35th Hemophilia Symposium Hamburg 2004: Epidemiology;Risk of Infections and Inhibitors in Hemophilia; Chronic lic Synovitis and Long-term Results of Orthopedic ... Hemostaseology;Free Lectures

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35th Hemophilia Symposium Hamburg 2004 Editors: I. Scharrer, W. Schramm Presentation: Epidemiology Risk of Infections and Inhibitors in Hemophilia Chronic Synovitis and Long-term Results of Orthopedic Treatment Laboratory Diagnostics: Coagulation Factors, Inhibitors, Monitoring Pediatric Hemostaseology Free Lectures

Scientific Board: I. Scharrer, Frankfurt/Main W. Schramm, Munich

Chairmen: G. Auerswald (Bremen); U. Budde (Hamburg); M. von Depka Prondzinski (Hanover); L. Gürtler (Greifswald); W. Kreuz (Frankfurt/Main); K. Kurnik (Munich); A. Kurth (Frankfurt/Main); I. Scharrer (Frankfurt/Main); R. Schneppenheim (Hamburg); W. Schramm (Munich); R. Zimmermann (Heidelberg)

Professor Dr. med. Inge Scharrer Hemophilia Center, Dept. of Internal Medicine University Hospital Theodor-Stern-Kai 7 D-60590 Frankfurt am Main Germany

Professor Dr. med. Wolfgang Schramm Dept. of Hemostaseology University Hospital Ziemssenstr. 1a D-80336 München Germany

ISBN-10 ISBN-13

3-540-28543-1 Springer Berlin Heidelberg New York 978-3-540-28543-4 Springer Berlin Heidelberg New York

Library of Congress Control Number: 2005930815 A catalog record for this book is available from Library of Congress. Bibliographic information published by Die Deutsche Bibliothek. Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the Internet at http://dnb.ddb.de This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. Springer is a part of Springer Science+Business Media springeronline.com © Springer Verlag Berlin Heidelberg 2006 Printed in Germany The use of designations, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publisher cannot guarantee the accu-racy of any information about dosage and application contained in this book. In every individual case the user must check such information by consulting the relevant literature. Editor: Dr. Ute Heilmann, Heidelberg, Germany Desk editor: Meike Stoeck, Heidelberg, Germany Production: Goldener Schnitt, Sinzheim, Germany Typesetting: cicero Lasersatz GmbH & Co. KG, Dinkelscherben, Germany Print: Druckerei Stürtz, Würzburg, Germany Printed on acid-free paper

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Contents

Johann Lukas Schoenlein Prize 2004 I. Scharrer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XXXI

I. Epidemiology HIV Infection and Causes of Death in Patients with Hemophilia in Germany (Year 2003/2004 Survey) W. Schramm, H. Krebs, on behalf of the participating German hemophilia centers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3

Hemophilia Registry of the Medical Committee of the Swiss Hemophilia Association – Update and Annual Survey 2004 S. Hartmann, K. Locher . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13

II. Risk of Infections and Inhibitors in Hemophilia Update on the Safety of Clotting Factors, Mainly Regarding the Risk of Transmission of vCJD (Variant Creutzfeldt Jakob Disease) L. G. Gürtler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

21

nvCJD and Blood Products in the UK P. L. F. Giangrande . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

29

Mutation Type Dependent Inhibitor Risk – a Single Center Study on 432 Patients with Severe Hemophilia A J. Oldenburg, J. Schröder, R. Schwaab, C. Müller-Reible, E. Seifried, P. Hanfland, H.-H. Brackmann . . . . . . . . . . . . . . . . . . . . . . .

33

Inhibitor Incidence in Previously Untreated Patients (PUPs) with Hemophilia A and B. A Prospective Multi-Center Study of the Pediatric Committee of the German, Swiss and Austrian Society for Thrombosis and Hemostasis Research (GTH) W. Kreuz, G. Auerswald, U. Budde, H. J. Klose, H. Lenk and the GTH-PUP-Study-Group . . . . . . . . . . . . . . . . . . . . . . . . .

34

VI

Contents

ADVATE Inhibitor Risk Profile: 18 Months post-Licensure E. Gomperts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

38

EACH-Registry: An European Registry for Acquired Hemophilia A. Huth-Kühne, P. Lages, R. Fischer, R. Zimmermann . . . . . . . . . .

42

Elective Orthopedic Surgery for Hemophilia Patients with Inhibitors P. L.F. Giangrande . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

44

III. Chronic Synovitis and Long-term Results of Orthopedic Treatment Results after Total Knee and Hip Replacement in Patients with Hemophilia A – A Single Center Experience R. Klamroth, Ch. Heinrichs, S. Gottstein, R. Koch . . . . . . . . . . .

