17 th Congress of the European Hematology Association

Bleeding disorders 0458 PLASMA CONCENTRATION OF Z AND PROTEIN Z-DEPEND - ENT PROTEASE INHIBITOR IN PATIENTS WITH HAEMOPHILIA A 0457 L Bolkun 1, M Galar 2, D Lemancewicz 2, K Mazgajska-Barczyk 2, E Cichocka 2, J Kloczko 2, J Piszcz 2 NINE NOVEL ADAMTS13 MUTATIONS IN CONGENITAL PREGNANCY 1University Hospital, Bialystok, Poland ASSOCIATED ADULT ONSET TTP 2Haematology Department, Bialystok, Poland K Langley , E Heelas, M Underwood, J Iseppi, I Mackie, S Machin, M Scully UCL, UK, United Kingdom Background. The potential role of alterations in protein Z concentrations in the pathogenesis of has been investigated in several studies which, ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type I however, yielded conflicting results. Protein Z deficiency may induce bleeding domains 13) deficiency is associated with thrombotic thrombocytopoenia pupu - as well as prothrombotic tendencies and it might occur as an inherited disor - ra (TTP), a rare, life threatening thrombotic microangiopathic anaemia. TTP der. Aims. The purpose of the present study was to explore the concentration may be acquired, usually due to the presence of autoantibodies, or congeni - of protein Z and protein Z-dependent protease inhibitor in patients with tal. The congenital form can present in childhood, adolescence or adulthood, haemophilia A. Additionally; it examined the correlation between PZ/ZPI plas - but in adults usually occurs secondary to haemostatic challenge. The differ - ma concentration and bleeding rate and joint bleeding per year. Materials and ences in age of onset are likely to result from the individual repertoire of muta - Methods. The study was based on fifty male patients: 25 with severe, 15 with tions in the ADAMTS13 , located on 9.DNA sequencing was moderate and 10 with a mild form of haemophilia A. Median age at the time of performed on nine patients with pregnancy associated, adult onset TTP, two of samples taking was 29 (range 19-55 years). None of these patients had under - whom were sisters. Eight of the patients were Caucasian and one of Asian ori - gone prophylaxis of bleeding with FVIII (factor VIII) before the study. All stud - gin. All the patients presented with a markedly reduced ADAMTS13 activity of ied subjects received FVIII on demand, doses depending on the kind of bleed - < 5% (FRETs NR 60-123%) and negative for anti-ADAMTS13 IgG autoantibody. ing. Haemophilia A patients with confirmed hepatitis B or C and with inhibitor Using population data from the 1000 Genomes Project and Hapmap we con - of factor VIII had been excluded from the study. The control group consisted firmed the presence of 3 single nucleotide polymorphisms (SNPs), 10 synony - of 90 healthy male blood donors at median age of 30 (range 19-52). The year - mous SNPs, 5 missense mutations, 4 frame shift mutations and 4 intronic ly bleeding rate and joint bleeding rate were calculated on the basis of data from mutations.Six novel exonic mutations and 3 novel intronic mutations were iden - medical records and home-treatment reports from 2005 to 2011.Quantitative tified. These included two exonic missense mutations: R497C and R1219Q. assessment of protein Z and ZPI in the plasma was performed using commer - Four frame shift mutations were found: p.A111QfsX18, p.N667TfsX31, cial test (Asserachrom Protein Z Elisa Kits, Diagnostica Stago, France) with p.M486VfsX47 and p.I1339SfsX21, one of which occurred in both of the two expected mean value (±SD), 1.56±0.61 μg/mL) and Enzyme-linked Immunosor - siblings. The intronic mutations detected were c.L987+11, c.L1786+90 and bent Assay Kit for Protein Z Dependent Protease Inhibitor (ZPI) (USCN Life Sci - c.Q1245+81. Some SNPs had a higher prevalence in our patients than antic - ence Inc, China). Quantitative determination of FIX in the plasma was done ipated from normal population frequencies, for example R7W is present in 7% using Stago® Deficient IX (Stago,France), with expected normal range of fac - and A1033T is present in 3% of the general Caucasian population, but were tor IX between 60-150%. Active form of FVIII was tested by means of standard found in 7 of the 8 Caucasian patients studied. Exons 4,6,13,15 and 24, exclu - methods. Results. In haemophilia A patients mean plasma concentrations of sively in our cohort, demonstrated several differences from wild type, where - PZ and ZPI were significantly higher than in healthy individuals: PZ (1.87±0.68 as exons 2,3,7,9,10,14,20,22,25,27 showed no variation from wild type in any μg/mL vs 1.49±0.54 μg/mL) and ZPI (5.02±1.11 μg/mL vs 4.22±0.55 μg/mL), of the patients investigated. Notably, seven of the nine patients had both the with p=0.02 and p=0.03, respectively. In the subgroup with severe haemophil - R7W and A1033T SNPs in conjunction with the R1060W mutation. The patients’ ia A, an in-depth analysis revealed a tendency to modulating effect of the PZ full mutational profile (including synonymous mutations) was examined in the (r=-0.53; p=0.072) and a statistically significant one in the case of ZPI (rho=- context of their clinical features. One patient with a moderate clinical profile; only 0.79, p=0.002) on the bleeding rate. It simultaneously disclosed a statistically one heterozygous missense mutation (R1060W) and relatively few exonic dif - significant correlation between the number of bleeds to the joints (20.18±14.1), ferences from wild type (R7W, A900V, A1033T, V970V) however had three PZ (r=-0.72; p=0.04) and ZPI (rho=-0.88, p=0.001). With reference to this par - intronic mutations. It is not yet understood how these may interact to influence ticular group of patients, the study also showed some other statistically mean - the clinical picture, or how their position on the allele may contribute. In con - ingful correspondences: between PZ and ZPI (rho=0.65, p=0.02), PZ and FIX clusions in conjunction with further novel mutations in our cohort, we have also (r=-0.61, p=0.04), as well as ZPI and FVIII (rho=0.78, p=0.002).No such link in documented a higher frequency of SNPs and intronic mutations in association patients with mild and moderate haemophilia A was established. Conclusions. with synonymous SNPS, further work will focus on how these new findings Despite the fact that FVIII deficiency is undoubtedly the main mechanism of result in changes in expression and function of ADAMTS13 in relation to TTP. bleeding in haemophilia A patients, the activity of PZ/PZI complex may play some modulating role in the matter. Further research is required to fully eluci - date the haemostatic role of ZP/ZPI complexes in haemophilia A patients.

