37 4 1 0 0 3 0 0

0.36 0.005 0.3 – 0.4 1.0 0.07 0.4

40 11 1 4 3 1 2 0

0.98 0.79 0.3 0.0004 0.4 0.2 0.7 0.21


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11 (11%) patients with DVT/PE with these deficiencies (P = 0.02). The prothrombin gene mutation (PGM) was significantly higher in patients with CVT 4 (29%) vs. 9 controls (9%); (P = 0.05). The JAK2 mutation was only associated with SVT (P = 0.0004). We identify a heterozygote factor V Leiden mutation in three patients with UEDVT; five patients with UEDVT had indwelling catheters/pacemakers and two had thoracic outlet syndrome. There were no differences with respect to pregnancy/puerperium, or female hormone use between female patients with UVT and DVT/PE. Conclusion: Our study showed that thrombophilias were less common in patients with UVT than in those with DVT-PE. However PGM was more common with CVT and JAK2 mutations with SVT. Selected thrombophilia screening should be considered according to the location of the thrombosis Disclosure of Interest: None declared. Keywords: deep vein thrombosis, pulmonary embolism, thrombophilia diagnosis.

PE09 One year event rate of serious thrombosis and bleeding in Japanese patients with myocardial infarction, stroke and atrial fibrillation

PE07 Micropartical-associated thrombin generation in patients with postthrombophlebitic syndrome

Objectives: Event rate of serious thrombosis and bleeding in Japanese patients with myocardial infarction, stroke and atrial fibrillation in real world practice is still to be elucidated. Image/Graph:

Matvienko O*, Namestnikov Y, Golovina O and Saltykova N Russian Research Institute of Hematology and Transfusiology, Saint-Petersburg, Russian Federation Objectives: The postthrombophlebitic syndrome (PTS) is accompanied by increase venous pressure, disrupt microcirculation and activation of blood cells that can lead to microparticles (MP) release. MP express tissue factor (TF) and procoagulant phospholipids on their surface that can enhance prothrombogenic potential of blood plasma. Our aim was estimation significance MP for thrombin generation in the group of patients with PTS. Methods: This research included 34patients with PTS. Reference blood samples were obtained from 54 healthy controls. Thrombin generation was measured in platelet-free plasma with Calibrated Automated Thrombinogram Assay method (CAT). Following reagents were used: «MP-reagent», containing negatively charged phospholipids (4 lM), «PRP-reagent», containing rTF (1 pM), «FluCa‑kit» and CTI (40 mg/ mL). For plasma samples following parameters were derived: endogenous thrombin potential (ETP, nM-min), peak thrombin activity (Peak, nM), and rate of thrombin generation (R, nM/min). Results: ‘PRP-reagent’ and ‘MP-Reagent’ allow to estimate thrombin generation which depends on procoagulant phospholipids and TF, respectively. Significant differences were found when were used both of that reagents. Conclusion: MP enhance thrombin generation and increase prothrombogenic potential of patients with PTS due to TF and procoagulant phospholipids expressed on their surfaces. Disclosure of Interest: None declared. Keywords: microparticles, postthrombophlebitic syndrome

Table. Thrombogram parameters in the group of patients and control group (Me, 50% CI). PRP-Reagent Patients Parameters (N = 31)

MP-Reagent Controls (N = 30)

Patients (N = 25)

Controlss (N = 24)

ETP 813.3** 471.84 1132.0** 566.5 (nM-min) 585.8–1042.1 384.25–564.00 758.0–13.0 462.3–708.0 Peak 25.1** 18.40 126.6** 46.3 (nM) 19.3–35.4 12.29–20.26 77.5–172.7 32.9–67.2 R(nM/min) 2.2* 1.59 27.3* 7.3 1.5–3.1 1.19–2.01 10.9–45.1 5.2–12.6 *P < 0.01, **P < 0.0001 for difference with controls. © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

Toda E1,*, Uchiyama S2, Shimada K3, Origasa H4, Kobayashi H5, Kawamura A6, Nagao T7, Ikeda Y8, Goto S9 and on behalf of The Japan Thrombosis Registry for Atrial Fibrillation Coronary or Cerebrovascular Events (J-TRACE) 1 Cardiology, Tokai University School of Medicine, Hachioji; 2 Neurology, Tokyo Women’s Medical Uniersity, Tokyo; 3 Cardiology, Jichi Medical University Hospital, Tochigi; 4Toyama Medical and Pharmaceutical Univertisy, Toyama; 5Tokai University School of Medicine, Isehara; 6Cardiology, Keio University Hospital; 7Neurology, Tokyo Women’s Medical University; 8Waseda University, Tokyo; 9Cardiology, Tokai University School of Medicine, Isehara, Japan

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Methods: We have analyzed the data fromJ-TRACE, which is a nationwide prospective cohort of stable outpatients with a history of myocardial infarction (MI: n = 2.291), stroke (n = 3.554), and/or atrial fibrillation (AF: n = 2.242). One-year follow-up data were available for 7513 of 8087 patients. We defined stroke and MI as serious thrombotic events. Investigator decided serious bleeding events were used as serious bleeding events. Results: The rates of fatal stroke, fatal MI, non-fatal stroke, and non fatal MI within 1 year were 0.15 (95%CI: 0.08–0.27), 0.06 (95%CI: 0.02–0.14), 1.85 (95%CI: 1.55–2.19), and 0.33 (95%CI: 0.21–0.49) per 100 person-years, respectively. The rate of fatal bleeding was 0.05 (95%CI: 0.01–0.14). Investigator decided serious non-fatal bleeding was 0.15 (95%CI: 0.08–0.27) per 100 person-years. Conclusion: In Japan, the rate of serious thrombotic event (especially stroke) in patients with MI, stroke and AF is higher than serious bleeding events in real world practice, which indicate the further needs for antithrombotic intervention, especially in the prevention of stroke. Disclosure of Interest: None declared. Keywords: atrial fibrillation, bleeding events, Myocardial infarction, Stroke, thrombosis.

PE10 The value of D-dimer as a marker for cardiovascular and arterial thrombotic events in patients with peripheral arterial disease: a systematic review Kleinegris M-C*, ten Cate-Hoek A and ten Cate H Department of Internal Medicine, Laboratory for Clinical Thrombosis and Haemostasis, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands

PE11 Venous thromboembolism prophylaxis in elderly patients admitted to Italian internal medicine wards Marcucci M1,*, Violi F2, Iorio A3, Tettamanti M4, Pasina L4, Nobili A4, Djade CD4, Marengoni A5, Salerno F6, Corrao S7, Mannucci PM8 and REPOSI (REgistro POliterapie Societ a Italiana di Medicina Interna) Investigators 1 Department of Internal Medicine, University of Perugia, Perugia; 2 Department of Internal Medicine and Medical Specialities, Sapienza, University of Rome, Rome, Italy; 3Department of Clinical Epidemiology and Biostatistics, McMaster University, Hamilton, ON, Canada; 4Mario Negri Institute for Pharmacological Research, Milan; 5Geriatric Unit, Ospedali Civili, Department of Medical and Surgery Sciences, University of Brescia, Brescia; 6Internal Medicine, IRCCS Policlinico San Donato, Department of Medical and Surgery, University of Milan, Milan; 7Biomedical Department of Internal Medicine, University of Palermo, Palermo; 8Department of Medicine and Medical Specialties, IRCCS Maggiore Hospital Foundation, Milan, Italy Objectives: Pharmacological thromboprophylaxis (TP) is generally known to reduce Venous Thromboembolism (VTE) in medical inpatients but the by-risk -driven prescription, safety and impact on mortality are still debated. Image/Graph:

Objectives: Peripheral Artery Disease (PAD) is associated with an increased risk for cardiovascular events. D-dimers have been established as a marker for hypercoagulability and are linked with thrombotic events in patients with venous as well as arterial thrombosis. The predictive value of plasma D-dimer levels in relation to cardiovascular events in patients with PAD is not unambiguously established. Aim of this review was to synthesize evidence evaluating the value of D-dimer as a predictor of arterial thrombotic events in claudicants and patients with PAD. Methods: The Pubmed, Embase, and Cochrane databases were searched with English and German as language restrictions (January 1980 to February 2012) and 65 abstracts were found. The strategy was supplemented with manual review of reference lists. Case-control, cohort or prospective cohort studies that measured fibrin D-dimer in patients with PAD, were included. Studies were excluded if there was no follow-up for arterial thrombotic events or when no specific information on D-dimer was available. Results: The search yielded 10 studies for our analysis, consisting of 2420 patients with PAD, with a total of 1052 cardiovascular events in 10599 patient years. Two studies with a follow-up of 1 year show that fibrin D-dimer predicts both deterioration of PAD and subsequent thrombotic events. Five out of six studies with a median follow-up of 2–4 years reveal that an increased D-dimer is predictive of various arterial thrombotic events including mortality. Two studies with a longer follow-up (over 6 years) do not show an independent association between increased D-dimer levels, arterial thrombotic events and CVD mortality. Conclusion: In patients with peripheral arterial disease increased fibrin D-dimer is an independent predictor for near-term cardiovascular thrombotic events and mortality. This research was supported by the Center for Translational Molecular Medicine and the Dutch Heart Foundation (INCOAG). Disclosure of Interest: None declared. Keywords: arterial thrombotic events, D-dimer, Peripheral arterial disease.


ABSTRACTS Methods: We analyze data on elderly (> 65 years old) patients admitted to the Italian internal medicine wards participating in REPOSI project (year 2010) to investigate the rate of TP during the hospital stay and which demographic and clinical (acute and chronic) factors were related to its prescription. Multivariate logistic regression, Area Under the ROC Curve and CART analysis were performed to find independent predictors of TP prescription. Patients on heparin/fondaparinux for other reasons and patients on VKAs were excluded from the analyses. Association between TP and VTE, bleeding and death, in-hospital and during the 3 months post-discharge follow up was explored by logistic regression and propensity score analysis. Patients who were prescribed with TP at discharge were sub-analyzed for outcomes at follow up. Results: Out of 1121 patients enrolled, 171 (162 on low molecular weight heparin, 9 on fondaparinux) were on TP during the hospital stay (15.2%). Barthel Index was found as the most important independent predictor of prescription in multivariate analyses; the CART figure depicts the hypothetical flow chart which drove the choice of the physicians. Rate of fatal and non-fatal VTE or bleedings during and after hospitalization did not differ between TP and not-TP patients. Overall in-hospital and post-discharge mortality was significantly higher in patients on TP (P ≤ 0.001) but TP was not an independent predictor. Otherwise TP at discharge was independently related to death for bleeding. Conclusion: Although aware of the limits due to sample size and observational design, we found a not widespread use of TP among elderly medical patients, prescribed primarily according to the degree of patient disability; it appeared to not reduce in-hospital and post-discharge overall mortality. Disclosure of Interest: None declared. Keywords: Elderly, medical patients, Mortality, prophylaxis.

PE12 Readmission and death rates from venous thromboembolism within 90 days of hospital admission: what can we learn from measuring real outcomes at a national level? Lester WA1,*, Khosla S2, Ray D2, Richardson M3, Pagano D4 and Quality and Outcomes Research Unit, University Hospitals Birmingham 1 Haematology; 2Informatics, Queen Elizabeth Hospital; 3 Statistics, University of Birmingham; 4Cardiothoracic Surgery, Queen Elizabeth Hospital, Birmingham, UK Objectives: It is essential that efforts to reduce hospital acquired venous thromboembolism (VTE) should be monitored using real rather than surrogate outcomes. Methods: Analysis of all patients admitted to NHS Hospitals in England between April 2009 and May 2011 with ICD10 codes for VTE, or with pulmonary embolism as a primary cause of death within 90 days of a prior hospital admission, using Hospital Episode and Office of National Statistics data. Results: 20% of all VTE admissions occurred within 90 days of a prior hospital admission. 1/3rd of these previous admissions are 10 fold difference in VTE readmission rates between hospitals. The absolute number of VTE readmissions to NHS hospitals in England within 90 days of hospital admission have reduced from late 2010 in some regions of the country but not all. After interrogating the main contributors to the original admissions, it was identified that ‘non-specific’ leg and chest pain represent a common preceding diagnosis, probably representing missed diagnosis of VTE. Lower limb fracture is a more common primary diagnosis than fractured neck of femur. Mortality from pulmonary embolism after discharge from hospital (approx 4000 events per year) is sustained and then reduced across all regions from January to May 2011 but further data is required to confirm whether this observation is significant and © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

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sustained. The profile of admission types prior to fatal PE show some differences compared to those contributing to non-fatal VTE readmission. Hip and other limb fracture represent the commonest reason for prior hospital admission. Non-specific leg pain remains a common preceding diagnosis. Conclusion: National guidelines and policies aimed at reducing hospital acquired VTE in England may be having an impact on VTE readmission and mortality rates. Analysis of the relative contribution of specific types of hospital admission to risk of post discharge VTE could help prioritize appropriate preventative strategies. Disclosure of Interest: W. Lester Consultant for: Bayer, Boehringer Ingelheim, Speakers Bureau: Sanofi Aventis, Bayer, Boehringer Ingelheim, S. Khosla: None Declared, D. Ray: None Declared, M. Richardson: None Declared, D. Pagano: None Declared Keywords: hospital acquired venous thromboembolism.

PE13 Coffee consumption and a reduced risk of venous thrombosis: mediated through hemostatic factors Roach RE*, Siegerink B, le Cessie S, Rosendaal FR, Cannegieter SC and Lijfering WM Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, The Netherlands Objectives: Moderate coffee consumption has been found to reduce the risk of venous thrombosis (VT), but the role of confounding and the pathophysiology behind these findings are unclear. Methods: From a large population based case-control study, performed in the Netherlands between 1999 and 2004, 1669 patients with a first VT (cases) and 1669 partner controls were included. Choosing partners as controls had the benefit that both known and unknown confounding could be taken into account. Using conditional logistic regression, the risk of VT was estimated by calculating Odds Ratios (OR) and 95% Confidence Intervals (95%CI) across categories of increasing coffee consumption. In addition, we calculated mean differences in levels of hemostatic factors between these categories in control subjects. Results: Daily intake of 1–4 or 5–9 cups of coffee yielded a reduction in the risk of VT compared with no coffee consumption: OR 0.7 (95% CI: 0.5–0.9) and 0.6 (95% CI: 0.5–1.0), respectively. Adjustment for several confounders (age, sex, BMI, smoking, statin use, aspirin use, comorbidity, physical activity, food supplement use and alcohol intake) did not change these findings (OR 0.7 (95% CI: 0.5–1.0) and 0.7 (95% CI: 0.3–1.3)). Results were similar for provoked and unprovoked events, and for deep vein thrombosis and pulmonary embolism. In controls, we found a decrease in fibrinogen, factor VIII and von Willebrand factor for increasing levels of coffee consumption. After adjusting the ORs for these coagulation factors, the protective effect of coffee consumption diminished: OR 0.9 (95% CI: 0.6–1.3) and 1.0 (95% CI: 0.5–1.9), respectively. There was no effect of coffee consumption on anticoagulant (protein S, protein C, antithrombin) or fibrinolytic markers (clot lysis time). Conclusion: Our study confirms a reduction in the risk of VT for increasing levels of coffee consumption, which seems to be mediated through lower levels of factor VIII, fibrinogen and von Willebrand factor. Disclosure of Interest: None declared. Keywords: coffee consumption, epidemiology, Factor VIII, haemostatic factors, risk factors, venous thrombosis.

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PE14 Venous thrombosis in a patient with congenital afibrinogenemia: clinical implications and management Chapin J* and DeSancho M Division of Hematology-Oncology, New York Presbyterian Hospital-Weill Cornell Medical School, New York, NY, USA Objectives: Congenital afibrinogenemia is a rare coagulopathy resulting from a fibrinogen mutation that prevents clot formation. We present a 33-year-old female patient with congenital afibrinogenemia who developed pleuritic chest pain and dyspnea on exertion. A port-catheter had been placed 3 weeks prior due to poor venous access. A CT angiogram revealed bilateral segmental and subsegmental pulmonary emboli (PE). Six months earlier she was diagnosed with PICC-line related right subclavian and axillary thrombosis. The PICC line was removed and the thrombosis resolved in 1 month without anticoagulation. Methods: Case Report. Results: Her past medical history was significant for recurrent intracranial hemorrhages and menorrhagia. Three years prior to her presentation, she was started on human fibrinogen concentrate (RIASTAP; hFC) 50 U/kg after a subdural bleed and was maintained on bi-weekly infusions. Previously she had received cryoprecipitate and fresh frozen plasma and acquired transfusion-related hepatitis B and C. Her fibrinogen deficiency resulted from a homozygous base pair insertion in FGA exon 5 (FGA g.3808-3809 ins T) causing a frameshift mutation in the fibrinogen a-chain. Her baseline fibrinogen activity level was 60 s, and aPTT>150 s. Pharmacokinetic studies for hFC are shown in Figure 1a.During her hospitalization for PE, she was continued on hFC, and was transitioned from unfractionated heparin (UFH) to enoxaparin (Figure 1b). She completed 4 months of anticoagulation with enoxaparin 1 mg per kg twice daily without bleeding complications. A follow up CT angiogram showed complete resolution of the PE. She has been switched to prophylactic dose enoxaparin on the days that she receives hFC. Image/Graph:

Conclusion: In conclusion, congenital afibrinogenemia is rare bleeding disorder; both the disease and treatments with hFC can contribute to venous thromboembolism. Anticoagulation in these patients is challenging and requires careful monitoring. Disclosure of Interest: None declared. Keywords: afibrinogenemia, fibrinogen, pulmonary embolism, Thrombosis.

PE15 Acquired and genetic risk factors assessment for deep venous thrombosis in 389 of hospitalized Chinese patients: a single center experiences Deng M-Y*, Zhang G-S and Luo Y-Y Hematology, The second Xiangya Hospital, Central South University, Changsha, China Objectives: To identify the frequency of acquired risk factors and genetic risk factors including Hyperhomocysteinaemia, antithrombin activity deficiency, protein C activity deficiency, decreased total protein S concentration,prothrombin 20210A allele,antithrombin Cambridge II and FV Leiden mutation in Chinese patients with deep venous thrombosis Methods: In the present study, we analyzed retrospectively acquired risk factors in 389 of hospitalized patients with DVT and investigated mainly potential genetic risk factors in 120 patients.The clinical datas of 389 patients with deep vein thrombosis were analyzed retrospectively.Antithrombin activity and protein C activity were measured with a chromogenic method on automatic analyzer .Total protein S concentration was determined by polyclonal enzyme-linked immunosorbent assay .Plasma homocysteine levels were determined by enzymatic cycling assay.For the identification of FV Leiden,antithrombin Cambridge II and prothrombin 20210A allele genetic mutations, we used the method of restriction fragment length polymorphism analysis. Results: Among the identifiable acquired risk factors, Major acquired risk factors included surgery (41.1%), malignancy (22.92%), major trauma(19.8%); gestation(12.6%);varicosity(12.6%).In both DVT patients and healthy controls, no any mutation was detected for FV Leiden, prothrombin 20210A and antithrombin Cambridge II mutations. Antithrombin deficency, as well as protein C deficency, were an independent risk factor for DVT (OR 3.35, 95% CI: 1.20–9.39; and OR 2.92, 95% CI: 1.03–8.29, respectively). No significant correlation was found between protein S deficiency and DVT (OR 1.36, 95% CI: 0.43–4.29) or between hyperhomocysteinaemia and DVT (OR 1.54, 95% CI: 0.50–4.76). Conclusion: Our results suggest that major acquired risk factors for DVT patients in Chinese population are surgery operations and malignant neoplasm;while main genetic risk factors are the deficiencies of antithrombin and protein C. Disclosure of Interest: None declared. Keywords: Antithrombin, Deep vein thrombosis, homocysteine, protein C, protein S, risk factors.