49

Motion Analysis Epidemiology in Hemophilic Children A. Seuser, G. Schumpe, T. Wallny, H.-H. Brackmann . . . . . . . . . . .

52

Radiosynoviorthesis in Hemophilic Arthropathy – A Single Center Experience S. Gottstein, R. Klamroth . . . . . . . . . . . . . . . . . . . . . . . . . . 57 IV. Laboratory Diagnostics: Coagulation Factors, Inhibitors, Monitoring Individual Therapy of Hemophilia – New Laboratory Methods Considering Platelets Th. Siegemund, A. Siegemund, S. Bassus, W. Wegert, S. Petros, U. Scholz, L. Engelmann . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

61

Epitope Mapping during FVIII Inhibitor Elimination with Rituximab Reveals Conformational Epitopes on FVIII and Identifies Small Molecules Blocking Inhibitor and Targeting B Cells C. Kessel, C. Königs, R. Linde, C. Escuriola-Ettingshausen, J. Roland, H. Stoll, T. Klingebiel, U. Dietrich, W. Kreuz . . . . . . . . . . . . . .

66

Lack of Factor VIII Expression Represents a Novel Mechanism Leading to Hemophilia A O. El-Maarri, H. Singer, H.-H. Brackmann, J. Schröder, J. Graw, C. R. Müller, W. Schramm, R. Schwaab, P. Hanfland, J. Oldenburg . .

81

Contents

VII

V. Pediatric Hemostaseology Effects of the Factor V G1691A Mutation and the Factor II G20210A Variant on the Clinical Expression of Severe Hemophilia A (< 2%) in Children – Results of a Multicenter Study K. Kurnik, C. Escuriola-Ettingshausen, S. Horneff, C. Düring, R. Schobess, C. Bidlingmaier, S. Halimeh, H. Pollmann, N. Bogdanova, U. Nowak-Göttl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

87

Continuous Infusion of Factor Concentrates in Children with Hemophilia A in Comparison to Bolus Injections C. Bidlingmaier, M. Deml, K. Kurnik . . . . . . . . . . . . . . . . . . . . 95 Decreased Clotting Factor Activity (VIII, IX, XI, and XII) due to Inhibitors with Lupus-like Activity in Childhood V. Aumann, L. Wiens, G. Lutze, U. Mittler . . . . . . . . . . . . . . . . . 102 VI. Free Lectures Effect of Activated Recombinant Coagulation Factor VII on the Function of Glycoprotein IIb/IIIa-Inhibited Platelets in Vitro M. Udvardy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

109

Mutation Analysis in Hereditary Angioedema Identifies Patients at Risk for Developing Acute and Life Threatening Edema T. Förster, A. Kocot, J. Schröder, W. Kreuz, E. Aygören-Pürsün, I. Martinez-Saguer, K. Bork, I. Scharrer, C. Müller, J. Oldenburg .

120

Recurrent Mutation in ADAMTS13 Gene as a Cause of a Hereditary Thrombotic Thrombocytopenic Purpura in the Czech Republic I. Hrachovinová, P. Salaj, J. Suttnar, Sˇ. Rittich, P. Jinoch, T. Sˇuláková, ˇ ulícˇek, T. Seeman . . . . . . . . . . . . . . . . . . . . . . . . . . 123 J. Pták, P. D Clinical Manifestations of Patients with Dysfibrinogenemia W. Miesbach, V. Catania, M. Boehm, Th. Vigh, I. Scharrer

. . . . . . .

126

Case Report of a FXIII Inhibitor in a 77-year-old Patient W. Miesbach, M. Boehm, Ch. von Auer, I. Scharrer . . . . . . . . . . . .

130

VII. Poster a) Hemophilia and Hemorrhagic Disorders The ABC’s of Hemophilia German Working Group of Hemophilia Nurses . . . . . . . . . . . . . . . .

135

VIII

Contents

The Long Journey to Being Diagnosed as a Carrier of Hemophilia A – A Woman with Abnormally Prolonged Bleeding after Myocardial Infarction W. Miesbach, B. Putz, Ch. von Auer, I. Scharrer . . . . . . . . . . . . .

136

Clinical Investigation of Orthopedic Outcome in Patients with Severe Hemophilia – Advantage of an Early Prophylactic Treatment? S. Meister, K. Christensen, A. Weidenhammer, T. Spranger, G. Auerswald . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

138

The Relevance of Thrombophilic Risk Factors on Bleeding Tendency in Hemophilia A Patients M. Krause, A. Hahn, F. Peyvandi, I. Scharrer . . . . . . . . . . . . . . .

142

Magnetic Field Therapy in Patients with Severe Hemophilia – Motion Analysis and Quality Control A. Seuser, G. Schumpe, T. Wallny, H.-H. Brackmann . . . . . . . . . . .