0459

WITHDRAWN

186 | haematologica | 2012; 97(s1) Amsterdam, the Netherlands, June 14 – 17, 2012

0460 0461

LOCAL EXPERIENCE IN REVERSAL DABIGATRAN USING ACTIVATED A NOVEL FLOW CYTOMETRY-BASED AGGREGATION ASSAY PROTHROMBIN COMPLEX CONCENTRATES (FEIBA) THAT DISCRIMINATES BETWEEN GLANZMANN THROMBASTENIA AND KC Cheung , S Rodgers, S Mcrae LAD-III SYNDROME. IMVS, Adelaide, Australia L Gutiérrez 1, I De Cuyper 1, M Meinders 1, E Van de Vijver 1, A Gerrits 1, D De Kor - te 1, L Porcelijn 2, M De Haas 2, K Seeger 3, A Verhoeven 4, T Kuijpers 5, T van den Background. Dabigatran etexilate (Pradaxa, Boehringer Inhgelheim) is a pro- Berg 1 drug of dabigatran, a reversible direct inhibitor of that exhibits its effect 1Sanquin Research and Landsteiner Laboratory, Amsterdam, Netherlands by binding directly to the active site of thrombin. The major advantage of dabi - 2Sanquin Diagnostics, Amsterdam, Netherlands gatran over is that it produces a predictable pharmaco-dynamic effect 3Otto-Heubner-Center for Pediatric and Adolescent Medicine, Charité-Univer - which renders routine laboratory monitoring unnecessary. Several studies sitätsm, Berlin, Germany demonstrated that the efficacy of dabigatran is at least comparable to that of 4AMC, Amsterdam, Netherlands current therapies in preventing stroke related to atrial fibrillation and venous 5Emma Children’s Hospital, AMC, Amsterdam, Netherlands following hip and knee replacement surgeries.Like other anticoag - ulants, Dabigatran is associated with a risk of major bleeding with an expect - Background. The main function of is to maintain normal hemostasis. ed rate of 1-2% per annum receiving therapeutic anticoagulation. The optimal Several platelet function tests have been developed to date to be used in the management of a major bleeding complication occurring in patients receiving clinic and in experimental animal models. In particular, platelet aggregation is Dabigatran remains uncertain, and current recommendations regarding the use routinely measured in an aggregometer, which requires normal platelet counts of reversal agents are based on a combination of pre-clinical data, anecdotal and significant blood sample volumes, making difficult analyses in thrombocy - case reports and the absence of effective alternatives. Aims. To determine the topenic patients or infants. For the same reason, analysis of platelet aggrega - usefulness of the CAT in determining the effect of dabigatran at therapeutic con - tion in small rodents can be tedious. Patients with Glanzmann thrombasthenia centrations, and to measure the effect of activated prothrombin complex con - or Leukocyte Adhesion Deficiency-III syndrome (LAD-III or LAD-1/variant) pres - centrate (FEIBA®, Baxter AG, Vienna, Austria) in reversing the haemostatic ent with increased bleeding tendency because of the lack or dysfunction of the defect induced by dabigatran. Methods. The thrombin generation assay was receptor GPIIb/IIIa (integrin αIIb β3), respectively. Although the bleed - performed in platelet poor plasma (PPP) using the Calibrated Automated ing disorder is more severe in LAD-III patients, classic aggregometry or perfu - Thrombogram (CAT) (Thrombinoscope). Commercial plasmas containing a sion of Glanzmann or LAD-III platelets over collagen-coated slides under phys - range of concentrations of dabigatran (30 to 500 µg/L) were tested. Different iologic shear rate does not discriminate between these 2 conditions because it therapeutic concentrations of FEIBA (0.2 - 3 U/ml) were added to a test plas - requires functional integrin αIIb β3 as a readout. Aims. To develop a novel test ma containing approximately 200 µg/L dabigatran. Commercial PPPLOW of platelet aggregation, in which lower platelet counts or volumes can be used, reagent (1 pM with 4 µM phospholipids) and Fluorogenic substrate and that would allow dissection of single receptor contribution to the platelet (FluCa) were used (Thrombinoscope) according to manufacturer’s manual. aggregation process. Methods. Platelet labeling with fluorescent dyes or con - Results. Dabigatran induced a concentration-dependent increase in lag time jugated antibodies, flow cytometry. Results. We have developed a novel flow and time-to-peak, as well as a decrease in peak height and ETP at higher con - cytometry test of platelet aggregation, in which 10-25-fold lower platelet counts centrations (>130 µg/L). Addition of FEIBA to the test sample decreased the lag- or volumes can be used, either from platelet-rich plasma or whole blood (includ - time and time-to-peak in a concentration-dependent manner between 0.2 and ing murine embryonic blood) from human subjects or mice. Furthermore, this 1U/ml FEIBA, with no further decrease with 2 and 3 U/ml FEIBA. At all concen - novel test is able to discriminate between Glanzmann thrombasthenia and LAD- trations both times remained prolonged in comparison to normal. Peak height III aggregopaties. We were able to measure collagen-dependent aggregate and ETP was increased to normal values by 0.2 U/ml FEIBA, with further formation in Glanzmann platelets, whereas LAD-III platelets were not able to increases above normal up to 1 U/ml, and no further increase with higher con - form aggregates upon this stimuli. These aggregates required functional centrations of FEIBA. Conclusions. Our results demonstrated that FEIBA par - GPIa/IIa (integrin α2β1) instead of integrin αIIb β3, thus explaining the clinical - tially reversed dabigatran mediated inhibition of thrombin generation (i.e. short - ly more severe bleeding manifestations in LAD-III patients, in which all platelet ening of delay in lag time and increasing peak response). Future experiments integrins are functionally defective. These findings provide genetic evidence for should be conducted to determine the in-vivo effect of FEIBA in reversing dabi - the differential requirements of platelet integrins in formation and gatran in overdosed patients, correlated with clinical outcomes. demonstrate that correct integrin function assessment can be achieved with a combination of diagnostic methods . Summary and Conclusions. This set-up can be applied to test in small assay volumes the influence of a variety of stim - uli, drugs and plasma factors, such as antibodies, on platelet aggregation. The presented principle stands as a promising user-friendly tool, which allows analy - sis of platelet aggregation in thrombocytopenic patients or infants, and facilitates studies in platelets obtained from experimental animal models without the need of special devises but a flow cytometer.