PE16 Quality of life in acute deep vein thrombosis and pulmonary embolism Hogg K1,*, Kimpton M2, Carrier M1, Coyle D3 and Wells P1 1 Thrombosis department, Ottawa Hospital Research Institute; 2 Department of Medicine; 3Department of Epidemiology, University of Ottawa, Ottawa, ON, Canada Objectives: Thrombosis medicine has seen a proliferation of antithrombotic and diagnostic agents in recent years. Health funding bodies frequently rely on cost-utility analysis to inform decision mak-


ABSTRACTS ing. There is only one published study reporting the utility of acute deep vein thrombosis (DVT) and pulmonary embolism (PE), performed on 106 women from the community. We aimed to measure the utility of health states required for cost-utility analyses of thrombosis treatments, using people with personal experience of venous thromboembolism (VTE). Methods: We performed 30 min interviews with thrombosis clinic patients who had a history of VTE. Utilities were measured for acute DVT, acute PE, gastrointestinal (GI) bleeding, minor intracranial haemorrhage and major intracranial haemorrhage using the standard gamble technique. Median utility values were reported. Results: Two hundred and fourteen patients were interviewed (mean age 56, 52% male). 21% were within 4 weeks of diagnosis of VTE. 58% had one prior episode of VTE, 26% two and 16% three or more episodes of VTE. 23% had prior cancer-associated VTE. The median utilities for acute DVT was 0.81, acute PE 0.75, GI bleed 0.65, minor intracranial bleed 0.75 and major intracranial bleed 0.15, during the respective health state. For patients within 4 weeks of diagnosis, the utilities for VTE were lower: for DVT 0.75 and PE 0.65. For cancer patients these were 0.75 (DVT) and 0.71 (PE). The differences between subgroups were not significant. Conclusion: The quality of life utility for acute DVT and PE is essential for thrombosis related economic analyses and has not been well researched. Our study is the largest study to date, and reports these utilities, taken from patients with experience of the condition. The results suggest that patient reported quality of life estimates might be influenced by proximity to diagnosis and the presence of cancer. Disclosure of Interest: None declared. Keywords: deep vein thrombosis, pulmonary embolism, QALY.

PE17 Explicit assessment of thromboembolic risk and prophylaxis for medical patients in Switzerland (estimate): preliminary results Spirk D1, Aujesky D2, Hayoz D3, Beer J4, Husmann M5, Frauchiger B6, Korte W7, Wuillemin W8, Bounameaux H9 and Nendaz M9,* 1 Medical Department, Sanofi-Aventis (Suisse) SA, Meyrin; 2 Division of General Internal Medicine, University Hospital Bern, Bern; 3Department of Internal Medicine, Cantonal Hospital Fribourg, Fribourg; 4Department of Internal Medicine, Cantonal Hospital Baden, Baden; 5Clinic of Angiology, University Hospital Zurich, Zurich; 6Department of Internal Medicine, Cantonal Hospital Frauenfeld, Frauenfeld; 7Department of Internal Medicine, Cantonal Hospital St. Gallen, St. Gallen; 8Department of Internal Medicine, Cantonal Hospital Lucerne, Lucerne; 9 Department of Internal Medicine, University Hospital Geneva, Geneva, Switzerland Objectives: Thromboprophylaxis in hospitalized medical patients remains underused and often inadequate, and there is an urgent need to validate risk assessment tools for venous thromboembolism (VTE). We aimed to evaluate whether the Geneva Risk Score (Chopard P, Spirk D, Bounameaux H. JTH 2006;4:915–16) is suitable for the identification of patients at high risk of VTE. Methods: In the prospective Explicit ASsessment of Thromboembolic RIsk and Prophylaxis for Medical PATients in SwitzErland (ESTIMATE) study, 1478 consecutive patients admitted to internal medicine wards within a 12-month period in eight Swiss hospitals were classified as having a high (Geneva Risk Score≥3) or low (Geneva Risk Score < 3) risk of VTE. The primary endpoint was VTE-related death or symptomatic VTE complication at 90 days. Results: Overall, the mean age was 65 17 years, 962 (65%) patients were at high risk and 516 (35%) were at low risk of VTE. The mean Geneva Risk Score was 5.2 2.4 in high-risk and 1.3 0.8 in low© 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

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risk patients (P < 0.001). Upon hospital admission, thromboprophylaxis was administered to 596 (62%) high-risk patients and 245 (48%) low-risk patients (P < 0.001). The primary endpoint occurred in 3.2% high-risk vs. 0.6% low-risk patients (P = 0.002). Among patients with prophylaxis, the rate of primary endpoint was 3.0% in high-risk vs. 0.4% in low-risk patients (P = 0.026). Among patients without prophylaxis, this rate was 3.5% in high-risk vs. 0.8% in low-risk patients (P = 0.029). Geneva Risk Score ≥ 3 independently predicted the occurrence of primary endpoint (HR 5.51, 95% CI: 1.66–18.29; P = 0.005) after adjustment for the use of thromboprophylaxis. Conclusion: The Geneva Risk Score can help physicians to reliably identify acutely ill medical patients at high risk of VTE. Disclosure of Interest: D. Spirk Employee of: Sanofi-Aventis (Suisse) SA, Meyrin, Switzerland, D. Aujesky: None Declared, D. Hayoz: None Declared, J. Beer: None Declared, M. Husmann: None Declared, B. Frauchiger: None Declared, W. Korte: None Declared, W. Wuillemin: None Declared, H. Bounameaux: None Declared, M. Nendaz: None declared. Keywords: Geneva Risk Score, Medical patients, Risk assessment model, Thromboprophylaxis, Venous thromboembolism.

PE18 Prophylaxis guidelines as assessed retrospectively on a cohort of hospital-acquired venous thrombosis medical patients without thromboprophylaxis Ng HJ1,*, Leong YH1, Siow LK2, Yeang SH2, Wong SY2 and Lee LH1 1 Department of Haematology; 2Department of Pharmacy, Singapore General Hospital, Singapore, Singapore Objectives: Pharmacologic prophylaxis against venous thromboembolism (VTE) in medical patients is effective and guidelines are available to identify high-risk patients and recommend appropriate prophylaxis. Unlike compliance, the performance of such guidelines in adequately covering the highest risk patients has not been well studied. Methods: A cohort of medical patients with hospital-acquired VTE (HaVTE) was identified from a hospital-based prospective VTE registry using predefined criteria. They had received no pharmacologic prophylaxis. Criteria from our hospital and American College of Chest Physician (ACCP) VTE prevention guideline were retrospectively applied to determine the likelihood of this cohort receiving pharmacologic prophylaxis. Results: Of 57 832 patients admitted to medical disciplines over a 17month period, there were 84 patients who fulfilled criteria for a HaVTE with no prophylaxis given. Among patients admitted for three or more days, the rate of symptomatic HaVTE was 0.21% (95% CI 0.05). Conclusion: Therefore, TF+MPs detecting by FCM is useful for the diagnosis of hemostatic disorder, especially in the evaluation of patiens with APL, which may also suggest an important pathogenic role in the evolution of overt DIC. Disclosure of Interest: None declared. Keywords: Acute promyelocytic leukemia, Disseminated intravascular coagulation, Flow cytometry, Tissue factor-positive microparticles.

PE20 Significantly higher level of lipoprotein (a) among acute coronary syndrome (ACS) than stable angina pectoris (SAP) patients or healthy subjects in Indonesian population

respectively, P = 0.005) but there was no difference of Lp(a) level between SAP group and healthy group (15.6 mg/dl vs 11.7, P = 0.408). Acute Coronary Syndrome group had higher glucose level than SAP and healthy group (130 mg/dL, 125 mg/dL, 106.5 mg/dL respectively, P = 0.001). However, the HDL median level was significantly lower than healthy group (37.2 mg/dl vs 51.8 mg/dL, P = 0.001). SAP group had significantly higher triglyceride level than healthy group (144.0 mg/dL vs 78.5 mg/dL, P = 0.001). Other laboratory result showed no differences among ACS, SAP, and healthy group. Conclusion: Lipoprotein(a) of ACS group is higher than SAP and healthy group in Indonesian population. Other parameters that may be associated with this difference are age, glucose, HDL and triglyceride. Disclosure of Interest: None declared. Keywords: Acute Coronary Syndrome, Lipoprotein(a), Stable Angina Pectoris.

PE21 IL-18 and vascular mineralization in coronary artery disease patients ~a A1,2,*, Valente-Acosta B1, Reese-Parada J3, De La Pen ~a-Duque M5, ~or A4, Quintanar-Trejo L1, Pen Contreras-Villasen Martinez-Rios M5, Herrera-Alarcon V3, Linares-Lopez C6, ~ezDelgado-Granados H6, Flores-Garcia M1 and Santiban Escobar F3 1 Biologia Molecular, Instituto Nacional de Cardiologia; 2 Farmacologia, Universidad Nacional Autonoma de Mexico; 3 Cirugia; 4Instituto Nacional de Cardiologia, Mexico, Mexico; 5 Hemodinamica, Instituto Nacional de Cardiologia; 6Instituto de Geofisica, Universidad Nacional Autonoma de Mexico, Mexico, Mexico Objectives: IL-18 is implicated as an inflammatory promoter, which enhances atherosclerosis and could leads to vascular mineralization. Increased IL-18 serum levels have been associated with diabetes type 2, metabolic syndrome and the severity of atherosclerosis. Our objetive was to identify the plasma concentration of IL-18 and the amount and type of minerals that accumulate in the aortic and mammary artery vessels of patients with atherosclerosis. Image/Graph:

Puspitawati I1,*, Setyawati S1, Anggrahini DW2 and Herningtyas EH1 1 Department of Clinical Pathology; 2Department of Cardiology, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia Objectives: There was still controversy of Lipoprotein a (Lp(a)) role as a risk factor for Coronary Heart Diseases in different population and ethnics, thus, we were triggered to investigate Lp(a) level among two spectrum of Coronary Heart Diseases; the Acute Coronary Syndrome (ACS) and the Stable Angina Pectoris (SAP) in Indonesian population. Methods: This cross sectional study was conducted in Sardjito General Hospital, Yogyakarta, Indonesia consisted of 40 participants in each disease group and 36 participants as healthy group. The Lp(a) was measured once by turbidimetric immunoassay and other laboratory results was obtained from medical record. Data were analyzed using Kruskal Wallis test and Mann-Whitney test. Results: We found that ACS group had higher median value of Lp(a) than SAP and healthy subjects (24.3 mg/dL, 15.6 mg/dL, 11.7 mg/dL,


ABSTRACTS Methods: We performed a cross-sectional study in 34 patients, 56 2.5 years old, with two and three-vessel coronary artery disease (CAD), undergoing coronary artery bypass surgery. IL-18 was determined by an ELISA kit, following manufacturing directions. Aortic artery specimens were analyzed using a scanning electron microscope with an energy dispersive X-ray spectrometer. Results: The average IL-18 was greater in CAD patients, 272.63 23.9 pg/mL than those obtained in 43 blood bank donors, 53 1 years old, 131 25 pg/mL, (P < 0.05). The amount of minerals in the aorta was greater in the aorta than that in the mammary-vessel (258 54.62 vs. 58.36 12.81 particles per 500 lm2, P = 0.002). Also we found a positive correlation between the particles of phosphorus in the aorta and the IL-18 (r = 0.599, P = .040), we were unable to find a correlation with calcium or iron. Conclusion: Our study demonstrates that the aorta is more affected by mineralization compared to the mammary artery. We observe a correlation between IL-18 and phosphorus deposits. Disclosure of Interest: None declared. Keywords: IL-18, Vascular mineralization.

PE22 Study on gene markers of thrombosis in coronary heart disease by multi-analyte suspension array technology He W1,*, Hu Y1, Yu J2, Guo T1 and Zhang X1 1 Institution of hemotology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan; 2Institution of cancer, second affiliated hospital, Zhejiang University, Hangzhou, China Objectives: Coronary heart disease is caused by multiple factors, which also include genetic factors. The main objective of this study was to detect seven gene polymorphisms in acute myocardial infarction (AMI) patients by liquid gene chip technology (MASA technology), and positive findings were verified, so as to find out validity of MASA technology in detecting gene variations. Methods: Thirty-four patients with acute myocardial infarction admitted to department of cardiology in Wuhan Union Hospital were enrolled in this trail. DNA was amplified by PCR technology after being extracted, nucleic acid probes of seven gene variations (FV Leiden G/A mutation, Fbg Beta -455 G/A mutation, Fbg Beta -148 C/T mutation, Fbg Beta 448 G/A mutation, FVII -323 10 bp deletion/insertion mutation, prothrombin 20210 G/A mutation and MTHFR 677 C/ T mutation) were combined with microspheres (beads) by covalent bonds, the latter were coded with specified fluorescence, PCR products was added after various beads were mixed, the mixture was detected by Luminex 100TM Multi-Analyte Suspension Array, finally, positive findings were verified by Sanger dideoxynucleotide chain termination. Results: There were 19 patients with gene variations, including four patients with FV Leiden mutation, 14 patients with MTHFR 677 C/T mutation, five patients with fibrinogen b gene 448 G/A mutation, among them there were one patient with FV Leiden and Fbgb448G/A mutation, 1 with FV Leiden and MTHFR677 C/T mutation 2 with MTHFR677 C/T mutation and Fbgb 448G/A mutation, the total accuracy of MASA in positive findings was 73.9%. Conclusion: Liquid gene chips technology is feasible in detecting gene variations in AMI patients, nevertheless, the MASA technology needs to improve. Disclosure of Interest: None declared. Keywords: Acute myocardial infarction, gene chips, Multi-Analyte Suspension Array, mutation, polymorphism


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PE23 Results from the national registry of thrombophilic states in Slovakia ov Stasko J*, Plamen a I, Sokol J, Chud y P, Dobrotov a M and Kubisz P Department Hematology and Transfusiology, Jessenius Faculty of Medicine, Comenius University, Martin, Slovakia Objectives: Primary objective of our project was to find out prevalence of the most frequent inherited TS in families of patients with thrombosis in Slovakia using National Registry of Thrombophilia States (NRTS). Methods: Patients from all regions of Slovakia with a history of thrombosis and their relatives were examined for the inherited and acquired thrombophilia. All patients were informed and agreed to participate at the database of NRTS. Results: On 25th January 2012, overall 5356 patients have been registered in NRTS. Factor V Leiden (FVL) was found as the most frequent thrombophilia with 44.7%, followed by prothrombin mutation (G20210A) 11.7%, protein S (PS) deficiency 9.1%, antiphospholipid syndrome (APS) 5.5%, sticky platelet syndrome (SPS) 4.3%, acquired resistance to protein C (APC-R) 3.5%, increased FVIII 3.1%, antithrombin deficiency 3.1%, hyperhomocysteinemia 2.6%, protein C (PC) deficiency 2.5%, decreased FXII 2.2%, plasminogen activator inhibitor (PAI-1) abnormalities 2.2% and dysfibrinogenemia 1.6%. The other TS were considered as very rare. Venous thromboembolism (VTE) was proved in 52% of TS (45% venous thrombosis and 7% combination of venous thrombosis and pulmonary embolism), arterial thrombosis was present in 5% and other complication in 3% of patients. Interesting finding seems high prevalence of SPS in patients with unexplained thrombosis (25%). Conclusion: Data from NRTS suggest that TS are common causes of thrombosis in Slovak population. The most frequent TS in families of patients with thrombosis were recognized FVL, prothrombin mutation and PS deficiency. Acknowledgement: Supported by the project CEPV II (ITMS 26220120036), project CEVYPET (ITMS 2622012053), grant Vega 1/ 0029/11 and 1/0016/12. Disclosure of Interest: None declared. Keywords: arterial thrombosis, national registry, pulmonary embolism, thrombophilia, venous thrombosis

PE24 Venous thromboembolism: a refined United States cost model with long-term attack rates Mahan CE1,*, Woersching AL2, Borrego ME2, Federici R3 and Spyropoulos AC4 1 Outcomes Research, New Mexico Heart Institute; 2 Pharmacoeconomics, Epidemiology and Pharmaceutical Policy and Outcomes Research Graduate Program, University of New Mexico College of Pharmacy; 3Cardiology, New Mexico Heart Institute, Albuquerque, NM; 4Division of Hematology/Oncology, University of Rochester Medical Center, Rochester, NY, USA Objectives: To model United States (US) costs for venous thromboembolism (VTE), comprised of deep vein thrombosis (DVT) and pulmonary embolism (PE). Total costs were calculated as well as hospital-acquired, ‘preventable’, and indirect VTE costs. Methods: A literature review determined VTE incidences, morbidity probabilities, case-fatality rates, and direct medical and indirect costs including the present value of lifetime earnings (PVLE) lost due to premature death. Authors developed decision trees for PE and DVT outcomes and cost models in Microsoft Excel. The decision trees contain primary VTE characteristics and outcomes: hospital- or communityacquired; fatal vs. non-fatal; readmission/recurrence vs. none; and instant vs. non-instant death. Initially surviving patients are at risk for associated morbidities: minor and major bleed; heparin-induced

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thrombocytopenia; chronic thromboembolic pulmonary hypertension; post-thrombotic syndrome; and no/partial resolution of symptoms, or death. The average patient’s cost, the sum of each decision tree pathway’s probability-weighted cost, were multiplied by low and high annual PE and DVT incident events to determine baseline total cost ranges. Two multi-way sensitivity analyses were conducted: the first with higher probabilities and costs and the second with long-term attack rates (LTAR), pooling incident and recurrent events. All costs were adjusted to 2011 US dollars. Results: Results are presented in the table. PE comprises the majority of VTE costs. Indirect costs due to PVLE represent at least 34% of total VTE costs. Image/Graph:

Conclusion: The US could avoid substantial costs ($4.5 to 39.3 billion), morbidity and mortality if appropriate VTE prophylactic strategies were universally implemented. This attainable goal would contribute to important health care reform. Given appropriate cost, incidence, and probability inputs, this model may also be utilized to estimate costs in other countries, or groups of countries such as the European Union. Disclosure of Interest: C. Mahan Consultant for: Sanofi S.A., Boehringer Ingelheim, Polymedix Inc. and Janssen Pharmaceuticals, Employee of: New Mexico Heart Institute, A. Woersching: None Declared, M. Borrego: None Declared, R. Federici Employee of: New Mexico Heart Institute, A. Spyropoulos: None Declared. Keywords: None.