144

The Clinical Course of two Patients Receiving High Dose Factor VIII – Replacement Therapy K. H. Beck, M. Mohrmann . . . . . . . . . . . . . . . . . . . . . . . . . . .

150

Determination of the Factor VIII Plasma Activity of Hemophilia A Patients Treated with a New Recombinant Factor VIII Concentrate K.-W. von Pape, R. Klamroth, U. Kyank, J. Bohner . . . . . . . . . . . . .

153

Socio-Economic Aspects of Hemophilia Treatment in Romania D. Mihailov, M. S¸erban, B. Lippert, W. Schramm, S. Arghirescu . . . .

157

Immunosuppressive Treatment in Acquired von Willebrand’s Syndrome H.-H. Wolf, A. Harba, A. Frühauf, H.-J. Schmoll . . . . . . . . . . . . . .

165

HCV-Infection in HIV-Infected and Non-Infected People with Hemophilia – A Retrospective Study: Medical Aspects H. Krebs, M. M. Schneider, W. Schramm . . . . . . . . . . . . . . . . . .

168

HCV-Infection in HIV-Infected and Non-Infected People with Hemophilia – A Retrospective Study: Psychosocial Aspects M. M. Schneider, H. Krebs, W. Schramm . . . . . . . . . . . . . . . . . .

173

Testing Factor VIII Activity by Using the Chromogenic Assay in Carriers of Hemophilia A W. Miesbach, Th. Vigh, I. Scharrer . . . . . . . . . . . . . . . . . . . . .

179

Hip Replacement in Hemophilic Patients – A 30 Years Single Center Experience A. Kurth, Ch. Eberhardt, L. Hovy, M. Krause, I. Scharrer . . . . . . .

183

Contents

IX

b) Inhibitors in Hemophilia Successful Therapy with anti CD20 Monoclonal Antibody Rituximab in Patients with Acquired Hemophilia against Factor VIII M. Krause, Ch. von Auer, I. Stier-Brück, I. Scharrer . . . . . . . . . .

187

Unusual Prolonged Course of an Immune Tolerance Therapy (ITT) in a Patient with Severe Hemophilia A and a High-Titer Inhibitor Development S. Meister, T. Spranger, K. Christensen, H.-H. Brackmann, G. Auerswald . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

190

Successful Major Surgery with Minimal Dosage of rFVIIa in a Hemophilia A Patient with High Level of Alloantibodies to Factor VIII M. S¸erban, P. T˛epeneu, S. Arghirescu, R. Badet˛i, C. Jinca, D. Mihailov, D. Lighezan, W. Schramm . . . . . . . . . . . . . . . . . . . . . . . . . . .

194

Inhibitor Development after Continuous Infusion of Factor VIII: A Retrospective Study in Germany Ch. von Auer, J. Oldenburg, M. Krause, W. Miesbach, I. Scharrer and CI-Study Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

198

Inhibitors in PTP’S: A Retrospective Study in Germany Ch. von Auer, J. Oldenburg, M. Krause, W. Miesbach, I. Scharrer and PTP-Study Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

201

Elective Orthopedic Surgery in Inhibitor Patients – the Frankfurt Concept A. Kurth, U. Stumpf, B. Habermann, M. Krause, I. Scharrer . . . . . .

205

c) Clinic and Casuistics The Frequency of Venous Thromboembolism in Women with FV Leiden in Association with Pregnancy and Puerperium P. Dulícˇek, J. Maly´, M. Pecka, M. Beránek . . . . . . . . . . . . . . . . . .

209

d) Diagnostics Spectrum of Molecular Defects and Mutation Detection Date in Patients with Severe Hemophilia A N. Bogdanova, A. Markoff, U. Nowak-Göttl, R. Eisert, C. Wermes, H. Pollmann, A. Todorova, A. Eigel, B. Dworniczak, J. Horst . . . . . . 215 Hemophilia Patients and Prothrombotic Gene Mutation A. Hlusˇí, V. Krcˇová, P. Novák, L. Slavík . . . . . . . . . . . . . . . . . . .

219

X

Contents

gAla82Gly Represents a Common Fibrinogen Chain Variant in Caucasians V. Ivaskevicius, E. Jusciute, M. Steffens, Ch. Geisen, P. Hanfland, Th.F. Wienker, E. Seifried, J. Oldenburg . . . . . . . . . . . . . . . . . .