0462

NOVEL VON WILLEBRAND GENETIC MUTATION (C.4097T>G) ASSOCI - ATED WITH VON WILLEBRAND DISEASE TYPE 2M A Danaee , D Bevan, J Cutler, M Mitchell Guys and St Thomas’ Hospital, London, United Kingdom

We report a previously unreported (vWF) gene mutation associated with an unusual phenotype of von Willebrand Disease ( probable vWD Type 2M) characterised by markedly reduced vWF Ristocetin co-factor activity (vWF:RCo) in the presence of normal or high von Willebrand Factor anti - gen (vWF:Ag), von Willebrand Factor Collagen Binding Acitivity (vWF:CBA), Figure 1. Partial reversal of delay in lag time in Thrombinoscope by Feiba in and FVIII:C activity.The proband C (female, 55yrs) and both her daughters (J, test sample. 26yrs; L, 28yrs) were referred for investigation of a bleeding disorder. The father of J and L was already known to have mild haemophilia A. C was found to have normal plasma vWF:Ag (120iu/dL; nr 45-160iu/dL) and vWF:CBA (95u/dl; nr 35- 140u/dL) but reduced vWF: RCo of 25 iu/dl (nr 54-202iu/dL). Her blood dis - played delayed closure times in both collagen/ADP and collagen/epinephrine cartridges in the PFA-100® system. Low resolution Von Willebrand Factor mul - timeric analysis (Phast system) showed a normal distribution with no deficit in HMW multimers. Her factor VIII:C was 140iu/dl (nr 50-150iu/dL).DNA analysis of the von Willebrand Factor (VWF) gene detected a heterozyogus substitution in exon 28 of her VWF gene (c.4097T>G). This mutation corresponds to the replacement of native phenylalanine at codon 1366 with a cysteine

haematologica | 2012; 97(s1) | 187 17 th Congress of the European Hematology Association