PE25 Coagulation factor XIII-B His95ARG polymorphism and the risk of coronary artery disease in a Croatian population Bronic A1,*, Ferencak G2, Bernat R3, Dumic J4 and Dabelic S4 1 Clinical Institute of Chemistry, Traumatology Clinic, Clinical Hospital Center ′Sestre Milosrdnice′, Zagreb, Croatia; 2 Outpatients Clinic Medicol, Zagreb; 3Magdalena Special Hospital for Cardiac Surgery and Cardiology, Krapinske Toplice; 4 Department of Biochemistry & Molecular Biology, University of Zagreb, Faculty of Pharmacy & Biochemistry, Zagreb, Croatia Objectives: A relatively common A?G point transition in the codon 95 of the gene coding coagulation factor XIII B subunit (FXIII-B) results in histidine to arginine substitution (His95Arg FXIII-B) in the FXIII-B second Sushi domain and has been associated with an increased dissociation rate of the FXIII tetramer. There are controversial data regarding His95Arg FXIII-B impact on the pathogenesis of myocardial infarction (MI) and coronary artery disease (CAD). Our aim was to investigate the prevalence of this polymorphism and its association with CAD and MI risk among Croatians since these could appreciably vary between different populations. Methods: Association analysis was carried out in 208 subjects with angiographically proven disease (CAD+) and 142 subjects in whom disease was angiographically excluded (CAD–). CAD+ patients had >50% stenosis in at least one of the major coronary vessels. A diagnosis of MI was ascertained in 108 CAD+ patients, and an additional subgroup analysis was performed. FXIII-B genotypes were determined by restriction fragment length polymorphism of the PCR-amplified fragments of DNA isolated from the whole blood.

Results: Overall, AA genotype was recorded in 54.0% (53.8%CAD+ vs. 54.2%CAD ), AG in 39.1% (39.9%CAD+ vs. 38.0% CAD-), and GG in 6.9% (6.3%CAD+ vs. 7.7% CAD ) of subjects. There was no significant difference in genotype distribution between studied CAD groups (P > 0.05) as well as in the rare G allele prevalence (26% CAD+ vs. 27% CAD ; P = 0.954). On logistic regression analysis His95Arg FXIII-B genotype was not significantly associated with the CAD even when we assumed that the G allele is dominant and the heterozygous and homozygous subjects were combined (OR: 1.01, 95% CI: 0.80–1.27; P = 0.944). Similar results were obtained in subgroup analysis according to the history of MI. Conclusion: Obtained data do not point an association between His95Arg FXIII-B polymorphism and CAD risk in sampled Croatian subjects. Disclosure of Interest: None declared. Keywords: FXIII, coronary artery disease, His95Arg FXIII-B.

PE26 No correlation between mean platelet volume and the risk of recurrent venous or arterial thrombosis in patients with antiphospholipid syndrome ska E, Zajdel K, Ciepłuch K, Rupa-Matysek J*, Gil L, Wojtasin Lewandowska M and Komarnicki M Department of Haematology with Transplantation Unit, Poznan , Poland University of Medical Sciences, Poznan Objectives: Mean platelet volume (MPV) is associated with platelet reactivity and is a predictor of cardiovascular risk. Several reports have showed that an increased MPV correlates with venous thromboembolism (VTE). The aim of our study was to assess, if MPV could be an indicator of platelet activation in antiphospholipid syndrome (APS). The secondary objective was to identify the correlation between the value of MPV and thrombosis recurrence. Methods: A total of 226 patients (pts; median age 38, range 18–77) with a clinical suspicion of APS were analysed. According to the clinical probability of lupus anticoagulant (LA) detection, we found 6/21 (29%) pts from low, 42/135 (31%) pts from moderate and 41/117 (35%) pts from high risk group (Pengo V. et al. 2009). APS was diagnosed according to the updated Sapporo classification criteria with laboratory assays for LA, anticardiolipin antibodies and anti-B2 glycorptein-I (Siemens Healthcare Diagnostic). Finally, APS was diagnosed in 66 pts. A full blood count was analysed on automate analyser within 90 min of collection by CELL-DYN 3700SL analyzer (Abbott Diagnostics). Results: In the studied cohort, MPV was statistically significantly higher in males (7.9 1.43 vs 7.47 1.28, P = 0.029). The correlation analysis revealed that there was a significant inverse relation between platelet count and MPV in moderate and high group risk for LA diagnosis, r = 0.521 and r = 0.596; respectively (P < 0.001). MPV had a positive correlation with an increased ratio of LA1/LA2 (P = 0.013). In univariate analysis, history of pulmonary embolism, hereditary thrombophilia (presence of F5G1691A or F2G202010A) and male sex significantly correlated with thrombosis recurrence. No correlation was found between MPV in patients with or without APS and recurrence of venous or arterial thrombosis. Conclusion: Our results do not support the observation that MPV may be a potentially useful prognostic biomarker for prediction of recurrent VTE or arterial complications in patients with APS. Disclosure of Interest: None declared. Keywords: mean platelet volume, antiphospholipid syndrome, venous thromboembolism, thrombosis recurrence.


ABSTRACTS PE27 Developing animal models of hemolytic uremic syndrome Stearns-Kurosawa D*, Leibowitz C, Mayer C and Kurosawa S Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, MA, USA Objectives: Hemolytic Uremic Syndrome (HUS) is characterized clinically by thrombocytopenia, thrombotic microangiopathy and hemolytic anemia, giving rise to acute kidney injury and failure. HUS is a potentially fatal complication of Shiga toxins from enterohemorrhagic E. coli which infects150 million patients annually. New virulent strains are emerging, such as the German 2011 outbreak, and no toxin-specific drugs exist. Animal models are needed that develop HUS to permit screening (murine) and pre-clinical (nonhuman primate) testing of potential therapeutics. Methods: C57B/6J mice were challenged with endotoxin free Stx2 (1 ng/20 g i.p.; n = 5/group) on days 0, 3 and 6. Mice were weighed daily and blood drawn on alternate days. Baboons (Papio; 4–6 kg) were anesthetized and challenged with Stx1 or Stx2 (10–100 ng/kg; i.v.) +/- anti-toxin peptide (TVP; cell permeable) given i.v. at 6 and 24 h post-challenge. Tissues were collected at necropsy or upon euthanasia for pathology and EM microscopy. HMGB1 was measured by ELISA. Results: C57B/6J mice challenged with Stx2 had 4–6 days survival accompanied by progressive weight loss, increased plasma BUN and creatinine, but not thrombocytopenia. Anesthetized baboons challenged iv with Stx1 or Stx2 elicited HUS, renal failure and 3–6 days survival. Baboons were rescued from lethal Stx2 with TVP anti-toxin peptide with preservation of renal function, reduced thrombocytopenia, but prolonged anemia. Pathology analysis (EM) revealed more severe thrombotic glomerular endothelial damage after Stx1 (thickening, double contours), but apoptosis of mesangial cells only after Stx2. DAMP biomarkers in plasma (HMGB1, mtDNA) and urine (HMGB1) increased after Stx1 challenge earlier than traditional BUN or creatinine levels. Conclusion: Mice do not develop full HUS after Stx challenge, but are still useful for screening protective therapeutics. Nonhuman primates develop full-spectrum HUS and are a valuable animal model of HUS for pre-clinical testing of therapeutics. Disclosure of Interest: None declared. Keywords: Animal model, Enterohemorrhagic E. coli, HUS, Hyaline thrombus, Shiga toxin, Thrombocytopenia, Thrombotic microangiopathy.

PE28 Complications associated with picc insertion in a hospital for high level of complexity of the city of medellin – Colombia Combariza JF*, Roldan S, Galvez K, Olaya V, Ballesteros L and Palacios V Medellin Antioquia, Hospital Pablo Tobon Uribe, Medellin, Colombia Objectives: Identify for complications with peripherally inserted central (PICC). The PICC has thrombotic, infectious and traumatic complications being the biggest concern when deciding on the implantation of the same. Methods: This study was conducted as a retrospective observationalcutting between the months of January 2011 to November 2011. Data were obtained from electronic medical records with the approval of the ethic and research committee. We analyzed data from 200 patients who underwent insertion of a PICC Results: In this study of 200 patients found a median age of 42.4 years, 22 IPCC presented infectious complication for 11% of PICC collected [6 Infections caused by Klebsiella spp (3%), 5 Sthaphylococus spp © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

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infections (2.5%) 4 Proteus spp infections (2%), 6 Escherichia spp infections (3%) and 1 infection by Pseudomonas spp (0.5%)], thrombotic events were 2 (1%). The indications PICC placement were difficult venous access 53 catheters (26.5%), Chemotherapy 55 catheters (27.5%), TPN 26 catheters (13%), prolonged antibiotics 41 catheters (20.5%), hypokalemia 7 catheters (3.5%), No data eight catheters (4%), other 10 catheters (5%). The average days of stay for PICC were 29 days. The PICC that presented complications were removed in average 40 days of insertion and only three of them before day 10 mortality complications associated with PICC insertion was 0.0%. Conclusion: Complications associated with PICC insertion are more than 10% prevalence, infectious complications being the most frequently encountered in our center. It also shows that the PICC catheter stay exceeding 10 days is a identified risk factor for the submission of such complications, and despite of the elevated complications evidenced, there is no association with mortality in this group of patients. Disclosure of Interest: None declared. Keywords: picc, Thrombosis

PE29 Using haemostatic and rheologic factorials as diagnostic and prognostic markers of acute myocardial infarctions in Nigerians Ajayi IO1,*, Nwaeke G2 and Ihejirika OE2 1 Physiology, University of Benin; 2Haematology, University of Benin Teaching Hospital, BENIN CITY, Nigeria Objectives: The advent of MI and its diagnosis as a major cardiovascular problem is moderately recent in this part of the world. Therefore, researches into the responses of rheological, lipid and fibrinolytic parameters are modestly new and on-going.We therefore aimed to highlight basic information on haemorheologic and fibrinolytic parameters with a view to indicate their possible use as management indices in MI. Methods: Ten acute myocardial infarction (AMI) patients were studied longitudinally together with 20 age and sex –matched apparently healthy subjects as controls. Blood samples were taken at the point of admission (Day 0), on the 4th and 7th day respectively after treatment has commenced. Rheologic and fibrinolytic indices such as complete blood count (CBC), erythrocyte sedimentation rate (ESR), Plasma Fibrinogen concentration (PFC), D-dimer concentration (DDC), Euglobulin lysis time test (ELT) and Plasma viscosity (PV) as well as lipids were measured using standard methods. Results: We recorded a significantly reduced values of Haematocrit and fibrinolytic activity coupled with significantly increased D-dimer levels, PFC, ESR and PV in AMI patients on admission compared with controls (P < 0.05, respectively). However, PFC, DDC and PV became significantly lowered from the 4th day of admission while all the parameters became significantly reduced from the 7th day of admission and treatment (P < 0.05, respectively). On the other hand lipids such as Cholesterol and HDL remained relatively stable from admission and throughout the duration of the study. Conclusion: We conclude therefore that hyperfibrinogenaemia coupled with hypofibrinolytic activity and high plasma viscosities could be likely associated risk factors of thrombosis in Nigerians with AMI and their reduction during treatment are positive indicators and as factorials in its pathogenesis/prognosis. The role of lipids in AMI pathogenesis in Nigerians seem to be unpronounced. Disclosure of Interest: None declared. Keywords: Hyperfibrinogenaemia, Hypofibrinolysis, Myocardial infarction, Rheologic indices, Thrombosis.

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ABSTRACTS

PE31 Novel hemostatic markers for thromboembolic risk in patients with prostate cancer Toukh M1,*, Black A2, Siemens RD2 and Othman M1 1 Biomedical and Molecular Sciences, Queens University; 2 Department of Urology, Kingston General Hospital, Kingston, ON, Canada Objectives: To investigate the role of Thromboelastography (TEG) in assessing hemostatic status of patients with various stages of prostate cancer under different systemic therapies and to investigate whether TEG and/ or plasma procoagulant microparticles can help identification of those with higher risk for developing thromboembolic complications. Methods: We studied 36 patients diagnosed with prostate cancer aged 58–88 years and eight age and sex matched controls. With the exclusion of four patients for being on anticoagulant prophylaxis, 32 patients were divided according to their cancer status and line of management into: group A: watchful wait (11 patients), group B: androgen deprivation therapy (10 patients) and group C: castration resistant cancer (CRPC; 11 patients). We performed thromboelastography using citrated recalcified whole blood samples and flow cytometry analysis of plasma tissue factor- carrying procoagulant microparticles (TF-MPs) using FITC- Annexin V and PE-anti-human tissue factor antibodies. Results: Overall, 81% of patients showed hypercoagulable TEG traces. One way ANOVA showed significant difference in groups B and C with respect to R time and coagulation index CI values (P < 0.05). Plasma TF- MPs were significantly elevated in patients compared to the control (P = 0.014) with significant variations in groups B and C (P < 0.05). Prostate specific antigen (PSA) was found to be correlated with hypercogulability with a significant reversed correlation with each of CI (r = 0.535; P = 0.012) and TF-MPs (r = 0.38; P = 0.035) and a significant direct correlation with R time (r = 0.449; P = 0.011). Conclusion: To our knowledge, this is the first report to assess TEG and procoagulant plasma microparticles in relation to variuos stages of prostate cancer and to correlate these markers to PSA levels.TEG and TF-MPs have potential to hel P risk stratification and may guide towards the use of anticoagulant prophylaxis in these. Disclosure of Interest: None declared. Keywords: Thromboleastography, PSA, Tissue factor, VTE and PE.

PE32 Fibrinogen-like protein 2 (FGL-2) – a novel biomarker for cancer Inbal A1,*, Rabizadeh E2, Lederfine D2, Rosenbaum E3 and Brenner B3 1 Hematology; 21Hemato-Oncology Laboratory; 32Oncology Institute, .Beilinson Hospital, Rabin Medical Center, Petah Tikva, Israel Objectives: Hematologic and solid tumors are associated with hypercoagulability the reason for which has not been delineated. Prothrombinase named fibrinogen-like protein 2 (FGL-2) is a 70 kD transmembrane protein that was found to have a quality of a serine protease capable of directly cleaving prothrombin to thrombin. FGL-2 is synthesized by monocytes, T-lymphocytes and endothelial cells. FGL-2 protein and its mRNA have been previously found within different tumor cells. We studied the role of FGL-2 in patients with prostate and colon cancer. Our hypothesis is that upregulation of FGL-2 activity in cancer patients may contribute to tumorigenesis via generation of thrombin leading to increased angiogenesis and spread/metastasis of tumor cells. Methods: Thrombin generation reflecting FGL-2 activity was measured in homogenized peripheral blood mononuclear cells (PBMC) from patients with prostate and colon cancer after addition of prothrombin and compared to that of normal controls. mRNA of FGL-2

was measured in PBMC and malignant cell lines by quantitative RTPCR analysis. Results: Above 3-fold increase in FGL-2 activity in PBMC from patients with prostate or colon cancer was observed (P < 0.0001 for each) compared to that of normal controls. Similarly, immunoprecipitated FGL-2 caused 3-fold increase in thrombin generation. Increased mRNA of FGL-2 was found in PBMC from patients with colon cancer and from human prostate adenocarcinoma line (PC-3). Following treatment of PC-3 with INF-c the amount of mRNA increased even further. In line with this finding an increase in thrombin generation in PC-3 cell line (reflecting FGL-2 activity) was observed as well Conclusion: The results of this research suggest that FGL-2 may serve as a new biomarker of cancer in particular prostate and colon cancer. In the future FGL-2 activity may be used as a biomarker at diagnosis and during follow up. Disclosure of Interest: None declared. Keywords: cancer biomarker.

PE33 Case series: patients with JAK2 positive myeloproliferative neoplasms associated with thrombotic events Combariza JF*, Olaya V, Galvez K and Ballesteros L 1 Medellin Antioquia, Hospital Pablo Tobon Uribe, Medellin, Colombia Objectives: Describe the thrombotic events in a myeloproliferative neoplasms jak 2 positive. Methods: Retrospective description. Results: We present a case series of seven patients with a diagnosis of chronic myeloproliferative neoplasms associated with thrombotic events, which had performed the diagnostic test for the mutation of JAK 2, and the result was positive. We reviewed the clinical records of these patients, to extract information for analysis of different variables such as age, gender, hemoglobin, platelets and leukocytes, general symptoms, type of myeloproliferative neoplasms and outcome of bone marrow. Finding the following results: Of the seven patients 6 (86%) were women, showing a mean age of 57 years (SD + / - 10), hemoglobin 12 g/dL (SD + / - 2) with a count of platelets and leukocytes 662 714 (SD 266 887) and 20 286 (SD 11,521) respectively. Only 1 (14%) patient had splenomegaly, none referred sweats, weight loss or fever. Arterial thrombosis was documented in 3 (43%) patients, of which 100% was thrombosis of the middle cerebral artery; regarding venous thrombosis there were five cases documented (71%), of which 2 (40%) events thrombotic committed only the portal vein, 1 (20%) case committed the hepatic vein, 1 (20%) case of porto-mesenteric thrombosis, 1 (20%) patient had multiple events including thrombosis porto-mesenteric, pulmonary thromboembolism, venous thrombosis of upper limb. One of these patients presented arterial and venous thrombosis. The types of neoplasms were: polycythemia vera 2 (29%) patients, essential thrombocythemia in 3 (43%) patients and primary myelofibrosis in 2 (29%) patients. Conclusion: Most of these events occurred in female patient, with high counts of leukocytes and platelets, being the most frequent sites of the middle cerebral artery thrombosis and portal vein, found in most bone marrow fibrosis, with increased cellularity and megakaryocytic hyperplasia. Disclosure of Interest: None declared. Keywords: jak 2, myeloproliferative neoplasms, thrombosis.