221

A Novel Mutation (Asp36Tyr) in the Vitamin K Epoxide Reductase Complex Subunit 1 Gene (VKORC1) Causes Increased Phenprocoumon Requirement C. Geisen, G. Spohn, K. Sittinger, S. Rost, M. Watzka, P. Lages, A. Huth-Kühne, R. Zimmermann, C. R. Müller, E. Seifried, J. Oldenburg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

226

Denaturing High Performance Liquid Chromatography Represents an Efficient Technique for Detection of Heterozygous Large Deletions in Antithrombin Gene A. Pavlova, O. El-Maarri, B. Luxembourg, E. Lindhoff-Last, D. Delev, M. Watzka, E. Seifried, J. Oldenburg . . . . . . . . . . . . . . . . . . . .

230

e) Miscellaneous Recurrent Coronary Stent Thrombosis in a Patient with Combined Aspirin and Clopidogrel Resistance Ch. Templin, A. Schaefer, B. Stumme, H. Drexler, M. von Depka Prondzinski . . . . . . . . . . . . . . . . . . . . . . . . . .

235

Coagulation Factor XIII Mutation Profile: Update 2004 V. Ivaskevicius, R. Seitz, H.-H. Brackmann, W. Eberl, K. Kurnik, W. Kreuz, R. Klamroth, H. Rott, R. M. Loreth, F. Herrmann, E. Seifried, J. Oldenburg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 Site-Directed Mutagenesis of VKORC1, the Target Protein of Coumarin-Type Anticoagulants S. Rost, A. Fregin, M. Hünerberg, C. R. Müller, J. Oldenburg . . . . .

242

Treatment of Dilution Coagulopathy by Fibrinogen and Platelet Concentrates C. De Lorenzo, M. Spannagl, B. Heindl . . . . . . . . . . . . . . . . . . . 245 Pathogenesis of Hepatic Veno-Occlusive Disease in Patients Undergoing Hematopoietic Stem Cell Transplantation M. Dávid, O. Tóth, Á. Nagy, B. Meng, J. Tábori, H. Losonczy . . . . . . .

247

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

259

List of Participants

ABESADZE, N., Frau Dr. Universitätsklinikum Hamburg-Eppendorf, Abt. für Hämatologie/Onkologie, D-Hamburg ANDRITSCHKE, K., Frau Universitätskinderklinik, Hämophilie-Ambulanz 31-1, D-Frankfurt ANTONY, CH., Frau Dr. Universitätsklinikum Ulm, Abt. Innere Med. III, D-Ulm ARENDS, P., Dr. Krankenhaus Güssing, A-Güssing ASPÖCK, G., Prim. Dr. Krankenhaus Wels, A-Wels AUERSWALD, G., Dr. Klinikum Bremen-Mitte gGmbH, Prof. Hess-Kinderklinik, D-Bremen AUMANN, V., Dr. Otto-von-Guericke-Universitätsklinik, Zentrum für Kinderheilkunde, D-Magdeburg AYGÖREN-PÜRSÜN, E., Frau Dr. Universitätsklinikum, Zentrum für Kinderheilkunde, D-Frankfurt/Main BAIER, K., Frau Dr. Universitätsklinik Graz, A-Graz BALLEISEN, L., Prof. Dr. Evangelisches Krankenhaus Hamm, D-Hamm BARTHELS, M., Frau Prof. Dr. D-Hannover

XII

List of Participants

BAUMGARTNER, CH., Dr. FMH Kinder- und Jugendmedizin, CH-Gossau BECK, K.H., Dr. Med. Dienst der Krankenkassen, D-Lahr BECK, CH., Frau Dr. D-Berlin BECKER, TH., Dr. Interessengemeinschaft Hämophiler e. V., D-Bonn BECKER, D., Frau Universitätsklinik Hamburg-Eppendorf, Päd. Hämatologie/Onkologie, D-Hamburg BEESER, H.P., Prof. Dr. Institut für Qualitätsmanagement in Hämostaseologie und Transfusionsmedizin, D-Teningen BEHA, L., Frau Praxis Dr. Wolfgang Mondorf, D-Frankfurt/Main BEHNISCH, W., Dr. Universitätsklinik, Abt. für Kinder und Jugendmedizin, D-Heidelberg BERGS, B., Frau AOK Hessen – Die Gesundheitskasse, D-Eschborn BERTHOLD, B., Dr. Dietrich-Bonhoeffer-Klinikum, D-Neubrandenburg BEUTEL, K., Frau Dr. Universitätskinderklinik Hamburg-Eppendorf, Abt. für pädiatr. Hämatologie/Onkologie, D-Hamburg BIDLINGMAIER, CH., Dr. Klinikum der Universität München, Dr. v. Haunersches Kinderspital, D-München BLICKHEUSER, R., Dr. DRK-Kinderklinik, D-Siegen BOEKHORST, J., Dr. University Medical Center Nijmegen, NL-Nijmegen