(p.Phe1366Cys). This mutation has not previously been reported but in silico 0464 analysis strongly supports its being pathogenic and it is in the same region as several other mutations associated with a Type 2M phenotype (E1359K, INDIVIDUALIZED REPLACEMENT OF FACTOR VIII ACCORDING TO THE V1360A, K1362T and F1369I). The associated phenotype suggests that this IN VIVO RECOVERY IN HEMOPHILIA A PATIENTS DURING SURGERY mutation results in a highly selective effect on vWF binding to its platelet recep - KS Lee 1, YJ Shim 2, JK Suh 1 tor, demonstrated by low ristocetin cofactor activity in the washed-platelet ris - 1Kyungpook National University Hospital and School of Medicine, Daegu, tocetin co-factor assay without evidence of reduced vWF function in other South-Korea modalities.Both daughters inherited this mutation, and the associated Type 2M 2Kyungpook National University Medical Center and School of Medicine, von Willebrand Disease phenotype, from their mother, in addition to inheriting Daegu, South-Korea their father’s factor FVIII mutation (p.Tyr551Cys). This resulted in J manifest - ing a mild bleeding disorder with the combined phenotype of Type 2M vWD Backgroud and Aims. Although patients’ individual pharmacokinetics is con - (vWF:RCo 12.5iu/dL; vWF:Ag 61iu/dL) and mildly-affected carrier of sidered very important in hemophilia, there have been few reports about the Haemophilia A (FVIII:C 40iu/dL).L. had a FVIII:C level of 64iu/dl (indicating practical result after direct application of individual in vivo recovery (IVR) to the non-affected carrier status for Haemophilia A), vWF:RCo of 19iu/dl, vWF:Ag surgery. Thus we investigated the correlation between individual IVR and the 79iu/dl. Both sisters also had normal vWF:CBA assays, normal vWF mulimer outcome of each operation. Methods. The hemophilia A patients who had composition and prolonged closure times in both cartridges of the PFA-100® blood tests for IVR just before each operations from January 2000 to January system. 2012 were reviewed. The patients with inhibitor (anti-FVIII) were excluded. The respective surgery cases were divided into 3 groups; A (IVR > 80% of expect - ed), B (IVR < 80% of expected, but they were managed by immediate addition - 0463 al replacement of FVIII), and C (IVR < 80% of expected and the extra supple - mentations of FVIII were delayed over 3 days). Their each consumption of FVI - CONGENITAL FXI DEFICIENCY: EVALUATION OF POST-SURGERY II and hospitalization periods was analyzed. The Mann-Whitney U test (SPSS BLEEDING PHENOTYPE AND CORRELATION WITH FXI ACTIVITY ver. 19) with bonferroni adjustment was used to compare inter-group differ - (FXI:ACT) ences.4. ResultsTwenty hemophilia A patients underwent total 27 various oper - C Santoro , R Di Mauro, E Baldacci, F Biondo, R Abbruzzese, P Pignoloni, R ations. Twelve patients were severe (FVIII:C < 1 IU dL -1 ), 4 patients were mod - Foà, MG Mazzucconi erate (FVIII:C 1-5 IU dL -1 ), and the remainder were mild (FVIII:C > 5 IU dL -1 ) Hematology, Rome, Italy hemophilia A. They were all males. Their mean age at the time of surgery was 32 (range from 0 to 69) years old, and mean number of surgery they received Background . The bleeding phenotype in FXI deficiency is variable and gen - was 1.4 (range 1 to 3) times. The total dose of FVIII was respectively 31,693 erally related to surgery/trauma. Moreover, there is a poor correlation between ± 22,858 (mean ± SD) IU in group A, 53,389 ± 48,778 IU in B, and 166,596 ± bleeding and baseline FXI:Act. Aims. To describe the post-surgery hemor - 149,327 IU in C. There was no statistical difference between them. The FVIII rhagic phenotype of our FXI deficient population and to relate the phenotype dose per patient’s body weight was respectively 481 ± 195 IU kg -1 in group A, with FXI:Act. Patients and Methods. Since 1973, we have been following 50 1,311 ± 283 IU kg -1 in B, and 3,502 ± 1,529 IU kg -1 in C. The mean value was FXI deficient patients: 26 F, 24 M; median age at diagnosis: 34 years, range lower in group A than B ( p < 0.001), and in group B than C ( p = 0.006). The 1.7-79.6; median follow-up: 1.9 years, range 0.1-36.2; positive family history: hospitalization period was respectively 14.5 ± 12.1 days in group A, 13.9 ± 4.0 18; median FXI:Act of all patients: 37% (range 