ABSTRACTS PE34 Fibrinogen, C-reactive protein and interleukin-6 in prediction of cardiovascular complications and death in type 2 diabetes: the advance study Lowe GD1,*, Hillis G2, Chalmers J2, Woodward M2 and Rumley A1 1 MVLS CAMS, University of Glasgow, Glasgow, UK; 2George Institute, Sydney, NSW, Australia Objectives: We studied the associations of plasma levels of fibrinogen and of the inflammatory markers C-reactive protein (CRP) and interleukin-6 (IL-6) with macrovascular and microvascular complications and total mortality in a large cohort of men and women with type 2 diabetes. Methods: Plasma fibrinogen and CRP (immunonephelometry, Dade Behring) and IL-6 (ELISA, R & D Systems) were determined in nested case-cohort studies (n = 3865) of macrovascular (coronary heart disease and stroke) and microvascular complications and total mortality, within 11 140 participants in the ADVANCE Study of blood pressure control and glycaemic control in men and women aged 55 years or over with type 2 diabetes. Results: Fibrinogen, CRP and IL-6 levels were each associated with risk of macrovascular events in analyses adjusting for age, sex and treatment groups [HRs 1.16 (1.07, 1.27); 1.17 (1.09, 1.26) and 1.77 (1.56, 2.02) respectively]; but only IL-6 was associated in multivariate analyses [HR 1.69 (1.43, 1.99); P 0.001]. Fibrinogen, CRP and IL-6 were associated with all cause mortality in initial analyses [HRs 1.25 (1.14, 1.36); 1.22 (1.13, 1.32) and 1.87 (1.64, 2.13) respectively]; but only IL-6 was associated in multivariate analyses [HR 1.67 (1.42, 1.96)]. Fibrinogen and IL-6 were associated with risk of microvascular events in initial analyses [HR 1.14 (1.02, 1.28) and 1.25 (1.07, 1.47) respectively; but CRP was not [HR 1.00 (0.92, 1.10)]; however none were associated in multivariate analyses. Conclusion: Fibrinogen and CRP levels are unlikely to be useful additions to clinical risk scores for prediction of cardiovascular complications or mortality in persons with type 2 diabetes. In contrast IL-6 levels merit further evaluation in potential prediction of macrovascular complications and all cause mortality (but not microvascular complications) in such persons. Disclosure of Interest: None declared. Keywords: Coronary Heart Disease, Diabetes, Fibrinogen, Inflammation, Stroke.

Thrombo-Haemorrhagic Issues in Women and Children PEWC01 The association of smoking with venous thrombosis and its modification by hormone replacement therapy Blondon M1,*, Wiggins KL2, Rice KM2,3, Heckbert SR1,2, Psaty BM1,2 and Smith NL1,2 1 Department of Epidemiology; 2Cardiovascular Health Research Unit; 3Department of Biostatistics, University of Washington, Seattle, WA, USA Objectives: The evidence for an association between smoking and venous thrombosis (VT) remains inconsistent, and the nature of this association has not been reported among users of hormone therapy (HT). Our aim was to estimate the risk of VT associated with smoking and its interaction by HT. Methods: We included 2125 women with a validated, incident VT and 5749 matched controls from a large healthcare system in Washington State, from 1995 to 2008. Smoking status (current, former, never) was assessed from medical record review and, for a subset, also by telephone interview. The agreement between the 2 measures was excellent © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

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(Kappa = 0.84). Current hormone use was calculated from pharmacy databases, which record information on dispensed drugs. We used multivariate logistic regression models, adjusting for race, diabetes, pregnancy and body mass index. Results: Our sample was composed of mostly postmenopausal white women, with a mean age of 65 years and a smoking prevalence of 10%. Current smokers were at higher risk of VT compared with never-smokers (OR 1.2; 95%CI 1.0–1.5). This risk did not differ between women not using and using HT: OR 1.2 (95%CI 1.0–1.5) and 1.2 (95%CI 0.8– 1.9), respectively (p for interaction = 0.73). However, among women who had started HT recently ( 1 year. Further studies are needed to elucidate the association of smoking with VT during the first year after initiating HT. Disclosure of Interest: None declared. Keywords: Effect modification, Hormone replacement therapy, Interaction, Risk factor, Smoking, Venous thrombosis.

PEWC02 Long term follow up of homozygote protein c deficiency following multi modal therapy Monagle KE1,*, Ignatovic V2, Hardikar W3, Newall F4 and Monagle P1 1 Paediatrics, University of Melbourne; 2Murdoch Childrens Research Institute; 3Gastroenterology; 4Clinical Haematology, Royal Childrens Hospital, Melbourne, Vic., Australia Objectives: Homozygous protein C deficiency is an extremely rare condition presenting in the neonatal period with purpura fulminans, ophthalmologic complications, as well as cerebral and renal thrombosis and is associated with very high rates of morbidity and mortality. Optimal treatment for this condition is highly complex, poorly understood, and, in many countries, still difficult to obtain for cost and supply reasons. Methods: We report a clinical case report. Results: The child was born at term to consanguineous parents, who presented 2 days after birth with purpura fulminans, on both of his feet and buttocks. Further examination revealed bilateral severe retinal damage but normal brain imaging. Until protein C concentrate became available he was treated with fresh frozen plasma and Unfractionated heparin, resulting in fluid overload. After surgical management of the original skin purpura, he was managed with a regime of daily warfarin (maintaining a TTR 4–5). Clexane and protein C were used as salvage therapy, for recurrent episodes of purpura fulminans. During this time the patient’s progress was complicated by glaucoma requiring bilateral lensectomies, and recurrent episodes of otitis media. This was continued until 2 years of age when daily subcutaneous protein C replacement therapy was used, before his liver transplant at 3 years of age. The patient is now 12 years of age, well, with blindness as his only long term deficit. Conclusion: This study is the first to report the sequential use of multimodal therapy, and follow up of a patient 10 years post successful liver transplant with normal levels of protein C antigen and activity. Furthermore, we describe here our use of lab parameters to guide the multimodal therapy. Disclosure of Interest: None declared. Keywords: Homozygous protein C deficiency, multi modal therapy.

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PEWC03 Administration of antithrombin concentrate in infants and children on ECLS improves anticoagulation

PEWC04 Clinical findings in a prospective cohort of women in childbearing age with thrombosis and/or pregnancy adverse outcomes

Ryerson L1, Bruce AA2,*, Massicotte PM3, Bauman ML4, Ghotra S5, Lequier L6 and Bauman ME3 1 Pediatric Cardiology and Critical Care; 2Pediatric Hematology, Oncology & KIDCLOT program; 3KIDCLOT program, Stollery Childrens Hospital, University of Alberta; 4Pediatrics, Stollery Childrens Hospital; 5Pediatrics, University of Alberta; 6Pediatric Critical Care, Stollery Childrens Hospital, Edmonton, Canada

S anchez-Luceros A1,2,*, Meschengieser SS1, Grosso SH1, Blanco A1, Ingratti M1, Alberto MF1 and Lazzari MA1,2 1 Thrombosis and Haemostasis, National Academy of Medicine; 2 IMEX, CONICET-ANM, Buenos Aires, Argentina Objectives: Thrombophilic (TF) disorders have increased their role in women’s health, presenting such as thrombotic events as pregnancies failures too. We evaluated prospectively childbearing age women with thrombosis and/or pregnancy adverse outcomes to determine the frequency of TF and the relationship to clinical findings. Methods: Three hundred and fifty-seven women (mean age 34.6) (JanDec 2010) were consecutively included (N): Group 1: arterial (37) and venous (56) thrombosis; Group 2: (a) early recurrent miscarriage (60); (b) 1 or more unexplained fetal deaths at or beyond the 10th week of gestation (126); (c) 1 or more premature births before the 34th weeks (2) and/or intrauterine fetal death (57), because severe preeclampsia/ placental insufficiency (20). Acquired and genetic TF screening was performed: anti-phospholipid antibodies (APA) (lupus anticoagulantLA, SSC-ISTH, IGG/IGM anti-cardiolipin antibodies-ACL), Protein S, Protein C, Antithrombin, polymorphism of IIG20210A (Prot), factor V Leiden (FVL). Venous occlusion test (VOT) (euglobulin clot lysis time) was done in Group 2. APA was confirmed to diagnosis Antiphospholipid Syndrome (APS). Details about obstetric and thrombotic history were analyzed Results: Results are described in Table. All FVL and Prot patients were heterozygous. LA* and ACL* refers to transient positive. Conclusion: There was no difference in the frequency of predisposing factors in TF vs. non-TF patients (p= ns) in Group 1, being hormones the most frequent one (41.3%). APS diagnosis is frequently associated to arterial and venous thrombosis in young women, regardless the presence of precipitating factors. In obstetric adverse outcome, the frequency of every TF disorder depends on obstetric history. However, FVL and Prot frequencies in this cohort were similar to our general population. Disclosure of Interest: None declared. Keywords: adverse pregnancy outcome, arterial thrombotic events, thrombophilia diagnosis, venous thrombosis.

Objectives: In infants and children the levels of antithrombin (AT) are lower than adults and AT may be consumed in illness. In order to achieve an anticoagulant effect an AT level of 30% is required. Unfractionated heparin (UFH) is required on extracorporeal life support (ECLS) to maintain circuit patency. When high dose UFH is inadequate to maintain an anticoagulant effect, AT concentrate (ATC) is a method of increasing AT levels and heparin effect. The supplier recommended dose is 50 l/kg/dose. The primary objective of this study was to review our experience giving a vial of ATC (~1000 U) to patients on heparin. Specifically, examining AT levels pre and post administration of high dose AT, evaluate the efficacy of high dose AT administration as measured by the anti-Xa levels, evaluate the sustainability of AT and adverse effects. Methods: A retrospective chart review of all infants and children on ECLS who received ATC between April 2003 and May 2011 at Stollery Children’s Hospital was performed. Medical charts were reviewed to assess the influence of ATC on AT levels and to determine the effect of ATC administration on the level of anticoagulation as measured by the anti Xa level and activated partial thromboplastin time (PTT). Descriptive statistics were used to evaluate our results. Paired ttests compare results pre and post-ATC administration. Patients were analyzed both as a total patient group and separately in those patients six months and > six months. Results: Eighty-nine doses of ATC were administered to 38 patients (median age 1.2 months (0.13–190 months) on ECLS. Median dose of ATC was 294U/kg (range 25–512 U/kg). Mean AT level pre administration was 0.42 and 0.89 post administration. Mean heparin level increased from 0.23 pre AT administration to 0.37 (P < 0.001). There were no associated adverse events including bleeding. Conclusion: The administration of high dose ATC in children and infants on ECLS is safe, and increases the binding sites for UFH as reflected by increased anti-Xa level. Disclosure of Interest: None declared. Keywords: Antithrombin, Children, extracorporeal life support, Heparin, Infants, unfractionated heparin.

PEWC05 Anticoagulation of cardiomyopathy in children Monagle KE1,*, Jones S2, King I3, Weintraub R4, Monagle P1 and Newall F2 1 Paediatrics, University of Melbourne; 2Clinical Haematology, Royal Childrens Hospital, Melbourne; 3Murdoch Childrens Research Institute, Melbourne; 4Cardiology, Royal Childrens Hospital, Melbourne, Vic., Australia Objectives: Cardiomyopathy is a common cause of heart failure, and heart transplantation in children. Altered heart function due to ventricle dilation, muscle hypertrophy or restrictive filling reduces intracar-

Group 1 Arterial Venous

Group 2

Normal Stroke Others Pulmonary Embolism DVT Unusual site abc-

19 0 5 21 8 34 70 51

APS 7 9 3 8 4 11 17 8

LA*

ACL*

FVL/Prot 1/0 1/0 0/1 3/0

4 9 5

3 4 4

0/1 2/3 5/3

PC/PS

Hypofibrinolysis

1/2 1/0

7 21 1


ABSTRACTS diac blood flow, which directly increases risk of thrombosis. Thus, warfarin is recommended by the American College of Chest Physicians for the treatment of children with Cardiomyopathy despite the lack of published evidence to support its use. Methods: A retrospective clinical audit to determine the safety and efficacy of oral anticoagulant therapy (warfarin) for primary thromboprophylaxis in a cohort of children with Cardiomyopathy. Relevant outcomes including thrombosis and major haemorrhage were defined a priori according to internationally accepted definitions. Results: Thirty-seven children (36.06 warfarin years) were examined, with 24.3% of children taking warfarin for > 1 year. Primary reasons for discontinuation of warfarin therapy were cardiac transplantation (n = 7), transition to VAD (n = 1), resolution of cardiomyopathy (n = 8), transfer of care (n = 6), and death (n = 2). The median age of children starting warfarin was 2.6 years (range 0.2– 15.2). 18.9% of all patients studied were under the age of 1 at the time of commencing warfarin. The most common Target Therapeutic range (TTR) for warfarin therapy was 2.0 – 3.0. TTR achievement was normally distributed and occurred in a mean 47.4% of all INR tests, and 51.3% of all tests in children over the age of 2 years. There were 0 warfarin related adverse events, including thrombosis or haemorrhage. Conclusion: The low rate of TTR achievement is consistent with previously reported TTR achievement rates for infants. In addition the low rate of TTR achievement was likely influenced by the clinical profile of this complicated condition in children. Nonetheless, this data shows that the clinical outcomes for this cohort are acceptable and warfarin therapy can be safe in children with cardiomyopathy. Disclosure of Interest: None declared. Keywords: anticoagulation, cardiomyopathy, children.

PEWC06 Predicting deep venous thrombosis in pregnancy: external validation of the ‘left’ clinical prediction rule Righini M1,* and Le Gal G2 1 Department of Angiology and Hemostasis, Geneva University Hospital, Geneva, Switzerland; 2Department of internal medicine and respiratory medicine, Brest University Hospital, Brest, France Objectives: Assessment of clinical probability is an important step in the management of patients with suspected deep vein thrombosis (DVT). The recently derived ‘LEFt’ clinical prediction rule combines three variables: symptoms in the left leg (‘L’), calf circumference difference above of two centimeters (‘E’ for edema) and first trimester presentation (‘Ft’). However, the rule has not been externally validated yet. Methods: The LEFt rule was computed among pregnant women with suspected DVT included in a prospective diagnostic management outcome study. The proportion of women classified into each clinical probability group was computed, as well as the proportion of confirmed DVT in these groups. Results: All variables needed to compute the rule could be retrieved in 156 out of the 167 pregnant women with suspected DVT. The prevalence of confirmed DVT was 15/157 (9.6%). The ‘LEFt’ rule was negative in 45 (29%) women. A DVT was diagnosed in 15/112 (13.4%, 95% CI: 8.3–20.9%) of women with at least one of the ‘LEFt’ criteria, as compared with 0/45 (0.0%, 95% CI: 0.0–7.9%) of women with none of the ‘LEFt’ criteria. Conclusion: A negative ‘LEFt’ rule accurately identified pregnant women in whom the proportion of confirmed DVT appears to be very low. Disclosure of Interest: None declared. Keywords: clinical prediction rule, deep vein thrombosis, pregnancy.


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PEWC07 The economic costs of routine inr monitoring in infants and children Gaw J1, Crowley S2, Monagle P1,3,*, Jones S1 and Newall F1,3,4 1 Clinical Haematology, The Royal Children’s Hospital; 2The Centre for Health Policy, Programs and Economics; 3Paediatrics; 4 Nursing, The University of Melbourne, Melbourne, Australia Objectives: The use of point-of-care (POC) devices within the home for routine INR monitoring has demonstrated their suitability and effectiveness in the management of infants and children requiring long-term warfarin therapy. However, the cost-effectiveness of using this method of management, compared to attending anticoagulation clinics has not been reported. This study examined the direct and indirect societal costs involved when using POC devices for routine INR monitoring compared to standard care. Methods: The study used a comparative before-and-after design of 60 infants and children (1–18 years of age) managed via the haematology department at a tertiary paediatric centre. Each participant was exposed to both modes of management at various times for ≥ 3 months. A purpose-built questionnaire, consisting of 25 questions was sent to families to complete and return via a reply-paid envelope. Data collected included; the frequency of monitoring, mode of travel to and from clinics, total time consumed, primary caregiver income level and employment status. Health sector costs were calculated using institutional records and local data. A human-capital approach was used to assign value to an individual’s time. Results were pooled together and statistically analyzed using student’s t-test. Results: The home monitoring cohort saved a mean total time of 1 h 19 min (P = 0.019) per test compared to attending anticoagulation clinics. There was a mean total cost saving to society of $66.83 (AUD) (P < 0.001) per test compared to standard care, incorporating health sector costs, travel expenses and lost time. Conclusion: The standard model of care requires a considerable investment of time per test from the child’s caregivers. This is the first study to report upon the economic consequences for infants and children requiring long-term INR monitoring. The recommendation of this study is that home INR monitoring in infants and children has greater societal economic benefits compared to standard care. Disclosure of Interest: None declared. Keywords: Children, Cost-effectiveness, Home INR monitoring, Point-of-care devices, Warfarin.

PEWC08 Cerebral te in Italian infants and children: data from the Italian registry of childhood thrombosis (RITI) Gentilomo C1,2,*, Saracco P3, Bagna R4, Bassi B5, Agostini M6, Giordano P7, Ramenghi LA8, Molinari AC9, Laverda AM2, Grassi M7, Putti MC10, Suppiej A11, Sartori S11, Simioni P12 and Italian Group of Childhood Thrombosis Registry 1 Pediatrics, Angel Hospital, Venezia-Mestre; 2Pediatrics, Padua University, Padua; 3Hematology, OIRM - S.Anna Children’s Hospital; 4Neonatal Intensive Care Unit, OIRM - S.Anna; 5 Pediatric Neurology, OIRM-S.Anna Children’s Hospital; 6 Pediatrics, OIRM-S.Anna, Torino; 7Hematology, Bari University, Bari; 8Neonatal Intensive Care Unit; 9Hematolgy, Gaslini Hospital, Genova; 10Hematology; 11Pediatric Neurology; 12 Department OD Cardiologic, Vascular and Thoracic Sciences, Padua University, Padua, Italy Objectives: Pediatric thromboembolism (TE) is an increasingly common problem, requiring high awarness by pediatricians. Data on epidemiology of pediatric TE in Italy are lacking. The Italian Registry of childhood Thrombosis (RITI) is the first national multicentre longitu-

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dinal registry, to determine the current epidemiology of pediatric TE in our country. Methods: RITI prospectively enrolls systemic and cerebral TE events in neonates (N: 0- 28 d) and children (P: 29 day–18 years). Launched by May 2010 it includes cases diagnosed since January 1st 2007. Results: Out of 352 TE events enrolled by RITI up to Sept 30th 2011, 239 refers to cerebral TE. Arterial ischemic stroke (AIS): 64 P (M 60%) and 46 acute N (M 71%) events were enrolled. 25% of P and 42% of N cases were defined idiopathic. Multiple risk factors were found in 35% of children, being vasculopathies (29%), heart diseases (27%) and infections (27%) the most frequent. Maternal disorders (39%) and perinatal complications (23%) were the main neonatal associated risk factors. Thrombophilic defects were found in 45% of investigated children and in 40% of newborns. Only 40% were diagnosed within 24 h, 20% within 6 h. Antithrombotic drugs were administered in 84% of P AIS, 7% of N cases. 64% of P and 52% of N presented neurological deficits at discharge, compared to 69% and 50% respectively, 12 months after. Sinovenous thrombosis (CSVT): 82 P (M 71%) and 37 N (57%) events have been enrolled. 26% of P cases and 57% of neonatal cases were defined idiopathic. Thrombophilic defects were found in 56% of P and 17% of Ncases. 26% of P and 48% of N cases presented neurological deficits at discharge; 35% of neonates 12 months after. Conclusion: RITI is becoming an important instrument to study pediatric TE in our country and hopefully it might rapresent an opportunity for investigators both Italians and from other registries to collaborate on multicentres studies. Disclosure of Interest: None declared. Keywords: cerebral thromboembolism, children, registry.