List of Participants

BOGDANOVA, N., Frau Dr. Institut für Humangenetik, KKM Münster, D-Münster BOHN, U., Frau Medizinische Hochschule Hannover, Innere Medizin/Hämatologie, D-Hannover BRACKMANN, CH., Frau Universitätskliniken Bonn, Institut für Experimentelle Hämatologie/Transfusionsmedizin, D-Bonn BRACKMANN, H.-H., Dr. Universität Bonn, Institut für Experimentelle Hämatologie und Transfusionsmedizin, D-Bonn BRAND, B., Frau Dr. Universitätsspital Zürich, CH-Zürich BRAUN, U., Frau Dr. DHG, CH-Zurzach BROCKHAUS, W., Dr. Gerinnungsambulanz am Klinikum Nürnberg, D-Nürnberg BUDDE, U., Prof. Dr. AK Laborbetriebsgesellschaft mbH, D-Hamburg CARL, H.D., Dr. Waldkrankenhaus St. Marien, Orthopädische Abteilung, D-Erlangen CASPARI, G., PD Dr. Institut für Transfusionsmedizin, D-Brandenburg CASTER, CH., Frau Universitätsklinikum des Saarlandes, D-Homburg CHRISTENSEN, K., Frau Dr. Prof. Hess Kinderklinik, Ambulanz Dr. Auerswald, D-Bremen CVIRN, G., Dr. Universitätsklinik Graz, A-Graz DANIELS, G., Frau Medizinische Hochschule Hannover, Dialyse, D-Hannover

XIII

XIV

List of Participants

DAVID, M., Frau Dr. Medical and Hygiene Center of Sciences, HU-Pécs DEHMEL, H. Med. Hochschule Hannover, D-Hannover DEML, M.-M., Frau Dr. Dr. v. Haunersches Kinderspital, D-München DITTMER, R., Frau Dr. Gemeinschaftslabor Prof. Arndt & Partner, D-Hamburg DOCKTER, G., Prof. Dr. Universitätsklinik für Kinder und Jugendmedizin, D-Homburg DRESAR-MAYERT, B., Frau Dr. AOK Hessen – Die Gesundheitskasse, D-Eschborn DRESSEL, B., Dr. A-Nüziders DÜRING, CH., Frau Universitätskinderklinik Münster, Päd. Hämatologie/Onkologie, D-Münster DWENGER, A., Frau Dr. BMGS, D-Bonn EBERL, W., Dr. Klinikum Braunschweig, Kinderklinik, D-Braunschweig EGGIMANN, H.-P. Deutsche Hämophilie-Gesellschaft e.V., D-Hamburg EICHERT-NAUMANN, A., Frau Deutsche Hämophilie-Gesellschaft e.V., D-Hamburg EIFRIG, B., Frau Dr. Universitätsklinikum Hamburg-Eppendorf, Med. Klinik II/Gerinnungsambulanz, D-Hamburg EL-MAARRI, O., Dr. Universität Bonn, Institut für Experimentelle Hämatologie und Transfusionsmedizin, D-Bonn ENDRES, G., Dr. Landeskrankenhaus Bregenz, A-Bregenz

List of Participants

ESCURIOLA-ETTINGSHAUSEN, C., Frau Dr. J.W. Goethe Universitätsklinikum, Zentrum für Kinderheilkunde, D-Frankfurt/Main FALGER, J., Frau Dr. Universitätsklinik für Kinder- und Jugendheilkunde, A-Wien FÄSSLER, H., Dr. Specialista FMH Medicina interna, CH-Chiasso FENGLER, V., Frau Klinikum Frankfurt (Oder) GmbH, Klinik für Kinder- u. Jugendheilkunde, D-Frankfurt/Oder FISCHER, B., Frau Abt. f. pädiatrische Hämatologie und Onkologie des Universitätskrankenhauses Eppendorf, D-Hamburg FÖRSTER, T., Frau Institut für Humangenetik, Biozentrum, Universität Würzburg, D-Würzburg FRANKE, D., PD Dr. Praxis Blutgerinnungsaufnahme, D-Magdeburg FRITSCH, P., Dr. Universitätsklinik Graz, Kinderklinik, A-Graz GABRIEL, M., Dr. Blutzentrale Linz, A-Linz GALLISTL, S., Prof. Dr. Universitätsklinik Graz, A-Graz GEISEN, U., Dr. Universitätsklinik Freiburg, Abteilung Klinische Chemie, Zentrallabor, D-Freiburg GERHARDT, A., Frau Dr. Universitätsklinikum Düsseldorf, Inst. für Hämostaseologie u. Transfusionsmedizin, D-Düsseldorf GIANGRANDE, P. L. F., Prof. Churchill Hospital, Oxford Haemophilia Center, GB-Oxford