0.25-60%; normal values: 70- days in B, and 45.8 ± 15.7 days in C. There was no extension of hospitaliza - 140); FXI:Act ≤1% in 4 patients, >1≤5% in 10, >5 ≤10 in 2, >10 ≤20 in 3, >20 tion in group B in comparison with A ( p = 1.000). The mean duration was longer in 31. Results. Eighteen patients experienced bleeding episodes not surgery- in group C than A and B (respectively, p = 0.015 and p = 0.006). Summary and related: ecchimoses in 14, hematomas in 7, epistaxes in 11, gastrointestinal Conclusions . The medical costs were much more expensive in low IVR groups hemorrhages in 7, meno-methrorrhagia in 1.Prior to diagnosis, 15 patients (B and C) than high IVR group (A) according to our expectation. However, the underwent 69 major/minor surgeries without prophylaxis: 21 hemorrhages were immediate extra supplementation of FVIII reduced the hospitalization period as reported. The median FXI:Act in the bleeders was: 28%, range 0.25-53%. Ten well as economized the FVIII consumption per body weight in low IVR group spontaneous deliveries (SD) and 5 caesarian sections (CS) were performed: (B). It is very important to check IVR on all occasions just before surgery and 1 post-partum hemorrhage occurred (patient FXI:Act 27%). Twenty-eight supply the FVIII according to the result to minimize the medical cost. patients underwent dental surgeries, 7 experienced hemorrhages (bleeders median FXI:Act: 6.9%, range 1-43%). Post-diagnosis, 13 minor/major surger - ies were performed, 8/13 with anti-hemorrhagic prophylaxis: plasma in 3 cas - 0465 es, plasma and tranexamic acid in 2 cases, tranexamic acid in 2 cases, desmo - pressin in 1: no bleedings were reported. Three dental exctractions were per - CASE STUDY OF THE USE OF INTRANASAL BEVACIZUMAB TO TREAT formed, 1 prophylactically treated with plasma and tranexamic acid: one bleed - RECURRENT EPISTAXIS DUE TO HEREDITARY HAEMORRHAGIC ing was reported in a patient who self-administered anti-inflammatory drugs TELANGECTASIA (FXI:Act 45%). One CS and 1 SD were performed: desmopressin was used as C Alderman , J Corlett, J Cullis prophylaxis in 1 case: no bleedings reported. Conclusions. We confirm the Salisbury District Hospital, Salisbury, United Kingdom wide variability in post-surgery bleeding events in FXI deficient patients, not related to the FXI:Act levels. Because of the low correlation between FXI:Act Background. Hereditary Haemorrhagic Telangectasia (HHT) is an autosomal and the phenotype, we highlight the need of laboratory-based prognostic fac - dominant condition with a prevalence of approximately 1:10,000 of the popu - tors for a better management of these patients. lation. Severe cases present with recurrent bleeding and may require regular blood transfusions or intravenous iron. Several genetic mutations have been associated with HHT and many of these encode that are involved in the vascular endothelial growth factor (VEGF) and transforming growth factor- β (TGF-ß) signalling cascades. Furthermore, plasma levels of VEGF and TGF- ß are increased in patients with HHT. Limited published data suggest that inter - ruption of VEGF signalling by Bevacizumab (a humanised monoclonal VEGF inhibitor) may represent a potential therapy for patients with HHT. Aims. Our aim was to assess the potential off label use of topical Bevacizumab to treat patients with severe epistaxis due to HHT. Methods. We treated a 65 year old male patient with a life-long history of severe epistaxis due to HHT. He was dependent upon regular iron infusions and had also required frequent blood transfusions as well as pulsed dye laser therapy given monthly for over 20 years. A history of pulmonary embolism and intracoronary stents precluded use of antifibrinolytic drugs or intravenous Bevacizumab.After informed consent was obtained, we injected a total dose of 100 mg of undiluted Bevacizumab via an intranasal atomization device given in 100 μL volumes to each nostril every five minutes until completion of the 4 mL ampoule. This was repeated on a monthly basis for three months. The severity of his symptoms were assessed using a normalised Epistaxis Severity Score (ESS) to give a value out of 10. Results. After three months, subjectively, his quality of life had improved remarkably. His average daily duration of epistaxis reduced from 17 to less than