PEWC09 Prospective study on incidence rate of neonatal and pediatric thromboembolism in a single tertiary care hospital in Italy during a 5-year period Saracco P1,*, Viano A2, Bagna R3, Bassi B4, Agostini M2, Agnoletti G5, Barisone E6, Farinasso D3, Cascarano MT7, Barberis L3, Magnetti F3, Gallo E3, Plazzotta C8, Bonaudo R9, Migliore G2, Gentilomo C10, Pollio B1, Simioni P11, RITI, Registo Italiano Trombosi Infantili and Infantili RIT 1 Hematology; 2Pediatrics; 3Neonatology; 4Neuropsichiatry; 5 Cardiology; 6Oncology; 7Cardiosurgery; 8Quality Management; 9 Nephrology, ASO Ospedale Regina Margherita S.Anna, Torino; 10 Pediatrics, University of Padova; 11Internal Medicine, University of Padova, Padova, Italy Objectives: Thromboembolism (TE) in neonatal (N) and pediatric (P) age is an emerging problem requiring utmost attention; epidemiological studies on incidence and risk factors are sparse. Recent retrospective studies report a dramatic increased rate of venous TE up to 70% (from 40 to 58/10.000 admissions) and of acute ischemic stroke=AIS up to 50% (3–4.2/10.000), in hospitalized children at tertiary care hospitals=TCH in US, in 2001–2007. This is the first prospective study assessing incidence rate and risk factors of both N and P TE at a single Obstetric and Pediatric TCH in Italy. Methods: All consecutive TE cases (STE = systemic, SVT = sinovenous and AIS), uniquely diagnosed at our TCH from 2007–2011 (41525 alive births, 2771 admissions to N Intensive Care Units = NICU and 35847 to P hospital) were prospectively registered and data included in the Italian National Registry - RITI. Discharge ICD-9-CM codes for TE were also analyzed. Results: A total of 85 cases, 25 N (6/10000 births) and 60 P (17/10.000 admissions) were reported. ICD-9-CM code analysis missed 30% of cases. Male sex was predominant (N 88%,P 58%). Incidence rate/ 10.000 admissions to Nursery was 2.65 (1.3 STE, 0.72 SVT and 0.72 AIS); to NICU was 36 (14.4 STE, 7.2 SVT, 4.4 AIS). Among P annual rate of TE during study period increased from 8.64 up to 29/10000

admissions (STE 9.62 to 20.53; SVT 4.94 to 6.84), except AIS (4.94 to 3.42). In 80% of cases at least one risk associated condition was present. N:maternal (52%), cardiopathy (48%), infections (44%) and CVL (24%). P:infections(29%), CVL (29%), surgery(24%), malignancy (17%)and cardiopathy(19%). 41%SVT was associated with malignancy and 37%AIS with cardiopathy. Inherited thrombophilia was found in 48% N and 31% P. Conclusion: Although significantly lower compared to US data, this study confirms increased incidence of STE in hospitalized children, in all age groups, in a single TCH in Italy. Understanding the cause of this increment is important for determining best prevention strategies. Disclosure of Interest: None declared. Keywords: epidemiology, neonatal, pediatric, thrombosis.

PEWC10 Obstetrics outcomes associated with an increased level of lipoprotein-a (LP-a) in thrombophilic patients Recchi D, Barros VV*, Baptista FS, Bortolotto MRDFL, Francisco RPV and Zugaib M Obstetrics and Gynecology, Clinics Hospital, University of Sao Paulo, Sao Paulo, Brazil Objectives: An increased level of Lipoprotein-A(Lp(a)) can be a new marker for adverse pregnancy outcome and/or thromboembolism risk during pregnancy. Our objective was to research Lp(a) levels in pregnant patients with thrombophilia and/or thromboembolism. Methods: The method used for Lp(a) measurement was immunoturbidimetry(reference value for women was up to 11.0 mg/dL). Values over 100 mg/dL were considered high. Results: We studied 300 patients diagnosed with deep vein thrombosis (current or past) and/or thrombophilia. They were divided into two groups: patients with increased Lp(a)(Group A), and patients with negative values (Group B). Group A (GA) had 94 patients and Group B (GB) had 206 patients. There was no difference in age or race between GA and GB. The average age was 29 years, the white group equaled 58% and black or brown equaled 27% in both groups. The diagnosis of fetal growth restriction was similar in both groups:GA (23%) and GB (19%). The newborns birth weights of GA and GB were similar. The reporting of previous gestational hypertension was 15% in GA (14/94p) compared with 8% in GB (17/206p),but it was highly significant the previous occurrence of eclampsia in GA (3/94p) and no reporting in GB(P < 0.05) .Placental abruption were higher in the group of patients with high titles of Lp(a)(6/19p, 31%) compared to the group with low titles of Lp(a) (11/75p, 14%)(NS). When comparing GA with GB for the incidence of placental abruption the difference was very significative(18% vs 2.9% in GB(6/2006)( P < 0.01). A significant finding was the higher occurrence of thombosis in pregnancy/ puerperium in GA (20p, 18%) than in GB (20p, 9%)( P < 0.04). The reporting of recurrent thrombosis were higher with high titles of Lp(a) (3/19p, 15%),when compared with GB (19/206, 9%)(NS). Conclusion: These findings suggest that elevated Lp(a) is a new thrombophilic risk factor in pregnancy and probably a new marker to be investigated in patients with severe forms of hypertension in pregnancy. Disclosure of Interest: None declared. Keywords: adverse pregnancy outcome, enoxaparin, lipoprotein-A, pregnancy, thrombosis, trombophilia.


ABSTRACTS PEWC11 Molecular basis of type I antithrombin deficiency in two women with recurrent venous thromboembolism in the first trimester of pregnancy Xia Y1,*, Ding Q2, Wang X2, Lu Y2 and Dai J2 1 State Key Laboratory of Medical Genomics,Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiaotong University School of Medicin; 2Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Objectives: Inherited antithrombin (AT) deficiency carries a 50% risk of venous thromboembolism (VTE) during pregnancy. Here, we investigated the molecular basis of type I AT deficiency in two women with recurrent VTE in the first trimester of pregnancy. Methods: Polymerase chain reaction (PCR)[A1] amplification of all exons and intron-exon junction regions of AT gene, followed by direct sequencing, was performed for the probands. In vitro expression experiments were performed in HEK293 or CHO cells transfected with either wild-type (WT) or mutant AT expression vectors. AT concentrations in the media and cell lysates were assayed by ELISA and western blot analysis. Intracellular localization of AT proteins was detected by immunofluorescence analysis. [A1]Although PCR is a commonly used abbreviation, please consider expanding it in the abstract. Results: Two novel heterozygous AT mutations were identified: g.7920 C>T resulting in a missense mutation (Trp225Cys) in case 1 and g.13863C>A causing an Ala404Asp mutation in case 2. In vitro levels of AT:Ag in Trp225Cys and Ala404Asp mutants were 4.8 0.4% and 7.9 0.6% of the AT-WT levels in the media, and 123.3 3.9% and 65.6 2.8% in cell lysates[A1], respectively. Immunofluorescence analysis revealed that the staining of AT-Trp225Cys in both endoplasmic reticulum (ER) and Golgi apparatus was similar to that of ATWT, and the staining of AT-Ala404Asp was mainly present in ER and was weaker than that of AT-WT. [A1]Please check if my revision conveys the intended meaning. Conclusion: The type I AT deficiency in two patients was caused by impaired secretion of the AT-Trp225Cys and AT-Ala404Asp mutant proteins, respectively. The two mutations are associated with a high risk of thrombotic onset and women with these AT mutations are prone to VTE in early pregnancy. Disclosure of Interest: None declared. Keywords: Antithrombin, Gene mutation, Pregnancy, Venous thromboembolism.

PEWC12 Higher doses of low molecular weight heparin (LMWH) are needed to achieve goal Anti-Xa levels in critically ill children Schloemer NJ1,*, Abu-Sultaneh S1, Hanson S1, Yan K2, Hoffmann RG2, Punzalan R3 and Havens P4 1 Pediatrics – Critical Care; 2Pediatrics – Quantitative Health Sciences; 3Pediatrics – Hematology; 4Pediatrics – Infectious Disease, Medical College of Wisconsin, Milwaukee, WI, USA Objectives: Critically ill patients have multiple confounding factors affecting drug dosing. Studies have suggested that critically ill premature neonates and adults may need higher doses of subcutaneous low molecular heparin (LMWH) to achieve a therapeutic Anti- Xa level of 0.5–1.0 IU/mL which may be related to impaired skin perfusion and edema. The optimal therapeutic dose of LMWH in critically ill children is unknown. We hypothesize that critically ill children require increased LMWH dosing compared to conventional dosing algorithms to achieve therapeutic Anti-Xa activity levels.


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Methods: This study is a retrospective chart review of children (1 day to 18 years) admitted to a tertiary level pediatric intensive care unit between January 2005 and December 2010 receiving LMWH Enoxaparin. Premature neonates (corrected gestational age < 37 weeks) and patients with renal failure on dialysis were excluded. Demographics, LMWH dosing, anti-Xa levels, and inotrope score (IS) were collected. Mann–Whitney test was used to compare the groups. Results: Patients (n = 261) with LMWH dosing q12 h and anti-Xa levels checked 3–6 h after dosing were included for analysis. Only 55.2% of patients with initial dose based on current dosing guidelines achieved therapeutic anti-Xa level from the initial dose. Patients who achieved therapeutic anti-Xa from the initial dose had lower IS (mean 0.33 0.12 vs. 0.87 0.20 for non-therapeutic, P = 0.015) and older age (median 99 (IQ range 5.3–191) vs. 7 months (IQ range 1.2–122) for non-therapeutic, P < 0.001). Patients on inotropes/vasopressors required higher LMWH dose 1.36 (IQ range 1.05–1.58) vs. 1.03 mg/kg (IQ range 0.98–1.46), P = 0.020. 18% of patients never achieved therapeutic anti-Xa levels during the hospitalization. Conclusion: Some critically ill children require higher doses of low molecular weight heparin to achieve a therapeutic anti-Xa level. Younger age and inotrope use are factors associated with need for higher dosing. Disclosure of Interest: None declared. Keywords: anti-Xa, CRITICAL CARE, low molecular weight heparin, PEDIATRIC.

PEWC13 The effect of antithrombin concentrates on angiogenesis in seriously ill children Karlaftis V1,*, Attard C1, Monagle P1,2,3 and Ignjatovic V1,2,3 1 Haematology, Murdoch Childrens Research Institute; 2Clinical Haematology, Royal Children’s Hospital; 3Paediatrics, The University of Melbourne, Melbourne, Australia Objectives: Antithrombin (AT), an important physiological anticoagulant exists in multiple isoforms. A conformational change in the native AT (NAT) protein can result in formation of the anti-angiogenic isoform, latent AT (LAT) that is also associated with the onset of severe and sudden thrombosis. Seriously ill children are administered AT concentrates (ATC) purified from adult plasma to compensate for the decreased AT activity in this population. As a direct result of this purification, ATC products contain up to 40% LAT. Due to the anti-angiogenic nature of LAT, administration of ATC may impair growth and development, particularly in neonates and children. This study aimed to establish the effect of ATC administration on markers of angiogenesis in seriously ill children. Methods: Blood samples were collected pre and post-ATC administration from seven children undergoing intensive care. The corresponding plasma samples were used in a multiplex ELISA to measure the concentration of 9 different markers of angiogenesis1. Results: There was a trend towards a decrease in three out of nine markers, namely angiopoietin-2, follistatin and IL-8. Overall, higher doses of ATC were administered to children < 1 year which was associated with a trend towards a decrease in markers of angiogenesis. In contrast, lower doses of ATC were administered to children > 1 year which was associated with a trend towards an increase in markers of angiogenesis. In a particular patient, subsequent administrations of ATC resulted in a trend toward a decrease in markers of angiogenesis upon the third dose of ATC. Conclusion: These results suggest a potential effect of ATC on angiogenesis, and hence growth and development in this vulnerable patient population. The clinical implications of these results require further investigation in a larger number of patients. Disclosure of Interest: None declared.

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Keywords: Angiogenesis, Antithrombin, Antithrombin concentrate, Children, Latent antithrombin.

PEWC14 The prevalence and role of elevated coagulation factors VIII, IX and XI in arterial thrombosis in children Avila M*, Zanette A, Canil T, Williams S, Kahr W, Brandao LR and Paediatric Haemostasis Thrombosis Paediatrics, The Hospital for Sick Children, Toronto, Canada Objectives: To investigate the prevalence of elevated FVIII, FIX, and FXI levels (>90th percentile for age) and their potential association to adverse outcomes (thrombus recurrence or lack of thrombus resolution). Methods: A retrospective cohort of subjects with radiologically proven ATE (excluding stroke), diagnosed between January 1996 and December 2008 was reviewed. Results: One hundred and thirty subjects (1.36M: 1F ratio) with radiologically proven ATE (excluding stroke) followed for a median of 2.6 years (IQR 1–4.6 years) was analyzed. Median age was 34 d (interquartile range [IQR] 0.3–7.2 mo; 60 neonates). 69 children (53%) had isolated ATE, and 61 patients (47%) had combined venous and ATE. Only five cases were idiopathic, all of which were exclusively ATE. Thrombi distribution encompassed upper vascular territory (28.5%), lower vessel territory (55.4%), and both territories (16.1%). Seventy four patients (57%) had occlusive thrombus at presentation; 127 patients (97.7%) received anticoagulant therapy and 10 cases (7.6%) had also received fibrinolysis. The overall prevalence of elevated FVIII, FIX, and FXI was 19.2%, 1.0% and 4.0%, respectively; for exclusive ATE, it was 24. 5%, 1.9%, and 5.8%, respectively. Nine patients (7%) had recurrent thrombosis and 71 (54.6%) showed total thrombus resolution on follow-up imaging. Univariate analysis showed no statistically significant association between elevated FVIII, FIX, and FXI and lack of resolution (Fischer’s exact test, P = 1.0, P = 0.39 and P = 0.06, respectively) and between elevated FVIII, FIX, and FXI and recurrence (Fischer’s exact test, P = 1, P = 0.24 and P = 0.22, respectively). Conclusion: In conclusion, FVIII elevation is the most prevalent finding among factor elevation in pediatric ATE, even when considering isolated ATE. However, factor elevation does not seem to be associated with unfavorable ATE outcomes in children. Disclosure of Interest: None declared. Keywords: arterial thrombosis, arterial thrombotic events, factor FVIII.

PEWC15 Assisted reproduction technologies failure and acquired thrombophilia: experience of a single specialized Argentinian center Begue G1,*, Sanchez-Luceros A1,2, Meschengieser SS1, Grosso SH1, Blanco A1, Alberto MF1 and Lazzari MA1,2 1 Hemostasis and Thrombosis Department, Academia Nacional de Medicina; 2IMEX, CONICET-ANM, Buenos Aires, Argentina Objectives: Thrombophilia has been lately associated with recurrent miscarriage and other pregnancy adverse outcomes. Unsuccessful implantation (UI) and placentation have been considered as potential explanations for the high failure rate of assisted reproduction technologies (ART). However, there is still conflicting evidence about the causative link between acquired thrombophilia (AT), UI and ART outcome. Methods: We have evaluated a prospective cohort of 81 consecutive infertile women (January to December 2011), from a single specialized Hemostasis and Thrombosis Department in Argentina, to determine

the relationship between ART and AT. Patients were included after at least one high complexity ART failure, in spite of good embryos quality. Lupus anticoagulant (LA), anticardiolipin antibodies (ACA), euglobulin clot lysis time (ECLT) with venous occlusion response were evaluated. LA was defined according to the SSC-ISTH criteria. ACA were detected using a commercial kit (ELISA INOVA ACA III). Results: The average age of the patients was 38 years old (29–49). 58 (71%) patients had primary infertility (never having achieved a positive Human Chorionic Gonadotropin blood test). Overall, 75.3% of women had other plausible cause of infertility (endocrinologic diseases 29.6%, endometriosis 22%, Fallopian tube obstruction 21%, ginecologic resection surgery 12%, smoking 9.8%, poor ovarian reserve 6%). 51 (62%) of the patients had normal test results. LA was identified in 15 patients, but its persistence could only be demonstrated in nine cases. All of six cases expressing ACA had low antibodies title, only two demonstrated persistent positivity. ECLT was prolonged in nine cases. No significant differences were found between primary and secondary infertility. Conclusion: LA was detected in 11% of the patients studied. Although we do not have a control group, LA seems to be more prevalent in this group than in general population. However, we can not confirm its causative association. Disclosure of Interest: None declared. Keywords: Assisted Reproduction Technologies, Infertility, Lupus Anticoagulant, Thrombofilia.

PEWC16 Hereditary antithrombin deficiency and pregnancy Desancho MT1,*, Levine R2, Cohen J3, Chapin J4, Kalish R5 and Shah S3 1 Medicine Hematology-Oncology, New York Presbyterian; 2 Transfusion Medicine, Lennox Hill Hospital; 3Medicine Hematology-Oncology, Stony Brook; 4Medicine HematologyOncology; 5Obstetrics and Gynecology, New York Presbyterian Hospital, New York, NY, USA Objectives: To describe eight natural pregnancies in five women with hereditary antithrombin (HAT) deficiency. Methods: We evaluated eight pregnancies in five women with HAT deficiency at a tertiary care center. Data collected included age, type of AT deficiency, personal and family history of venous thromboembolism (VTE), dose and adjustment of low molecular weight heparin (LMWH) based on anti-Xa activity, switch to unfractionated heparin (UFH) and postpartum anticoagulation. Antithrombin replacement, type of anesthesia and method of delivery were recorded. Maternal, infant complications and pregnancy outcomes were also evaluated. Results: Mean age was 37 years (32–41). Of the five women, three had type 1 AT deficiency and two had type 2 AT deficiency. Three had a history of VTE and all five had family history of VTE. Six pregnancies required therapeutic dose and 2 prophylactic dose LMWH. High-dose LMWH (120 mg every 12 h) was required on 4 pregnancies. Dose adjustments occurred in seven pregnancies; one was switched to UFH in the 3rd trimester. All women received AT concentrate at the time of delivery (6 plasma-derived AT and 2 recombinant-AT). Neuroaxial anesthesia was given in six pregnancies. Six of the pregnancies were delivered vaginally and two with cesarean section. All women received postpartum anticoagulation. Two women developed DVTs during the pregnancy (1 at day 3 and another at week 10 of pregnancy). One woman had premature contractions at week 29 and delivered at 36 weeks of pregnancy. Another woman had premature labor at 32 weeks and delivered within a few hours. Two women had transient visual abnormalities and one developed thrombocytopenia. One infant was born with a congenital diaphragmatic hernia and another infant was premature. All pregnancies resulted in live-births. Conclusion: Women with hereditary AT require close monitoring during pregnancy. High doses of LMWH are required for adequate anti-


ABSTRACTS coagulation. Administration of AT concentrates at the time of delivery minimizes the risk of peripartum complications. Disclosure of Interest: None declared. Keywords: anticoagulation, Antithrombin, pregnancy.