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XVI

List of Participants

GIEBEL, A., Dr. Medizinische Klinik Innenstadt der Ludwig-Maximilians-Universität, D-München GIERSDORF, G., Frau Kurpfalz-Krankenhaus Heidelberg, Hämophiliezentrum, D-Heidelberg GILDBERG, P.K. D-Flensburg GÖHAUSEN, M., Frau Hämophiliezentrum Münster, Praxis Dr. med. H. Pollmann, D-Münster GOLDMANN, G., Dr. Institut für Experimentelle Hämatologie und Transfusionsmedizin, D-Bonn GOMBERT, B., Frau Universität Rostock/Medizinische Fakultät, Klinik und Poliklinik für Innere Medizin, Abt. Hämatologie und Onkologie, D-Rostock GOTTSTEIN, S., Frau Dr. Vivantes Klinikum im Friedrichshain, Hämophiliezentrum, D-Berlin GROSS, J. CH., Dr. Universitätsklinikum des Saarlandes, Institut für Klinische Hämostaseologie, D-Homburg GÜLDENRING, H., Dipl.-Mediziner Städtisches Krankenhaus, Dresden-Neustadt/Kinderklinik, D-Dresden GÜRTLER, L., Prof. Dr. Friedrich-Loeffler-Institut für Medizinische Mikrobiologie, Ernst-Moritz-Arndt Universität, D-Greifswald GÜTHNER, CH., Frau Dr. Universitätsspital Zürich, CH-Zürich HABERLAND, H., Dr. Klinik für Kinder- u. Jugendmedizin Lindenhof, D-Berlin HALBMAYER, W.-M., Dr. A-Wien HALIMEH, S., Frau Praxis für Laboratoriums- und Transfusionsmedizin, D-Duisburg

List of Participants

XVII

HANFLAND, P., Prof. Dr. Institut für Experimentelle Hämatologie und Transfusionsmedizin der Universität Bonn, D-Bonn HARBA, A.-E. Universität Halle, Klinik für Innere IV, D-Halle HARTMANN, S., Frau Dr. CH-Chur HASCHBERGER, B., Frau Paul-Ehrlich-Institut Langen, D-Langen HAUCH, H. Universitätsklinikum Hamburg-Eppendorf, Abt. für Hämatologie/Onkologie, D-Hamburg HAUSHOFER, A., Doz. Dr. Zentralklinikum St. Pölten, A-St. Pölten HAWLINA-HESS, N., Frau Universitätsklinikum Bonn, Institut für Experimentelle Hämatologie und Transfusionsmedizin, Hämophilie-Zentrum, D-Bonn HEIDE, D., Frau Universitätskinderklinik Hämatologisch/Onkologische Ambulanz, D-Düsseldorf HEINE, S., Frau Dr. Universitätskliniken des Saarlandes, Klinik für Kinder-und Jugendmedizin, D-Homburg HEISTINGER, M., Dr. Landeskrankenhaus Klagenfurt, 1. Med., A-Klagenfurt HEMPELMANN, L., Dr. D-Berlin HENNEWIG, U., Frau Dr. Klinik für Kinderonkologie und -hämatologie, D-Düsseldorf HENSELER, D., Frau IHT, Hämophilieambulanz, D-Bonn HERRMANN, F. H., Prof. Dr. Dr. Ernst-Moritz-Arndt-Universität, Institut für Humangenetik, D-Greifswald

XVIII

List of Participants

HERZOG, D. AOK Schleswig-Holstein, D-Lübeck HILGER, A., Frau Dr. Paul-Ehrlich-Institut Langen, D-Langen HOFMANN, K., Dr. Klinikum Chemnitz gGmbH, D-Chemnitz HOFMANN, H., Dr. DRK Blutspendedienst, Berlin-Brandenburg, D-Potsdam HOFMANN, M., Dr. Klinikum der FSU, Klinik für Anästhesiologie und Intensivtherapie, D-Jena HOLZHÜTER, H., Prof. Dr. Hämophilie-Zentrum Nord-West, D-Bremen HUTH-KÜHNE, A., Frau Dr. Kurpfalzkrankenhaus Heidelberg, D-Heidelberg IMAHORN, P., Dr. Kinderspital Luzern, CH-Luzern IVASKEVICIUS, V., Dr. Institut für Transfusionsmedizin u. Immunhämatologie, D-Frankfurt/Main JENKINS, S., Frau Klinikum der Universität München Kinderklinik/Hämostaseologie, D-München JULEN, E., Dr. FMH Allgemeine Medizin, CH-Zermatt KADAR, J., Dr. habil. D-Köln KALNINS, W. Deutsche Hämophilie-Gesellschaft e.V., D-Hamburg KEHRL, B., Frau Prof. Dr. Klinik u. Poliklinik für Anästhesiologie, Uniklinikum Münster, D-Münster KEISKER, A., Dr. Universitätskinderspital Bern, CH-Bern