188 | haematologica | 2012; 97(s1) Amsterdam, the Netherlands, June 14 – 17, 2012

2 minutes. Objectively, his ESS has reduced from 10 to 3, and his need for 0467 pulsed dye laser therapy has diminished. Summary and Conclusions. Beva - cizumab can be sprayed intranasally to treat severe recurrent epistaxis due to ACQUIRED HAEMOPHILIA A-RETROSPECTIVE ANALYSIS OF 46 CASES HHT. We are planning to investigate further the potential of this therapeutic FROM A SINGLE CHINESE HAEMOPHILIA CENTER approach in further patients with severe epistaxis due to HHT. We anticipate Y Yang , F Xue, H Shi, H Wang, L Ji, R Yang that, in addition to the above therapeutic benefits, patients will require fewer Institute of Hematology and Blood Diseases Hospital, Tianjin, China blood transfusions and less intravenous iron support. It is, therefore, likely that there will also be a significant economic advantage to this novel treatment. Background. Acquired hemophilia A (AHA) is a rare disorder caused by the autoantibody directed against factor VIII in patients without previous histo - 0466 ry of a bleeding disorder. Severe bleeding occurs in a majority of patients with a high rate of mortality. The patients of AHA usually present to clinicians PLATELET FUNCTION DIAGNOSTIC LABORATORY INVESTIGATION IN without experience of this disease. As a result, the diagnosis and proper THALASSAEMIC PATIENTS treatment is often delayed. Aims and Methods. We retrospectively ana - S Theodoridou 1, M Economou 2, A Teli 1, E Vlachaki 1, D Kleta 1, A Karioti 1, A lyzed the characteristics and outcomes of 46 patients with AHA from 1994 Agapidou 1, N Kouri 1, S Vakalopoulou 2, V Garypidou 2, N Gompakis 2, F to2011 in our center. Results. The median age was 53 years ranging from Papachristou 2 14 to 78 years .The major peak of age was 60-70 years 16 of 46, 35% , the 1Hippokration Hospital, Thessaloniki, Greece second frequency peak was 20-30 years 9 of 46, 20% . The associated dis - 2Aristotle University, Thessaloniki, Greece ease was observed in 7 (16%) patients. Nine patients had medical conditions that were unlikely to be related to acquired inhibitors. The symptoms includ - Platelet function disorders constitute a rare cause of symptomatic bleeding. ed ecchymosis ( 44 of 46, 96%), muscle or soft tissue haematoma( 33 of Haemostatic disorders are reported in thalassaemic patients. The aim of this 46,77%), hematuria (12 of 46,28%), mucous bleeding (10 of 46, 21%), study was the platelet function investigation in thalassaemics and the detection hemarthrosis (7 of 46, 16%), intracranial bleeding (2 of 46, 5%). Twenty-four of any relation with the chelation treatment. The platelet function investigation patients with acute bleeding episode were treated with prothrombin complex consisted of the aggregation testing in platelet rich plasma with light transmis - concentrate (PCC) at a relative low dose of 30-50U/kg·d and achieved good sion aggregometry (LTG) by the use of 5 agonists (arachidonic acid, ADP, outcomes without adverse reaction. Corticosteroids alone or combined with adrenaline, collagen, ristocetin), and the global test of haemostasis by PFA-100 cyclophosphamide were used as first-line therapy to eradicate the inhibitors. (Dade Behring) whose principle is the in vitro platelet plug formation under In 37 evaluable patients, 33 (89%) cases achieved complete remission (CR). sheer stress with either collagen and epinephrine or collagen and ADP as ago - Four cases died of hemorrhage and 2 of them had neutropenia-associated nists. The P2Y12 receptor was also tested.39 thalassaemic patients (24 males, infections which conversely aggravated haemorrhage. The response time 15 females, aged 5 to 45 years) were included. Patients were on chelation was not related to FVIII:C and inhibitor titre. Two patients relapsed after 4.5 treatment with either deferasirox (18 patients), or deferiprone, desferioxamine months and 1 year respectively, but they achieved second CR after the sec - or both (21 patients). Eight were splenectomised and six received aspirin as pro - ond cycle of therapy. Conclusions. This study summarizes the experience phylactic treatment, seven patients had a liver biopsy. No haemostatic cover about AHA from a single Chinese hemophilia center where expensive was used and no untoward bleeding was reported. One patient had experienced bypassing agents such as recombinant activated factor Ⅶ and activated a peptic ulcer bleeding. Four cases with chronic viral hepatitis were prothrombin complex concentrate are not available . We find that PCC is included.RESULTS66% (26) of the patients showed reduced LTG with adren - effective and safe to control acute bleeding. Corticosteroids alone or com - aline and 20% (8) showed reduced LTG to two or more of the agonists used. bined with cyclophosphamide used as first-line therapy achieved good out - 34 patients were tested with PFA-100 and was prolonged in 73% (25) of them. comes with CR rate of 89%. The comorbidities and side-effect of immuno - 43,5% (17 patients) had both a defect in platelet aggregation and a prolonged suppressant in patients probablely have adverse impact on the prognosis. PFA-100 test, while 10,2% (4) had abnormal LTG and normal PFA-100 time. 20,5% (8) patients had normal aggregation and abnormal PFA-100 test, while 10,2% (4) had both normal aggregation and PFA-100 time. Three patients with chronic hepatitis had reduced LTG and one was found normal. The patients that had undergone liver biopsies had abnormal LTGs and 4 had prolonged PFA- 100. Among the patients that had undergone a splenectomy, we found five of them to have abnormal LTG and prolonged PFA-100, while two of them had nor - mal tests. The patient with GI hemorrhage had abnormal LTG and PFA-100. We found no correlation with either chelation regimen and LTG or PFA-100.Our patients’ results are indicative of a platelet defect similar to disorders of platelet secretion and signal transduction. The clinical manifestations though were few. The defective LTG can be explained by the release of ADP from the haemol - ysed red blood cells, which leads to defective platelet aggregation and second - ly the presence of two platelet populations in the circulation of the patients. The more active platelets present as circulating aggregates and are not detect - ed by in vitro study and the less active are poorly aggregable. An alternative explanation is the in vitro effect.In literature and clinical practice there is a lack of consensus about the best diagnostic tests concerning platelet function. In tha - lassaemics the reduced LTG and the prolonged PFA-100 closure time must not be overestimated due to the limited clinical manifestations. The administration of salicylates in splenectomised thalassaemics should not be ignored. Never - Figure 1. Age distribution. theless, bleeding can occur in patients with platelet defects of the above type.

haematologica | 2012; 97(s1) | 189 17 th Congress of the European Hematology Association