PEWC17 Venous thromboembolism in Italian infants and children: data from the italian registry of childhood thrombosis (RITI) Gentilomo C1,2,*, Saracco P3, Giordano P4, Bagna R5, Molinari AC6, Grassi M4, Putti MC7, Lasagni D8, Indolfi G8, Simioni P9 and Italian Group Of Childhood Thrombosis Registry 1 Pediatrics, Padua University, Padua; 2Pediatrics, Angel Hospital, Venezia-Mestre; 3Hematology, OIRM - S.Anna Children’s Hospital, Torino; 4Hematology, Bari University, Bari; 5Neonatal Intensive Care Unit, OIRM - S.Anna, Torino; 6Hematolgy, Gaslini Hospital, Genova; 7Hematology, Padua University, Padua; 8 Pediatrics, Meyer Children’s Hospital, Firenze; 9Department OD Cardiologic, Vascular and Thoracic Sciences, Padua University, Padua, Italy Objectives: Pediatric thromboembolism (TE) is an increasingly common problem, requiring high awareness by paediatricians and standardized protocols at children’s hospitals. Data on epidemiology of paediatric TE in Italy are lacking. The Italian Registry of childhood Thrombosis (RITI) is the first national multicentre longitudinal registry, to determine the current epidemiology of paediatric TE in our country. Methods: RITI prospectively enrols systemic and cerebral TE events in neonates (N: 0–28 day) and children (P: 29 day–18 years). Launched by May 2010 it has included cases diagnosed since January 1st 2007. Results: Out of 352 TE events enrolled by RITI up to September 30th 2011, 80 refers to systemic venous thrombosis (VTE): 56 P VTE (50% M) and 18 N events (M 67%). Age at onset of paediatric VTE shows two peaks between 1 and 5 yrs (32%) and 11–15 years (30%), and a few cases older than 15 yrs. VTE was mostly associated to iatrogenic factors (CVL, drugs and surgery). 43% o f N cases and 54% of P VTE were CVL-related. 4% of P cases and 12% of neonatal were defined idiopathic. At least one prothrombotic defect was found in 56% of P cases and 44% of neonatal cases. The superior venous system has been the most affected (42%) especially the jugular veins. 95% on children and 83% of neonates received antithrombotic drugs, with great variability in treatment duration. The overall mortality rate was 10% and the recurrence rate 11% among children. No deaths nor recurrences were observed among neonates. Systemic sequelae were observed in 28% of P (post-thrombotic syndrome in 17%) and 50% of N cases. Conclusion: Our series show an underrepresentation of adolescent patients. Being Paediatric Thrombosis and Haemostasis centres rare in Italy, adolescent might have been mainly referred to adult centres, and not included in RITI. RITI is becoming an important instrument to study paediatric TE in our country. Disclosure of Interest: None declared. Keywords: Children, registry, venous thrombosis.

PEWC18 Thromboembolic complications in Italian infants and chidren: comparison of data from RITI registry with a single centre consecutive serie Saracco P1, Gentilomo C2,3,*, Bagna R4, Viano A5, Agostini M6, Simioni P7 and Italian Group Of Childhood Thrombosis Registry 1 Hematology, OIRM - S.Anna Children’s Hospital, Torino; 2 Pediatrics, Padua University, Padua; 3Pediatrics, Angel Hospital, Venezia-Mestre; 4Neonatal Intensive Care Unit, OIRM - S.Anna; © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

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5

Pedaitrics; 6Pediatrics, OIRM-S.Anna, Torino; 7Department OD Cardiologic, Vascular and Thoracic Sciences, Padua University, Padua, Italy

Objectives: The worldwide institution of national and international registries of childhood thromboembolism (TE) is helping to define the epidemiology of neonatal (N) and pediatric (P) thromboembolism at various countries with different ethnicities. Methods: A prospective National Registry started in Italy (R.I.T.I.) in 2010, collecting data on N and P TE (STE=systemic, SVT=sinovenous, AIS=acute ischemic stroke) diagnosed since 1.1.2007. We report on incidence, risk factors and outcome of TE by analyzing RITI national data (323 TE at 31.12.2011) collected by more than 80 investigators at pediatric hospitals from most Italian regions and comparing them with those (85 TE) of a single obstetric-pediatric tertiary care hospital (Torino OIRM S.Anna). Results: Among 323 cases in RITI: 114 were N (35%), 193 P (60%) and 16 presumed perinatal (5%); 111 AIS, 108 SVT, 104 STE. Males represent 65% of the cohort. One or more underlying risk conditions (N: maternal 26%, CVL 12%, infections 17%, cardiac diseases 23%; P: infections 27%, CVL 18%, surgery 18%, malignancy 15%) were present in 64%. Thrombophilia was evidenced in 30% of screened cases (55.7%). Overall mortality and recurrence rate was 2% during hospitalization. Comparing data versus those of single centre (85 TE: 25 N(30%), 60 P (68%); 25 AIS, 17 SVT, 43 STE), the most significant differences were: overrepresentation of stroke cases in RITI (as most participating investigators during pilot phase being pediatric neurologists) and major incidence of neonatal risk factors at single centre (being an obstetric regional reference TCH). Conclusion: The RITI national registry by prospectively enrolling cases of N and P TE from various regional hospitals in Italy is providing correct epidemiological data and it will represent an opportunity for investigators of other registries to perform multicenter collaborative studies on childhood TE. Disclosure of Interest: None declared. Keywords: children, registry, Thrombosis.

PEWC19 Low levels of vitamin d in pregnant women with thromboembolism/thrombophilia GALLO L1, Barros VV1,*, Baptista FS1, Bortolotto MRDFL1, Francisco RPV1 and Zugaib M1 1 Obstetrics and Gynecology, Clinics Hospital, University of Sao Paulo, Sao Paulo, Brazil Objectives: A vitamin D deficiency in pregnant women could drive to pre-eclampsia, gestational diabetes and is associated to thrombophilia. Its deficiency and vitamin D receptor polymorphism are frequent in autoimmune diseases like lupus erythematosus systemic.Besides, antiphospholipid antibodies (antib2GPI) are pathogenic and can cause disease interfering with homeostatic reactions of the vascular cells surface and platelets.Vitamin D was able to inhibit the tissue factor expression induced by the anti b2GPI in vitro. It is speculated that vitamin D deficiency in vivo results in inhibition of expression of tissue factors promoting coagulation. Aims: establish the frequency of vitamin D inadequacy among thrombophilic patients from Obstetrics Department Ambulatory, University of Sao Paulo, Brazil. Methods: patients had their 25-hidroxyvitamin D serum levels analyzed by chemo-immunoassay method during three opportunities: without and with 400 UI or 2000 IU per day supplementation when still insufficient. Results: Seventy-six patients were studied. The diagnosis of diseases/ conditions associated most frequently were antiphospholipid antibody syndrome (42.7%), recurrent pregnancy loss(32%), superficial or deep vein thrombosis/ pulmonary embolism (37.3%), increased lipoproteinA (33.3%), systemic lupus erythematosus (21.3%), deficiency of factor V Leiden (6.7%), protein S/C deficiency (6. 7% each one), mutation of

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ABSTRACTS

MHFR hetero/ homozygous (6.7%), cerebral thrombosis/ stroke (5.3%). The results of vitamin d level are in Table1. Table1. Profile of serum 25 hydroxyvitamin D (ng/mL): three consecutive measurements: Mean (SD)/ median

Deficiency Insufficient Normal N (%) N(%) N(%)

1st dose (n = 76) 20.8 (6.9)/21.0 30 (39.5) 2nd dose (n = 52) 22.4 (6.2)/22.0 14 (26.9) 3rd dose (n = 34) 26.1 (6.7)/25 3 (9.1)

38 (50) 30 (57.7) 23 (69.7)

8 (10.5) 8 (15.4) 8 (24.2)

Conclusion: There was a high prevalence of inadequacy of vitamin D in patients with thrombophilic/autoimmunity disorders. Disclosure of Interest: None declared. Keywords: antiphospholipid syndrome, enoxaparin, lupus, osteopenia, pregnancy, trombophilia, vitamin D.

PEWC20 Placental abruption and thrombophilia Arissa K, Barros VV*, Baptista FS, Bortolotto MRDFL, Francisco RPV and Zugaib M Obstetrics and Gynecology, Clinics Hospital, University of Sao Paulo, Sao Paulo, Brazil Objectives: To investigate the presence of thrombophilias in cases of placental abruption. Methods: We have studied 22 patients who presented with placental abruption for the presence of the following thombophilic factors: factor V Leiden, prothrombin G20210A gene mutation, hyperhomocysteinemia, deficiencies of protein C, protein S, and antithrombin, antiphospholipid antibodies. Results: Eighteen patients had 22 pregnancies (4 had a recurrent abruption), the medium age was 29 years and 1 had a twin pregnancy. 19 newborn were studied, 4 (21%) were restricted. The medium gestational age were 36 weeks (range 31–40 weeks).Onewas listed as stillborn fetuses (5%), another died in the early neonatal period (5%) and one had tetralogy of Fallot (5%). All the others survived. An emergency c –section were done in all cases. A previous bad pregnancy outcome were present in 61% of the patients, one (5%) had systemic lupus erythematosus, five (27%) had hypertension in pregnancy where the detachment occurred, six (33%) had chronic hypertension, one (5%) sickle cell anemia, one (5%)diabetes, three (16%) were smokers, one (5%) had uterine fibroids. One (5%) patient developed thrombosis during pregnancy. Four (22%) had already had previous thrombotic events and two (11%) a cerebral stroke.Eleven (61%) had at least one thrombophilia. Of these, eight (72%) had only one abnormal test result, two (18%) had two changes and one (9%) of them had four changes simultaneously. None of the patients studied showed changes in the factor V Leiden, factor II or in homocysteine;one (9%) presented deficiency of antithrombin, two (18%) of protein C and two (18%) of protein S. Three (27%) patients had positive ANA. Two patients had antiphospholid syndrome (18%). Conclusion: The analysis of the data obtained in our department showed the presence of some factor of thrombophilia in eleven cases (61%). Deficiencies of protein C, protein S and antiphospholipid syndrome were equally present. Disclosure of Interest: K. Arissa: None Declared, V. Barros Speakers Bureau: Sanofi- Aventis, F. Baptista: None Declared, M. R. BORTOLOTTO: None Declared, R. FRANCISCO: None Declared, M. ZUGAIB: None Declared Keywords: FII G20210A, factor V Leiden, placental abruption, pregnancy, trombophilia.

PEWC21 Pediatric thrombosis educational tool (P-TET): an educational project in development for children receiving oral anticoagulation Cassis FR1, Carneiro JD2, D’Amico EA3 and Brand~ ao LR4,* 1 Hemophilia and Thrombosis Center, Hospital das Clinicas FMUSP,San Pablo, Brazil; 2Children’s institute,HCFMUSP; 3 Hemophilia and Thrombosis Center, San Pablo, Brazil; 4pediatric thrombosis, The Hospital for sick children, Toronto, Canada Objectives: Thrombotic events (TE) are becoming more common in children and a multidisciplinary approach for their treatment is required. The use of oral vitamin K antagonists (VKA), a common therapeutic option for children with TE, is particularly challenging within this population due to the narrow therapeutic window of VKA. Attractive educational tools that engage parents and children to improve their understanding about VKA-therapy and the disease are likely to benefit compliance, patient satisfaction, and ultimately, increase the patient’s time within therapeutic range (TTR). Additionally, affordable educational tools could be widely used by trained health care providers to improve the quality of care delivered. Objective: To develop a low cost educational instrument providing age-appropriate information related to VKA-therapy in children. Methods: An educational instrument containing visual flashcards and a short explanatory manual for children and their caregivers was developed. Areas of knowledge (ie. domains) pertinent to VKA clinical practice defined a priori by one adult and two pediatric hematologists were selected for its content. Results: P-TET flashcards encompasses five different domains (vascular anatomy, blood coagulation process, recognition of TE, anticoagulant management and anticoagulant side effects). A pre- and postassessment evaluation for knowledge retention, as well as comparison of TTR pre- and post-assessment will be conducted on two prospective pediatric cohorts in Brazil and Canada. Conclusion: The management of children receiving VKA is likely to benefit from the use of a new and accessible VKA-specific educational tool. This newly created visual flashcard instrument will be tested in two prospective cohorts of children with different economic and cultural backgrounds. Disclosure of Interest: None declared. Keywords: Educational tools, flashcards, VKA treatment, research, psychoeducation.

PEWC22 Thromboprophylaxis with two different dosages of lwmh (calcic nadroparin) in pregnant women with thrombophilia: a single center experience Samantha P*, Barillari G, Venturelli U and Turello M Center for Hemorrhagic and Thrombotic Diseases, University Hospital of Udine, Udine, Italy Objectives: Pregnancy is a hypercoagulable state in which coagulation is favorite and thrombolysis inhibited. When in pregnant women are also present inherited or acquired thrombophilia, risk of VTE is increased approximately six times compared with non-pregnant women of similar age. The aim of our study was to investigate the efficacy of thromboprophylactic therapy with two different dosages of LMWH in thrombophilic pregnant women with moderate or high risk for VTE. Methods: One hundred and fourty-six thrombophilic pregnant women were enrolled and divided in two groups. 1. Patients with moderate risk for VTE given the presence of hyperhomocysteinemia, ACE or PAI-I polymorphisms, MTHFR or FVH2R mutation and treated with LMWH 60–80 IU/kg/twice daily; 2. Patients with high risk for VTE given the presence of FV Leiden mutation, FII G20210A mutation,


ABSTRACTS LAC; previous VTE and treated with LMWH 80–100 IU/kg/twice daily. For both groups puerperal thromboprophylaxis was made at standard dosages 0.4 mL/day, until 42th day post-partum. Results: Mean age of our patients was 36 years. 46/146 patients presented a family history of VTE. Group 1. (68/146 pts): 17/68 (25%) had previous VTE: 22.1% DVT or SVT, 2.9% PE. 72% presented FV Leiden mutation, 26.5% FII G20210A mutation, 13% LAC. In this group, we have reported 2 abortions, 97% of successful pregnancies and no case of VTE. In 1/68 case the therapy was interrupted cause of allergy to LMWH. Group 2. (78/146 pts): no cases of previous VTE, 89.7% presented hyperhomocysteinemia, among these 46.6% had still MTHFR mutation, 21.8% presented FVH2R mutation, ACE polymorphisms or PAI-I polymorphisms were present respectively in 32% and 35.9% of patients. 97.6% were successful pregnancies. No cases of VTE were found. 1/78 case of mild PPH was resolved spontaneously. During puerpuerium in all 146 patients no adverse events were reported. Conclusion: In our study prophylaxis with LMWH may be considered for women with confirmed thrombophilia at moderate or high risk of VTE to reduce thromboembolic risk. Disclosure of Interest: None declared. Keywords: Thomboprophylaxis, pregnancy, thrombophilia.

PEWC23 The risk of venous thromboembolic during pregnancy Ouarhlent YY1,*, Boudjerra N2 and Bounecer H1 1 Hematology Service, CHU of Batna Algeria, BATNA; 2 Hematology Service, CPMC Alger, Alger Objectives: Thromboembolic disease is a pathologie quite frequent, the incidence of venous thromboembolic complications during pregnancy is between 0.1 to 0.9% depending on the studies include the postpartum period or not. Hyperhomocysteinemia as a factor favoring the occurrence of thrombosis heightens the thrombogenic state of pregnancy, knowing that hyperhomocysteinemia may be genetic or acquired. acquired by deficiency of essential co-factor in methionine metabolism (vitamin B12, folic acid) To assess the prevalence of thrombosis in pregnant deficient in vitamin B12 and / or folic acid. Methods: This is a prospective study at the centers of Maternal and Child Health between June 2009 and June 2010. The samples have been realized in 375 parturients from a total of 27 105 deliveries during this period. Parturients have benificied a blood count, serum ferritin, the determination of vitamin B12, serum folate, erythrocyte folate, hemostasis .But the Doppler ultrasonography has been practiced in some cases of suspicion of thromboembolic disease. Results: The average age of parturients was 30.11 6.9 years, the prevalence of iron deficiency, folate and vitamin B12 are respectively 49.3%, 37.6% and 5.2%. Four parturients had developed venous thrombosis of the lower limbs (left limb in 3 cases and one case, right-) three pregnant had vitamin B12 and fourth vitamin B9 deficiency .The prevalence of thrombosis in pregnant deficient in vitamin B12 is 15% and 0.7% of folic acid. The conduct was contention and mobilization in these patients because the risk was moderate, they were thrombocytopenic, with a curative treatement of vitamin deficiencies. Conclusion: Venous thromboembolic remains a major public health problem and the correct use of elastic compression has proven effective in patients with moderate risk. It is the constant wearing of an elastic adapted to measure the lower limbs during pregnancy and the long postpartum period Disclosure of Interest: None declared. Keywords: deficiency, folic acid, pregnancy, prevalence, Thromboembolic, vitamin B12.