List of Participants

XIX

KESSEL, CH., Dipl. Biol. J.W. Goethe Universität, Zentrum f. Kinderheilkunde III, D-Frankfurt/Main KLÄFFLING, CH., Frau Dr. J. W. Goethe-Universitätsklinikum, Med. Klinik I/Gefäßzentrum, D-Frankfurt/Main KLAMROTH, R., Dr. Vivantes Klinikum im Friedrichshain, Abt. Klinische Hämostaseologie, D-Berlin KLARE, M., Dr. Helios Klinikum Berlin, Klinikum Buch – II. Innere Klinik, D-Berlin KLIER, H., Dr. Steierm. Gebietskrankenkasse, A-Graz KLUGE, A., Frau Dr. Praxis für Innere Medizin und Gefäßkrankheiten, D-Magdeburg KNÖBL, P., Univ.-Prof. Dr. Universitätsklinik Wien, A-Wien KNÖFLER, R., Dr. Universitätskinderklinik Dresden, D-Dresden KÖHLER-VAJTA, K., Frau Dr. D-Grünwald KÖNIGS, CH. J.W. Goethe Universität, Zentrum für Kinderheilkunde III, D-Frankfurt/Main KONWALINKA, G., Dr. Mag. A-Gallneukirchen KÖSTENBERGER, M., Dr. Universitätsklinik Graz, A-Graz KÖTHEMANN, M., Frau Dr. Techniker Krankenkasse, D-Hamburg KRAUSE, M., Frau Dr. Hämophilie Ambulanz, J.W.Goethe-Universitskliniken, D-Frankfurt/Main

XX

List of Participants

KREBS, H., Dr. Medizinische Klinik Innenstadt der Ludwig-Maximilians-Universität, D-München KREMER, D., Frau Universitätsklinikum Bonn, Institut für Experimentelle Hämatologie und Transfusionsmedizin, D-Bonn KREUZ, W., PD Dr. Klinikum der Johann Wolfgang Goethe-Universität, Klinik für Kinderheilkunde III, Pädiatrische Hämatologie, Onkologie und Hämostaseologie, D-Frankfurt/Main KROHN, J., Frau Klinikum d. Med. Fakultät, Martin-Luther-Univ. Halle-W., D-Halle KURME, A., Dr. D-Hamburg KURNIK, K., Frau Dr. Ludwig-Maximilians-Universität, Dr. von Haunersches Kinderspital, D-München KURNIK, P., Dr. LKH Klagenfurt, A-Klagenfurt KURTH, A., PD Dr. Orthopädische Universitätsklinik, Stiftung Friedrichsheim, D-Frankfurt/Main KÜTING, A., Frau Universitätsklinikum Homburg, D-Homburg KYANK, U., Frau Dr. Universität Rostock, Kinder- und Jugendklinik, D-Rostock LAGES, P., Dr. Kurpfalzkrankenhaus Heidelberg, D-Heidelberg LANG, A., Dr. LKH Feldkirch, A-Feldkirch LECHLER, E., Prof. Dr. D-Esslingen LEHMANN, I., Frau Universität Leipzig, Med. Klinik I/Hämostaseologie, D-Leipzig

List of Participants

LENK, H., Prof. Dr. Universitätskinderklinik u. Poliklinik für Kinder- und Jugendliche Leipzig, D-Leipzig LENZ, A., Dr. Vorarlberger Gebietskrankenkasse, A-Dornbirn LESCHNIK, B., Frau Universitätsklinik Graz, A-Graz LESTIN, H.-G., Prof. Dr. Klinikum Schwerin, Institut für Labormedizin, D-Schwerin LIESE, B., Frau Medizinische Hochschule Hannover, Innere Medizin / Hämatologie, D-Hannover LIGHEZAN, D., Dr. RO-Timisoara LINDHOFF-LAST, E., PD Dr. J. W. Goethe-Universitätsklinikum, Med. Klinik I/Gefäßzentrum, D-Frankfurt/Main LOCHER, K., Frau EDV Spezialistin für SHG Register, CH-Weingarten LORETH, R. M., Dr. Westpfalz-Klinikum GmbH, Medizinische Klinik III, Klinische Hämostaseologie, D-Kaiserslautern LOSONCZY, H., Prof. Dr. University of Pécs, HU-Pécs LÜHR, C., Frau Medizinische Hochschule Hannover, Med. Poliklinik/Abt. für Hämatologie, D-Hannover LUTZE, G., Prof. Dr. Otto-von-Guericke-Universität, Institut für Klinische Chemie und Pathobiochemie, D-Magdeburg MAAK, B., Prof. Dr. Thüringen-Klinik »Georgius Agricola« gGmbH, Kinderklinik, D-Saalfeld