0468 0469

A SINGLE CENTRE EXPERIENCE OF INHIBITOR ERADICATION THERA - MANAGEMENT OF PATIENTS WITH HHT DURING PREGNANCY PY IN SEVERE HAEMOPHILIA A J Byrne , K Murphy G Ooi , C Owoeye, L O’Connell, E McLaughlin, M Kavanagh, J Smith, B Philbin, The National Maternity Hospital, Dublin, Ireland I Regan, O Smith, B Nolan Our Lady’s Children’s Hospital, Crumlin, Dublin, Ireland Background. Hereditary Hemorrhagic Telangiectasia (HHT) is a genetic dis - order of the blood vessels, which affects approximately 1 in 5000 people. Preg - Background. One of the most serious complications of haemophilia A is the nant women with HHT should be considered ‘high risk’. They should regularly development of factor VIII (FVIII) inhibitors. Immune tolerance induction (ITI) pro - attend multidisciplinary (Consultant Haematologist, Consultant Obstetrician, tocols use FVIII at varying doses with the addition of other immunomodulatory ther - Consultant Anesthetist and midwives) high risk clinics. At these clinics the apies in non-responders. This is a 20 year single centre experience of inhibitor women will be advised on the pregnancy-associated risks. The women should eradication therapy in severe haemophilia A. Methods. We reviewed the heath highlight if and when they experience any haemoptysis or sudden severe dys - care records of all patients with severe haemophilia A (FVIII: C <0.01 iu/ml) who pnoea, which can lead to immediate hospital admission. The outcome for any had developed a factor VIII inhibitor between 1990 and 2011. We identified 22 high-risk pregnancy is better when the high-risk multidisciplinary team is patients. Patients were excluded from the study if clinical details were inadequate informed and have a plan of care in place. Aims. We highlight the management or if the inhibitor was transient. All patients underwent ITI with recombinant FVIII of 3 women (five pregnancies) with significant issues pertaining to the manage - (rFVIII) ± immunomodulatory therapy. Immune tolerance was defined as 3 con - ment of HHT in pregnancy. Methods. PAVMsIdeally women planning a preg - secutive negative monthly inhibitor screens with FVIII recovery greater than nancy should have pre-pregnancy screening of PAVMs and treated according - 66%.ResultsWe report on 14 children (ten unrelated and two brother pairs) with ly. If there was no recent screening of PAVMs prior to conception then regular severe haemophilia A and FVIII inhibitor. Age at inhibitor detection ranged from 0.9 screening of PAVMs at the high-risk clinic and treatment of same during the months to 79 months and number of exposure days varied from 5 to 182 (Table pregnancy.MRIsA spinal MRI must be carried out during pregnancy ideally ear 1). Circumstance of inhibitor development is shown in Table 1. Time from first ly in the pregnancy to ensure there are no spinal AVMs and also to allow for exposure to inhibitor diagnosis was 0.5 to 79.1 months. 50% had high respond - regional anesthesia.A cerebral MRI must be carried out on women experienc - ing inhibitors [>10 Bethesda units (BU)] with peak inhibitor titre ranging from ing cerebral symptoms or a strong family history of cerebral hemorrhage, or 10 to 712 BU. All patients had received rFVIII. ITI was started when the inhibitor cerebral symptoms. Delivery. All women must receive prophylactic antibiotics titre was <10 BU in 11 patients. Three patients started ITI at inhibitor titres of during her labour. Prolonged second stage of labour should be avoided espe - 17, 26 and 256 BU respectively. ITI rFVIII schedules varied from low dose (50 cially in women where cerebral AVMs have not been excludedIf general anes - IU/kg 3 times weekly) to high dose (100 IU/ kg twice daily). Three patients with thesia is required, a modified induction regimen using opiates should be admin - high inhibitor levels (141, 276 and 712 BU) also received immunosuppressive istered. Postnatal. Refer women and baby back to the nearest HHT centre and therapies; Rituximab, vincristine, prednisolone and mycophenolate mofetil. All ensure family screening where not previously not done. Summary and Con - 14 patients had a central venous device (CVC) inserted. Eight had CVC relat - clusions. Most cases where HHT is known in pregnancy the outcome is favor - ed infection(s), necessitating CVC removal and subsequent replacement in able as the pregnancy proceeds normally. Due to the complications associat - seven of the eight patients. Breakthrough bleeding was treated with rFVIIa. ed with HHT these pregnancies should be considered high risk . Immune tolerance was achieved in all patients. Time to elimination of inhibitors ranged from 1.2-48.3 months (median of 9 months). All patients are now on rFVIII prophylaxis.ConclusionDespite the variability of the patient characteris - 0470 tics and the ITI schedules, all patients were successfully tolerised with rVIII. FREQUENCY OF BLEEDING DISORDERS IN WOMEN PRESENTING WITH Table 1. Summary of inhibitor development. Patients 1, 4 and 9 received MENORRHAGIA IN THE NORTH OF IRAN other immunomodulatory therapies. Patient 4 had 2 relapse needing fur - G Janbabai 1, S Borhani 1, M Rashidi 1, T Farazmandfar 1, R Shekarriz 1, M ther ITI. Khademloo 2 1Cancer Research Center, Sari, Iran 2Departement of Social Medicine,Sari,Iran, Sari, Iran

Background. Menorrhagia is a common presentation of bleeding disorders, especially VWD in women. Aims. We decided to determine the frequency of these disorders in women with menstruation problems. Methods. 208 patients in reproductive age with menorrhagia were investigated for bleeding disorders in two steps. Step one includes CBC, PT, PTT and BT tests which were per - formed for all patients; patients who had an abnormality in step one, the sec - ond step tests including VWF: A;VWF: RCo, factors level, RIPA and platelet aggregometry were performed. Results. Of 208 patients who were investigat - ed for bleeding disorders, 53 patients (25%) had abnormalities in coagulation tests or platelet counts. Frequencies of bleeding were as follows: VWD, 14(6.73%); thrombocytopenia,13(6.25%); factor II deficiency , 2(0.96%); fac - tor V,1(0.48%); factor VII,3(1.44%); factor VIII,2(0.96%); Factor XI,4(1.92%); factor XII,4(1.92%); Bernard Soulier,2(0.96%). Furthermore, we found that 18 patients (8.65%) had abnormal PT, PTT or BT with no definite diagnosis. Con - clusions. In this study, the most common bleeding disorder was VWD followed by thrombocytopenia. Although other bleeding disorders are rare, in our study a number of them were found. We recommend that, every woman with men - orrhagia should be offered the above coagulation tests.