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Haemorrhagic Disorders (Lab Studies) and Von Willebrand Disease VWF01 Detection of non inhibitory binding antibodies to von Willebrand factor affecting the clearance of VWF:AG in von willebrand disease Suiter T1,*, Mannucci PM2, Kempton CL3, Laffan M4, Romond EH5, Shapiro AD6, Birschmann I7, Ragni MV8, Gill JC9, Yee TT10, Klamroth R11, Horling FM1, Reipert BM1, Turecek PL1, Varadi K1, Chapman M1, Engl W1, Wong WY12, Ewenstein BM12 and rhVWF Study Team 1 Baxter Innovations GmbH, Vienna, Austria; 2The University of Milan, Milano, Italy; 3Department of Pediatrics, Div Pediatric Hematology, Emory University, Atlanta, GA, USA; 4 Hammersmith Hospital Department of Hematology, Imperial College School of Medicine, London, UK; 5Hemophilia Treatment Center, University of Kentucky, Lexington; 6Indiana Hemophilia and Thrombosis Center, Indianapolis, IN, USA; 7 Klinik f€ ur H€ amatologie, H€ amostaseologie, Onkologie und Stammzelltransplantation, Medizinische Hochschule Hannover, Hannover, Germany; 8Division Hematology/Oncology, 10University of Pittsburgh and Hemophilia Center of Western Pennsylvania, Pittsburgh, PA; 9Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA; 10Centre for Haemostasis & Thrombosis, St. Thomas Hospital, London, UK; 11Hemophilia Treatment Center, Vivantes Klinikum im Friedrichshain, Berlin, Germany; 12Baxter Healthcare Corp., Westlake Village, CA, USA Objectives: Von Willebrand Disease (VWD) is an inherited rare bleeding disorder caused by a deficiency of von Willebrand Factor (VWF). VWF 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 pdVWF/FVIII concentrates. Clinical impact of non-inhibitory antibodies to VWF has not been previously reported. To investigate the immunogenicity of a novel recombinant human VWF (rhVWF), total binding and inhibitory anti-VWF antibodies were assessed in a Phase 1 study. Methods: Neutralizing antibodies to the key functional activities of VWF (VWF:RCo, VWF:CB and VWF:FVIIIB) were measured using Bethesda-based assays (Nijmegen modification). To exclude false positive results, the detection limit for anti-VWF inhibitors was set to 1 BU/mL for all three assays. Samples were considered positive if they were at least 2 titer steps lower than the antibody titer detected in the screening assay. Results: 3/39 subjects had a pre-existing high titer non-neutralizing binding antibody (all 1/1280) to VWF; 1 of these 3 was excluded from study due to the presence of an inhibitory antibody to VWF:CB (1.3 BU/mL) at screening, and the other 2 were treated with rVWF and/or pdVWF concentrate during the study. The high titer binding antiVWF antibodies were associated with a significant decline in the VWF:Ag activity of either pdVWF or rVWF and consequential decreased activity of VWF:RCo, VWF:CB and FVIII:C. One of the subjects had a reduced VWF:Ag IR of 1.1 IU/dL/(U VWF:RCo/kg) (mean 1.6 IU/dL/[U VWF:RCo/kg]) and a reduced VWF:Ag t1/2 2.4 h (mean t1/2 25.3 h) post infusion of rVWF (50 IU VWF:RCo/kg). Conclusion: None of the study subjects developed an inhibitory antibody to VWF or FVIII and there was no impact on the subsequent treatment of bleeding episodes with commercial pdFVIII/VWF concentrates. The clinical relevance of non-neutralizing antibodies to VWF requires additional investigation. Disclosure of Interest: T. Suiter Employee of: Baxter Innovations GmbH, P. Mannucci Consultant for: Baxter Innovations GmbH, C.

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ABSTRACTS

Kempton Consultant for: Baxter Innovations GmbH, M. Laffan Consultant for: Baxter Innovations GmbH, E. Romond Consultant for: Baxter Innovations GmbH, A. Shapiro Consultant for: Baxter Innovations GmbH, I. Birschmann Consultant for: Baxter Innovations GmbH, M. Ragni Consultant for: Baxter Innovations GmbH, J. Gill Consultant for: Baxter Innovations GmbH, T. T. Yee Consultant for: Baxter Innovations GmbH, R. Klamroth Consultant for: Baxter Innovations GmbH, F. Horling: None Declared, B. Reipert Employee of: Baxter Innovations GmbH, P. Turecek Employee of: Baxter Innovations GmbH, K. Varadi Employee of: Baxter Innovations GmbH, M. Chapman Employee of: Baxter Innovations GmbH, W. Engl Employee of: Baxter Innovations GmbH, W. Y. Wong Employee of: Baxter Healthcare Corp., B. Ewenstein Employee of: Baxter Healthcare Corp. Keywords: von Willebrand Factor, von Willebrand Disease.

VWF02 Biochemical and molecular characterization of 10 Italians patients affected by von willebrand disease type 2A/IIE Pagliari MT*, Garcia Oya I, Stufano F, Cozzi G, La Marca S, Canciani MT, Peyvandi F and Baronciani L U.O.S. Dipartimentale per la Diagnosi e la Terapia delle Coagulopatie, A. Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione I.R.C.C.S. Ca’ Granda Ospedale Maggiore Policlinico, Universita delgli Studi di Milano and Luigi Villa Foundation, Milan, Italy Objectives: Von Willebrand disease (VWD) type 2A/IIE is due to a multimerization defect of Von Willebrand Factor (VWF) caused by dominant mutations in the D3 domain. It is characterized by normal/ relative decrease of high-molecular-weight multimers (HMWMs), absence of the triplet structure and often a mildly reduced binding of VWF to FVIII (VWF:FVIIIB). Recently, this variant has been reported to be relatively frequent in Germany (Schneppenhaim et al Blood 2010; 115, 4894). In this study 10 unrelated Italians VWD type 2A/IIE patients were identified and fully characterized. Methods: Conventional phenotype tests were used to evaluate patients plasma and platelets. Direct sequence analysis of exons 20–28 was performed using patient genomic DNA. RT-PCR and sequence analysis were performed using platelet VWF mRNA from patients 3–6. Image/Graph:

Results: Seven patients showed a reduced VWF:RCo/Ag ratio (T, whereas, in the fourth patient a novel mutation c.33802A>G led to the same alteration. Only two novel missense mutations were identified (p.C1142F and p.L1281R). Conclusion: This report shows that VWD type 2A/IIE is relatively frequent, also among the Italian patients. The heterogeneity of patients phenotype might mislead their classification. However, the appropriate use of multimer analysis at intermediate resolution, along with the molecular characterization, can be useful for a correct classification. Disclosure of Interest: None declared. Keywords: 2A/IIE, triplet structure, VWF.

VWF03 Two cases of congenital thrombotic thrombocytopenic purpura associated with pregnancy Bai X*, Su J, Yu Z, Wang Z and Ruan C Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, Suzhou, China Objectives: Congenital thrombotic thrombocytopenic purpura(TTP) is a homozygous or compound heterozygousautosomal recessive disorder caused by the deficient activity of ADAMTS13, a metalloprotease, which cleaves von Willebrand factor(VWF) under shear stress. And infusion of fresh plasma is the most effective treatment to this kind of disease. Methods: Here we report two casesof congenital TTP and treatment with or without infusion of fresh plasma. The ADAMTS13 activities were measured using the recombinant FRET-VWF73 during their acute episodes and their inhibitors were assayed by 9:1 mixture of patient and pooled normal plasma followed by ADAMTS13 activity assay using the VWF multimer electrophoresis. All 29 exonsof ADAMTS13 were amplified and sequenced with the correspondingprimers. Results: The ADAMTS13 activities of two cases were both less than 5%. and the absence of ADAMTS13 inhibitor was confirmed. The DNA sequencing results demonstrated that one case was homozygous for R692C combined with one polymorphism of S903L, while the other case carried compound heterozygous mutations in ADAMTS13 gene: one heterozygote mutation in exon 25 resulting in R1095W; one novel heterozygote splice mutation in intron 12 (c.1435 + 2T>G).Thrombocytopenia occurred during the second trimester in the case with R1095W/c.1435 + 2T>G in her first pregnancy, and she underwent termination of pregnancy followed by infusion of fresh plasma every two weeks until now. The other case with R692C/ S903L suffered from transient thrombocytopenia after she delivered her first child ten years ago and the second child ten years later though no plasma infusion was administed and she remains health. Conclusion: The mutation including R1095W/c.1435 + 2T>G and novel combination of mutation R692C and polymorphism S903L may be the pathogenic mechanism of congential TTP and treatment with fresh plasma may improve the disease. Disclosure of Interest: None declared. Keywords: thrombotic thrombocytopenic purpura(TTP),congenital, pregnancy, ADAMTS13, mutation.

VWF04 A new automated chemiluminescent method for the determination of von Willebrand factor ristocetin cofactor activity (VWF:RCO) and von willebrand factor antigen (VWF:AG) Friedman KD*, Jacobi PM, Shaw ME, Endres JL and Haberichter SL Hemostasis Reference Lab, Bloodcenter of Wisconsin, Milwaukee, WI, USA © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

ABSTRACTS Objectives: Automated methods using lyophilized platelets have reduced variability of the VWF:RCo assay, the ‘gold standard’ in VWD diagnosis and management. However, analytic sensitivity and precision remain issues. Chemiluminescent assays offer enhanced analytical sensitivity. We report our pilot evaluation of the analytical and clinical performance of a fully automated, paramagnetic particle-based chemiluminescent methods for determination of VWF:Ag and VWF: RCo. VWF:RCo assay is based on the binding of VWF to recombinant GPIba fragment in the presence of ristocetin. Methods: Thirty normal donors and 70 VWD patients (42 Low VWF, type 1 or type 2N VWD; 25 type 2A, 2B or 2M VWD; and 3 type 3 VWD) were tested with current methods (VWF:Ag by ELISA, VWF: RCo on Siemens BCS device) and with chemiluminescent VWF:RCo and VWF:Ag assays on an ACL AcuStarTM. CV was defined by repeat testing of two control plasmas and linearity was assessed by serial dilution of two samples from 1 to 64 fold. Results: AcuStar linearity was excellent for both VWF:Ag and VWF: RCo, R2 > 0.99. CV% of controls was 6.1–9.2% for VWF:RCo and 3.2–3.8% for VWF:Ag. Results obtained with the AcuStar panel were compared to clinical diagnosis. The area under the curve obtained by receiver operator characteristic (ROC) analysis of diagnostic accuracy for the ratio of VWF:RCo over VWF:Ag to detect types 2A, 2B and 2M VWD was 0.84 with suggested cut-off ratio of 0.7. Using this cutoff, the panel reported correctly 30/30 Normal, 36/42 Type 1 and 2N, 22/25 Type 2(A, B and M), and 3/3 Type 3 VWD samples. Misclassification occurred in 6/12 type 1 samples with VWF:Ag < 26 IU/dL and in 3/8 type 2M samples. Conclusion: In this pilot study, automated AcuStar von Willebrand factor assay showed excellent analytic linearity and precision for VWF:Ag and VWF:RCo. ROC analysis demonstrated diagnostic utility of the VWF:RCo to VWF:Ag ratio, however, accurate distinction between type 1 or type 2 VWD still requires integration of the ratio with other clinical data. Disclosure of Interest: K. Friedman Grant / Research support from: Instrumentation Laboratories, Consultant for: CSL, Alexion, Octapharma, P. Jacobi: None Declared, M. Shaw: None Declared, J. Endres: None Declared, S. Haberichter Consultant for: Instrumentation Laboratories. Keywords: assay, automation, von Willebrand Factor, von Willebrand Disease

VWF05 Head-to-head comparative study of the pharmacokinetic behavior of a high-purity factor ix concentrate (alphanineâ) and a recombinant factor ix (benefixâ) in patients with severe hemophilia B Aznar JA1,*, Moret A1, Cabrera N1, Matysiak M2, Zavilska K3, aez A6 and Gercheva L4, Antonov A5, Woodward MK6, P 7 Lissitchkov T 1 Hemostasis and Thrombosis, La Fe University Hospital, Valencia, Spain; 2Pediatric Hematology and Oncology Department, University Medical School, Warsaw; 3Hematology, Internal Medicine J. Strus Hospital, Poznan, Poland; 4Hematology, Sveta Marina University Hospital, Varna; 5Hematology, University

99

Hospital, Medical University, Pleven, Bulgaria; 6Clinical Trials and Pharmacovigilance, Instituto Grifols S.A, Parets del Valles, Spain; 7National Center of Hematology, Sofia, Bulgaria Objectives: Plasma-derived factor IX concentrates have consistently shown higher in vivo recovery (IVR) rates when compared to the only available recombinant FIX product. However, head-on comparative studies performed under standardized conditions are rarely conducted regardless of being a valuable instrument guiding healthcare providers towards better informed and cost-effective decisions Methods: The present study is a extension of a multicenter study that assessed the efficacy, safety, and pharmacokinetic profile (twice) of AlphaNineâ in 25 previously treated patients with severe hemophilia B (FIX activity ≤ 2%). After a washout period ≥ 7 days following the last PK performed with AlphaNineâafter a dose of 65–75 IU/kg, an identical PK study was performed with BeneFIXâ on 22 of the same patients. FIX activity was determined at baseline and at 0.25, 0.5, 1, 3, 6, 9, 24, 48, 72, and 74 hours postinfusion. The following parameters were evaluated with a model-independent method: IVR, half-life, area under the curve, mean residence time, and clearance. Results: The outcomes of the comparison of the selected PK parameters are summarized in Table 1. Conclusion: AlphaNineâ showed a comparable half-live but an IVR significantly higher than that of BeneFIXâ, despite BeneFIXâ displayed higher than expected IVR values. This dissimilarity may have implications on dosing requirements for both prophylactic and ondemand treatment regimes affecting optimal resource allocation. Disclosure of Interest: J. A. Aznar Grant / Research support from: Grifols provided support to this study, A. Moret Grant / Research support from: Grifols provided support to this study, N. Cabrera Grant / Research support from: Grifols provided support to this study, M. Matysiak Grant / Research support from: Grifols provided support to this study, K. Zavilska Grant / Research support from: Grifols provided support to this study, L. Gercheva Grant / Research support from: Grifols provided support to this study, A. Antonov Grant / Research support from: Grifols provided support to this study, M. K. Woodward Employee of: The author is employee at Grifols, A. Paez Employee of: The author is employee at Grifols, T. Lissitchkov Grant / Research support from: Grifols provided support to this study Keywords: AlphaNineâ, BeneFIXâ, comparison, Factor IX, FIX, hemophilia B, pharmacokinetics, recombinant, rFIX.

VWF06 Genetic diagnosis in sporadic haemophilia a families in China Lu Y* Department of Laboratory Medicine, Ruijin hospital, Shanghai, China Objectives: About 1/3 of haemophilia A families were sporadic. Genetic diagnosis in these families is necessary and it is important to determine the origins of the mutations. In this report, we made genetic diagnosis of 40 sporadic HA families. Methods: LD-PCR and PCR were adopted for the screening of the INV22 and INV1 respectively. The F8 coding and boundary

Parameter

AlphaNineâ PK1 (n = 25)

AlphaNineâ PK2 (n = 25)

AlphaNineâ Mean PK1 + 2

BeneFIXâ PK3 (n = 22)

In vivo recovery (IU/l:IU/kg) Half-life (h) Clearance (mL/min) AUC0 inf (IU h/dL) MRT0 nf (h)

1.0 (0.2) 35.5 (6.2) 0.058 (0.02)* 1556 (259) 33.7 (5.7)

1.3 (0.3)* 32.4 (8.8) 0.053 (0.02) 1684 (425) 31.7 (6.4)*

1.2 (0.2)* 33.9 (6.3) 0.056 (0.02) 1620 (306) 32.7 (5.6)*

1.0 (0.3) 36.0 (12.8) 0.046 (0.01) 1631 (467) 39.3 (13.9)

Results are expressed as Mean (SD), *P < 0.05 for the comparison of the AlphaNineâ group with the BeneFIXâ group. © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

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sequences were analyzed by sequencing. 7 STR sites related to F8 gene were combined together to apply to the linkage analysis. ABI PRISM SNaPshot ddNTP Primer Extension kit was used to analyze the chimerism. Results: These 40 HA families were caused by INV22 (13/40), small insertions (6/40), small deletions(5/40), missense mutations(10/40) and nonsense mutations(6/40). From the linkage analysis, we confirmed that the origins of the mutations. For INV22 families, eight were from the normal maternal grandfathers (MGF) and the others from the carrier maternal grandmothers (MGM). For the INV22 negative families, 13 index’s mothers were normal according to the sequencing, the other 14 were carriers, in which 6 of the mutations were from MGM and the others from the MGF. Mosaicism was identified in two families with the mutation of c.G96085T and c.T32874A, respectively. Somatic mosaicism was identified in both of the grandmother’s blood and oral cells with the percentage of 10.62% and 11.01% mutant (c.G96085T) respectively. 33% mutant (c.T32874A) was detected in the index’s mother’s blood cells while the sequencing demonstrated the normal results. We estimated the limit of the mosaicism detection as 2% mutant allele. Conclusion: Based on the study, we can reasonably suggest that intron 22 and 1 inversions screening combined with the linkage analysis using 7 STR sites are available for carrier detection and prenatal diagnosis in Chinese HA families. The extention primer PCR method is a good approach to detect the mosaicism and can give information which may prove valuable in determining the origin of new haemophilia mutations, and in counselling relatives as well. Disclosure of Interest: None declared. Keywords: genetic diagnosis, haemophilia A, mosaicism, sporadic.

VWF07 Prevalance and treatment outcome of transfusion transmitted infections in hemophilia A Alkindi S1, Kashoob M2, Balkhair A2, Al Obaidani N3 and Pathare A3,* 1 Department of Haematology, Sultan Qaboos University; 2 Department of Medicine; 3Department of Haematology, Sultan Qaboos University Hospital, Muscat, Oman Objectives: Our objective was to highlight the occurrence of transfusion transmitted viral infections [TTI] even today and assess the treatment outcomes in those who were treated. Methods: Retrospective analysis of Hemophilia A database. HIV-1 and 2, HCV and HBV were tested by ELISA followed by further testing with Western Blot in retrovirus positive samples. Viral load testing by PCR was available since 2002. Results: Out of the total of 93 Hemophilia A patients registered at our institution since 1992, 17 (18.3%) were observed to have developed TTI’s. Of these, 13(76.5%) had Anti-HCV positivity [6 HCV RNA PCR positive]; 2 (11.75%) were HIV-1 positive by western blot [both retrovirus RNA PCR positive]; and 2 (11.75%) were HBV positive [both HBV DNA positive]. Two of these patients were positive for all the three viruses together. Amongst the 6 HCV RNA PCR positive patients, 3 achieved a sustained virological remission[SVR] on treatment (2-Genotype 1a; 1 –Genotype 3), whereas, the remaining three did not qualify for treatment and are under close observation being asymptomatic with mildly elevated liver enzymes. The three treated patients received a combination of Ribavarin 1000 mg daily along with pegInterferon alpha 2a 180 lg/week for a period of 12– 18 months. Both the retrovirus positive cases, achieved SVR with undetectable retroviral RNA by PCR with standard HAART regimens. However, it took 32 and 27 months respectively to achieve SVR in these two patients. Both patients with HBV DNA positivity were also treated with either lamivudine or tenofovir as part of their HAART regimens and both had SVR.

Conclusion: The cumulative prevalence of TTI’s at our institution was 18.3% amongst the haemophilia A patients over the last 20 years. We report here SVR for HCV [3 ], retrovirus [2] and HBV [2] in the treated (100%) patients. Anti-HCV treatment was stopped 48 weeks after PCR negativity, whereas, anti-retrovirus treatment is ongoing according to the currently recommended treatment guidelines. Disclosure of Interest: None declared. Keywords: antiviral, HCV, hemophilia, HIV, transfusion transmitted infections.