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SCREENING BLEEDING DISORDERS IN ADOLESCENTS AND YOUNG WOMEN WITH MENORRHAGIA N Sarper , S Caki Kilic, E Zengin, S Aylan Gelen Kocaeli University, Kocaeli, Turkey

Background. Chronic menorrhagia causes anemia and impairment of life qual - ity. Aims. To screen bleeding disorders in adolescents and young women with menorrhagia. Methods. The study was performed prospectively by the pedi - atric hematologists. A form including demographic characteristics of the patients, bleedings other than menorrhagia, familial bleeding history and char - acteristics of the menorrhagia and impairment of the life quality due to menor - rhagia was filled by the researcher during face-to- face interview with the

190 | haematologica | 2012; 97(s1) Amsterdam, the Netherlands, June 14 – 17, 2012 patient. Pictorial blood assessment chart was also delivered for evaluation of blood loss. All the patients underwent pelvic examination and pelvic USG by the gynecologists. Whole blood count, peripheral blood smear, blood group, serum transaminases, urea, creatinine, ferritin, PFA-100, PT, aPTT, INR, TT, fibrino - gen, VWF:Ag, VWF:RCo, FVIII, platelet aggregation assays were performed. Platelet aggregations were studied by lumiaggregometer. Results. Sixty patients completed the study. Mean age was 20.68±10.34 (10-48) years and 65% (n=39) of the patients were younger than 18 years. In 18 (46%) of the ado - lescents menorrhagia subsided spontaneously. In 20% (n=12) of the patients, a bleeding disorder was detected (4 Von Willebrand disease, 3 Bernard-Souli - er syndrome, 2 Glanzmann thrombastenia, 2 immune thrombocytopenic pur - pura, 1 congenital factor VII deficiency). Summary and Conclusions. In patients with menorrhagia at least complete blood count, peripheral smear, aPTT, PT, VWF:Ag, VWF:RCo, and fibrinogen assays must be performed. When there is history of nose and gum bleeding, platelet function assay by lumiagregometer must also be performed. In nearly half of the adolescents, menorrhagia is dysfunctional and transient. Detailed coagulation assays can be postponed in adolescents if bleeding history other than menorrhagia and/or family history of bleeding and/or parental consanguinity is absent. All subjects with menorrhagia must be consulted with gynecologists and hematologists.

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THROMBIN GENERATION AND MICROPARTICLE-ASSOCIATED PROCO - AGULANT ACTIVITY AS NEW TOOLS TO EVALUATE THE HEMOSTATIC PROPERTIES OF PLATELET CONCENTRATES L Russo , M Marchetti, C Tartari, A Vignoli, E Diani, C Giaccherini, C Verzeroli, S Marziali, A Falanga Ospedali Riuniti di Bergamo, Bergamo, Italy

Introduction. Thrombin generation (TG) and platelet microparticles procoag - ulant activity (MP-PCA) are two emerging assays utilized to characterize the hemostatic profile of plasma samples. Aims of the study. In this study we eval - uate: 1) the applicability of TG and MP-PCA assays in assessing the procoag - ulant properties of platelet concentrates (PC) prepared for transfusion therapy; and 2. whether these assays are sensitive to the methodology used for PC preparation. Methods. PC were prepared from pooled buffy-coat of overnight- stored whole blood from healthy donors at the Immunohematology and Trans - fusion Medicine Dept of Bergamo (Italy). 10 PC were prepared with the Fenw - al System (FS) and 4 with Terumo TACSI System (TTS). PC samples were col - lected the day of preparation (D0) and after 3 days of storage (D3) at 22°C, and then processed to isolate supernatants of PC (S-PC) by two serial centrifuga - tions. S-PC were tested for their TG potential by the calibrated automated thrombogram (CAT) assay employing 1 and 5 pM TF concentrations (both with 4 uM phospholipids), and for MP-PCA by the P-PPL/1 kit (all reagent by Sta - go). Results of TG are expressed as lag-time, peak, area under the curve (ETP), and time-to-peak (ttPeak). Results of MP-PCA are expressed in seconds (sec). Results. At D0, TG of S-PC was directly proportional to the concentration of TF used as trigger, and similar to that generated in normal pool plasma (NPP). Lag-time of S-PC derived from PC prepared with TTS was significantly short - er compared to those prepared with FS (p<0.05), being the other TG parame - ters not different. Lag-time (+43%) and ttpeak (+24%) of S-PC collected after 3 days of storage were significantly (p<0.05) increased compared to D0, inde - pendently of the type of PC preparation. Differently, we could observe a signif - icant decrease of ETP in S-PC prepared with TTS (1478±238 vs 1058±216 nM*min; p<0.05), but not in S-PC prepared with FS (1501±240 vs 1440±216 nM*min). At baseline, MP-PCA in S-PC (80.34±9.21 sec) was similar to that found in NPP (82.5±10.01 sec; p=ns). The analysis according to method of PC preparation did not show any significant difference in MP-PCA at baseline. The MP-PCA of S-PC collected at D3 was significantly higher compared to D0 (p<0.05), as this time point the S-PC prepared with TTS (TTS: 84±6.5; FS: 78±10.01 sec) showed the highest procoagulant potential. Conclusions. Our data support the validity of CAT and P-PPL1 assays in the assessment of pro - coagulant activity of PC. The increased MP-PCA observed in samples collect - ed after 3 days of storage reveals the occurrence of platelet activation/apopto - sis, particularly in S-PC prepared with TTS. These results provide background for further evaluation of the usefulness of these new tests to assess the quali - ty of PC.

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