VWF08 The missing pieces for understanding fviii inhibitor development: hemophilia inhibitor pup study Hofbauer CJ1,*, Male C2, Brown D3, Santagostino E4, Oldenburg J5, Scheiflinger F1 and Reipert BM1 1 Baxter BioScience; 2Medical University, Vienna, Austria; 3 University of Texas, Houston, TX, USA; 4University of Milan, Milan, Italy; 5University Clinic Bonn, Bonn, Germany Objectives: The development of neutralizing antibodies against FVIII (FVIII inhibitors), as the major challenge in the treatment of hemophilia A patients with FVIII, has been studied for many years. Despite major progress in explaining the regulation of anti-FVIII immune responses in experimental animal models, we still do not understand how the immune system of a patient decides whether or not to develop FVIII inhibitors. The Hemophilia Inhibitor PUP Study (HIPS) searches for early biomarkers of the immune system that could indicate which patients develop FVIII inhibitors and why they develop inhibitors. Methods: HIPS will include 50 previously untreated patients (PUPs) suffering from severe hemophilia A, who will be monitored for up to 50 exposure days with a single FVIII product. Before first treatment and at regular intervals, blood samples will be taken for immune monitoring. Immune parameters to be studied include FVIII-specific antibodies (FVIII inhibitors, Ig isotypes and IgG subclasses of antibodies), affinity of FVIII-specific antibodies, FVIII-specific CD4 + T-cell signatures, and markers for general immune status as expressed by epigenetic signatures of regulatory T cells, NK cells and CD4 + T cells. The monitoring of immune parameters was optimized to require only a few milliliters of peripheral blood. Results: We will present ‘proof of principle’ data obtained from severe hemophilia A patients with and without FVIII inhibitors as well as healthy individuals with FVIII-specific antibodies. Our data indicate an association of high affinity IgG1 and IgG4 antibodies with FVIII inhibitors, low-affinity antibodies with a lack of IgG4 in patients without FVIII inhibitors and in healthy individuals, and an association of FVIII inhibitors with a FVIII-specific Th17/Tfh-like CD4 + T cell signature. Conclusion: We established technologies suitable to search for early biomarkers of the immune system during the course of FVIII inhibitor development in PUPs. Disclosure of Interest: C. Hofbauer Employee of: Baxter BioScience, C. Male: None Declared, D. Brown: None Declared, E. Santagostino: None Declared, J. Oldenburg: None Declared, F. Scheiflinger Employee of: Baxter BioScience, B. Reipert Employee of: Baxter BioScience Keywords: CD4 + T-cell signatures, FVIII-binding antibodies, Hemophilia A, Inhibitors, PUPs.


ABSTRACTS VWF09 Prenatal diagnosis for congenital factor VII deficiency: one case report Jing D* Shanghai Jiaotong University, School of Medicine, Ruijin Hospital, Shanghai, China Objectives: Congenital factor VII deficiency is a rare bleeding disorder with prevalence of around one individual per 500 000. It is an autosomal recessive desease. Severe bleeding manifestations tend to occur mainly in homozygous or compoundheterozygous individual. Here, a women asked for prenatal diagnosis for her second conception. Methods: factor VII activity detection, ELISA for factor VII antigen detection. factor VII gene including all exons and exon-intron boundary were amplified by PCR and sequencing the PCR product. Results: Biological assessment for this proband showed significant prolonged prothrombin time and the activity and antigen of factor VII were 2.8% and 3.62% respectively. Other factors of haemostasis (V, II and X) were in the normal range for age. Molecular diagnosis was performed by direct sequencing analysis of the factor VII gene. It revealed one homozygous missense mutation which is Gly360Asp substituation. Both parents were totally asymptomatic of blood disorders, but had partial FVII deficiency, with factor VII levels of 38.8 and 40.6% for the mother and the father respectively. Family investigation show that this couples are consanguineous marriage. Gene diagnosis resutls indicated that they were all heterozygous missense mutation carrier. So theoretically this couples will have another congenital factor VII deficiency patient with 25% probability. To avoid conceiv another patient fetus, fetal DNA was extracted from 10 to 15 mL amniotic liquid obtained by amniocentesis at 16–21 weeks gestation. Gender detect shows it is a femal fetus and gene analysis for factor VII indicated that this fetus have homozygous Gly360Asp missense mutation which is as same as its brother. Conclusion: To a conclusion, congenital factor VII deficiency disease can be avoided according to gene detection method. In this case, this fetus is a congenital factor VII deficiency female. Disclosure of Interest: None declared. Keywords: congenital factor VII deficiency, gene diagnosis, prenatal diagnosis, rare bleeding disorder.

VWF10 FXI deficiency: genotype-phenotype relationship Favuzzi G1,*, Tiscia GL2, Cappucci F2, Fischetti L2, Bafunno V3, Vecchione G1, Chinni E1, Vergura P2, Colaizzo D4, Villani M2, Margaglione M3 and Grandone E2 1 Thrombosis and Haemostasis, I.R.C.C.S. Casa Sollievo della Sofferenza; 2Thrombosis and Haemostasis, IRCCS ‘CASA SOLLIEVO DELLA SOFFERENZA’, S. Giovanni R. (FOGGIA); 3 Medical Genetics, University of Foggia, Foggia; 4Thrombosis and Haemostasis, IRCCS ‘CASA SOLLIEVO DELLA SOFFERENZA’, San Giovanni Rotondo (FG), Italy Objectives: To describe relationship between clinical history and molecular characterisation of three Italian patients carrying FXI deficiency Methods: FXI activity was determined by using an activated thromboplastin time (aPTT) based clotting test incorporating FXI deficient plasma. Direct sequencing analysis of the FXI gene was performed to identify the causative mutation. Results: Clinical and laboratory features of described patients and their relatives are shown in table. Patient 1. Italian woman with a clotting FXI deficiency diagnosed at 16th week of her first pregnancy, during a hospitalization because of menhorragia. At the 40th week she underwent a caesarean section because of a breech presentation. She was administered tranexamic acid and no bleeding episodes were observed also in the postpartum © 2013 International Society on Thrombosis and Haemostasis 11 (Suppl. 3) (2013) 1–105

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period. Molecular analysis showed two mutations never found combined. The Glu315 residue is located on the surface of the apple 4 domain and seems to be critical for an optimal secretion of FXI. Glu315Lys was previously expressed in BHK cells and revealed impaired secretion. Trp519 is conserved among all analysed species using OMA browser with a frequency ≥ 90%. Trp519X results in a premature termination of protein at the catalytic domain. Patients 2 and 3. Two female 6-years-old Italian twins without a personal history of bleeding. They were diagnosed by means of preoperative screening. DNA sequencing showed the presence of a missense mutation g.10819C>T (p.Arg162Cys). This mutation was previously found in another asymptomatic individual. Two computer-based algorithms, PolyPhen and SIFT, used to predict the pathogenicity of unpublished missense variants, showed that the substitution is likely to affect protein function. Image/Graph:

Conclusion: Our results strengthen the concept that bleeding manifestations in FXI-deficient patients are highly variable and poorly correlated with plasma FXI levels. Disclosure of Interest: None declared. Keywords: FXI deficiency, Genotype-Phenotype, Haemophilia C.

VWF11 A machine learning approach to merging and analyzing data from the condensed MCMDM1-VWD bleeding questionnaire James P1,*, Grabell J1, Bowman M2, Mollah S3 and Coller B4 1 Medicine; 2Pathology & Molecular Medicine, Queen’s University, Kingston, ON, Canada; 3Biomedical Informatics; 4The Laboratory of Blood and Vascular Biology, Rockefeller University, New York, NY, USA Objectives: Since its validation in 2008, the Condensed MCMDM1VWD Bleeding Questionnaire has been used in multiple studies - each published separately, the largest n = 259. To maximize diagnostic information, all legacy data from Queen’s University using this questionnaire was merged and analyzed using a detailed machine learning approach. Methods: Data cleaning and normalization was undertaken to successfully merge datasets. Attribute selection was then performed to identify features best able to distinguish affected and unaffected. The validity of the top 25 attributes was confirmed using classification analysis (Breadth-First Decision Tree Classifier) with 5-fold cross-validation. Results: Data from n = 735 were included (628 healthy controls, 67 Type 1s, 15 Type 2s and 25 with Type 3 VWD); 238 males and 497 females, mean age 45 years (range 12–88). Attribute selection showed that the overall bleeding score is better at predicting VWD than any

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single question, and better than the general question, ‘Do you have a personal history of excessive bleeding or bruising?’ A history of postdental extraction bleeding, as well as requiring medical attention for post-dental extraction bleeding, menorrhagia or bleeding from minor wounds also predicts VWD. Interestingly, feminine protection is also predictive, with affected females doubling up and using products with higher absorbency. Of great clinical importance, the above remain true when the analysis is limited to the controls and just those with Type 1 VWD. The classification analysis demonstrated a sensitivity of 99% and a specificity of 93% of the top 25 attributes. Conclusion: Our results show the benefit of the overall bleeding score and highlight the strength in pooling existing datasets to identify the most diagnostic questions. The results presented here are the first step in the creation of a system for merging and analyzing datasets derived using different bleeding questionnaires including the Condensed MCMDM1-VWD Bleeding Questionnaire and the ISTH-BAT. Disclosure of Interest: None declared. Keywords: bleeding questionnaire, bleeding score, Condensed MCMDM1-VWD Bleeding Questionnaire, von Willebrand disease.

VWF12 P.E1549K: a new mutation responsible of von willebrand disease type 2M Woods AI*, Sanchez-Luceros A, Kempfer AC, Bermejo EI, Calderazzo JC, Blanco AN, Meschengieser SS and Lazzari MA Hemostasia y Trombosis, Instituto de Investigaciones Hematologicas, Ciudad Autonoma de Buenos Aires, Argentina Objectives: von Willebrand disease 2M variant (VWD2M) is characterized by a loss of VWF binding to GP1b, VWF:RCo/VWF:Ag9 min; platelets=207 000/lL; FVIII=70 IU/dL; VWF:Ag=17 IU/dL; VWF: RCo=15 IU/dL; VWF:RCo/VWF:Ag=0.88; Slight absence of high molecular weight multimers; 0.4 mg/mL RIPA positive. Post DDAVP 1 h: BT>9 min; platelets: 154 000/lL; FVIII = 130 IU/ dL; VWF:Ag = 59 IU/dL; VWF:RCo = 40 IU/dL; VWF:RCo/VWF: Ag = 0.68. Mother (36–years old): BSS = 5. BT = 5.5 min; FVIII = 50 IU/dL; VWF:Ag = 30 IU/dL; VWF:RCo = 39 IU/dL; VWF:RCo/VWF: Ag = 1.3; platelets = 162 000/lL; 1.2 mg/mL 0.5 mg/mL RIPA: positive. Multimers: normal. Maternal aunt (25-years old): BSS = 3. BT = 7.5 min; FVIII = 40 IU/ dL; VWF:Ag = 26 IU/dL; VWF:RCo = 26 IU/dL; VWF:RCo/ VWF = 1.0; platelets = 205 000/lL; 0.4 mg/mL RIPA: positive. Multimers: normal. In all patients mixing tests were compatible with VWD2B. We found in this family the heterozygous p.M1304V but not in donors. It was reported by us to the ISTH SSC VWF database. In silico analysis by using PolyPhen-2 (http://genetics.bwh.harvard. edu/pph2/) predicted the p.M1304V to be probably damaging with a score of 0.994 (sensitivity: 0.68; specificity: 0.97). Conclusion: We described here a new missense substitution responsible for atypic VWD2B. Given that it was absent in the normal alleles and showed as probably damaging by Polyphen-2, we consider it is not a polymorphism. Disclosure of Interest: None declared. Keywords: atypical VWD 2B, new missense mutation.

VWF14 Comparison of recombinant FVIII utilisation in a cohort of UK patients following a switch from a full length to B-domain deleted FVIII: a retrospective analysis of homecare delivery data Verma S* and Gama J Baxter Healthcare plc, Compton, UK Objectives: B domain deletion increases recombinant factor VIII (rFVIII) manufacturing yield. However, it remains to be investigated if the change affects its utilisation. Regression analysis of a US specialty pharmacy database1 showed that after controlling for age, severity, treatment regimen and insurance type, patients receiving B domain deleted FVIII (BDD-rFVIII) utilised 33% more rFVIII compared to patients receiving full length FVIII (FL-rVIII). This study further explores the influence of product differences on utlisation of rFVIII.


ABSTRACTS Methods: A retrospective analysis of delivery data from a UK homecare database between October 2008 and September 2011 was conducted. The median monthly utilisation of rFVIII was compared using a Wilcoxon signed rank test in patients switched from a FL-rFVIII (rAHFPFM) to BDD-rFVIII who had received consecutive treatment with rFVIII for more than 6 months. Delivery data was used as a surrogate for utililisation data. Results: Sixty-one patients were switched from FL-rFVIII (rAHFPFM) to BDD-rFVIII. Forty-nine out of 61 (80%) received more than 6 months consecutive treatment. In the 44 prophylaxis patients (median age = 17), the median monthly usage per patient in IU of rFVIII increased by 20% from 16 993 IU with FL-rFVIII (rAHF-PFM) to 20 504 IU for BDD-rFVIII. The difference was statistically significant (P = 0.001). The median time on BDD-rVIII and FL-rFVIII (rAHF-PFM) was 17 and 16 months respectively. In the five on demand patients (median age=35), median monthly IU of BDD-rFVIII utilised was lower than than FL-rFVIII (rAHFPFM) and the difference was not statistically significant (0.893). Conclusion: This retrospective study has shown a statistically significant increase in monthly utilisation of rFVIII in patients on prophylaxis upon switching from FL-rVIII (rAHF-PFM) to BDD-rFVIII. This increase may have an important impact on the economics of care and should be investigated further. Reference: 1. Epstein, J et al. Haemophilia. 2012;18(2):187–92. DOI: 10.1111/j.1365-2516.2011.02636.x Disclosure of Interest: S. Verma Employee of: Baxter Healthcare, J. Gama Employee of: Baxter Healthcare. Keywords: BDD, FL, Haemophilia, haemophilia A, utilisation.

VWF15 Standardization of an APTT-based FVIII inhibitor screening assay Pinto P*, Shetty S and Ghosh K Department. of Haemostasis & Thrombosis, National Institute of Immunohaematology (Indian Council of Medical Research), Mumbai, India Objectives: Inhibitor development in congenital Haemophilia A patients is a serious complication of Factor VIII (FVIII) replacement therapy. In India, an increased post-operative inhibitor incidence has also been reported. FVIII inhibitor assays are still plagued with important unresolved issues such as inter-assay variability, lack of definition of a negative antibody titre, and interference by non-specific antibodies. The aim was to standardize a specific, sensitive, quick, and costeffective FVIII Inhibitor screening assay. Prompt and accurate diagnosis is critical, since early therapy can be life-saving. Methods: Recombinant B-domain deleted FVIII (Refacto, Wyeth) and full-length FVIII (Kogenate FS, Bayer), and plasma-derived monoclonal purified FVIII (Hemofil-M, Baxter), were each diluted to 100 IU/ dL (100%) FVIII:C, in Imidazole buffer (pH 7.3), and used instead of Normal Pooled Plasma (NPP) in mixing studies, at 37°C, at 1 and 2 h intervals. A total of 300 haemophilia A citrated plasma samples with a known FVIII inhibitor status (confirmed by the Bethesda assay), were analysed using Dadeâ Actinâ Activated Cephaloplastin reagent (Siemens) and CaCl2 (0.025 M). Results: APTTIncubated Mix < APTTSeparate Mix at both time intervals was observed in inhibitor negative samples, whereas APTTIncubated Mix > APTTSeparate Mix with a progressive increase in the difference, was observed in inhibitor positive samples. Hemofil-M showed a specificity of 98.27% as compared to Refacto (94.83%), NPP (75.91%) and Kogenate FS (42.86%), and was sensitive enough to detect clinically significant low-titre inhibitors. Conclusion: Hemofil-M was the most accurate in differentiating between inhibitor positive and negative samples. The assay was quick, economical, and drastically reduced false-positive results and the need


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for repeat confirmation assays. This screening assay would also enable clinicians to accordingly alter the treatment modalities in patients. Disclosure of Interest: None declared. Keywords: Haemophilia A, Inhibitors, Screening Assay.

VWF16 Inherited bleeding disorders in Pakistani women presenting with menorrhagia Hossain N1,*, shamsi T2, naz A2 and James A3 1 Obstetrics & Gynecology, Dow University of Health Sciences; 2 National Institue of Blood Diseases, Karachi, Pakistan; 3 Obstetrics & Gynecology, Duke University, Durham, NC, USA Objectives: To determine the frequency of inherited bleeding disorders in women presenting with menorrhagia: Methods: Prospective, observational study. This study was carried out at the Department of Obstetrics & Gynecology Unit 3 Dow University of Health Sciences and National Institute of Blood Diseases. The study period is from April 2008 to July 2009. All women, who were referred to as a case of menorrhagia, in whom endocrinological and pelvic pathologies were excluded, were evaluated for haemostatic abnormalities. Menorrhagia was defined as heavy and regular periods. Results: A total of 54 women with menorrhagia were identified. Haemostatic abnormalities were found in 17(31.5%) women. Mean age of women in the study group was 26 years. Of these 17 women, 6 (11%) were found to have von Willebrand disease (VWD). The mean VWF: Ag level was 42.98, and the mean VWF:RCo was 53.51. Four women had a low factor VIII levels mean (range 28–47%) Seven of these 17 women were found to have platelet function defects; there were two with Bernard Soulier Syndrome, three with collagen receptor defects, one each of epinephrine and ADP defect. Conclusion: Inherited bleeding disorders are an important cause of menorrhagia among women referred for evaluation. Disclosure of Interest: None declared. Keywords: None.

VWF17 Pharmacokinetics and safety profile of a single intravenous dose of recombinant factor XIII in paediatric patients with congenital factor XIII a-subunit deficiency Williams M1,*, Stenmo C2, Rosholm A3 and Tehranchi R4 1 Birmingham Children’s Hospital, Birmingham, UK; 2Clinical Pharmacology Biopharm; 3Biostatistics; 4Medical and Science, Haemophilia R&D Portfolio, Novo Nordisk A/S, Copenhagen, Denmark Objectives: Congenital Factor XIII (FXIII) deficiency is a rare autosomal recessive bleeding disorder, mostly caused by a mutation in the gene encoding the catalytic FXIII A-subunit. Because such patients have a significant bleeding tendency and in particular a high risk of potentially fatal intracranial haemorrhage, prophylaxis with a FXIIIcontaining product is recommended. Recently, a multi-national, openlabel, single-arm, phase 3 trial (mentorTM 1) demonstrated that recombinant FXIII (rFXIII) was safe and effective for prophylaxis in patients with congenital FXIII A-subunit deficiency with a dose of 35 IU/kg administered once every 4 weeks. Pharmacokinetic studies in child (> 6 years of age) and adult patients showed that rFXIII has a half-life of approximately 11 days (range 7.5–18.5 days). Whereas the concentrations of most haemostatic proteins are very close to adult values by 6 months of age in both term and preterm infants, adult levels for FXIII-A and FXIII-B are achieved by day 5 of life. However, it is known for other blood factors such as FVIII and FIX, rate of clearance is generally higher in young children compared with adolescents

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and adults, resulting in the need for higher dosing regimens of factor products in this age group. Therefore, the objective of this multinational, open-label, single-dose trial (mentorTM 4) was to investigate the pharmacokinetics and safety of 35 IU/kg rFXIII administered as an intravenous injection to paediatric patients (1 to

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