XXI

XXII

List of Participants

MALE, CH., Univ.-Prof. Dr. Universitätsklinik für Kinder- und Jugendheilkunde Wien, A-Wien MAREK, R., Dr. Mag. Wiener Gebietskrankenkasse, A-Wien MARTIN, G., Frau Dr. Hoˆpital des Enfants Genf, CH-Genf MAURER, M., Prof. D-Bernau MAYR, G., Dr. A-Walding MEDGYESSY, I., Dr. OVSZ Regionális Vérellátó Központ Debrecen, HU-Debrecen MEILI, E., Dr. Universitätsspital Zürich, CH-Wetzikon METZEN, E., Frau Universitätsklinikum Düsseldorf, Institut für Hämostaseologie u. Transfusionsmedizin, D-Düsseldorf MIESBACH, W., Dr. Universitätsklinikum Frankfurt, Hämophilie-Ambulanz, D-Frankfurt/Main MIHAILOV, D., Frau Dr. University of Medicine, RO-Timisoara MÖBIUS, D., Frau Dr. Carl-Thiem-Klinikum, Klinik für Kinder- und Jugendmedizin, D-Cottbus MONDORF, W., Dr. Praxis und Labor zur Diagnostik und Therapie von Blutgerinnungsstörungen, D-Frankfurt/Main MÖßELER, J., Dr. D-Dillingen MÜLLER, ST. Deutsche Hämophilie-Gesellschaft e.V., D-Hamburg MUß, N., Dr. Salzburger Gebietskrankenkasse, A-Salzburg

List of Participants

XXIII

NAGY, A., Frau Dr. University of Pécs, HU-Pécs NEMES, L., Dr. Országos Hemophilia Központ, HU-Budapest NEUMANN, S., Frau Klinikum Bremen Mitte gGmbH, Prof.-Hess-Kinderklinik, Ambulanz Dr. Auerswald, D-Bremen NIEKRENS, C., Frau Dr. Städtische Klinik Delmenhorst, D-Delmenhorst NIMTZ-TALASKA, A., Frau Dr. Kinderklinik Frankfurt/Oder, D-Frankfurt/Oder NOWAK-GÖTTL, U., Frau Prof. Dr. Universitätsklinik, Pädiatrische Hämatologie und Onkologie, D-Münster OLDENBURG, J., PD Dr. DRK Blutspendedienst, D-Frankfurt/Main ORLOVIC, A.M., Frau Dr. Klinikum im Friedrichshain, Hämostaseologie, D-Berlin PABINGER, I., Frau Prof. Universitätsklinik Wien, A-Wien PAVLOVA, A., Frau Dr. Institute of Transfusion Medicine and Immunohaematology, DRK Blood-Donor Service, Baden-Württemberg-Hessen, D-Frankfurt/Main PETER, K., Frau Dr. Inselspital Bern Hämostaselabor, CH-Bern PODEHL-KLOSE, J., Dr. Orthop. Fachkrankenhaus, Annastift Klinik e.V., D-Hannover POLEY-OCHMANN, S., Frau Dr. Paul-Ehrlich-Institut Langen, D-Langen POLLICH, CH., Dr. Vivantes Klinikum im Friedrichshain, Abt. Klinische Hämostaseologie, D-Berlin

XXIV

List of Participants

PRIESACK, J., Dr. Medizinische Hochschule Hannover, Abteilung Hämatologie, Hämostaseologie, Onkologie, D-Hannover RABENSTEIN, C., Frau Universitätsklinik Frankfurt, Zentrum für Innere Medizin, D-Frankfurt/Main RAMSCHAK, H., Dr. Universitätsklinik Graz, A-Graz REHBERGER, G., Dr. A-Frastanz REINHARD, H., Dr. Universitätskinderklinik Homburg/Saar, Pädiatrische Hämatologie und Onkologie, D-Homburg REITER, W. W., Dr. Facharzt für Innere Medizin, Hämatologie/Intern. Onkologie, D-Viersen RIEKE, M. D-Bergisch-Gladbach RINGKAMP, H., Frau Hämophiliezentrum Münster, Praxis Dr. med. H. Pollmann, D-Münster RISCHEWSKI, J., Dr. Universitätskinderspital, CH-Basel ROSCHITZ, B., Frau Dr. Universitätsklinik Graz, A-Graz ROST, S., Frau Institut für Humangenetik, Biozentrum, D-Würzburg ROTT, H., Frau Dr. Gemeinschaftspraxis Prof. Trobisch/Dr. Rott, D-Duisburg ROWOLD, CH., Frau Städtisches Klinikum Braunschweig gGmbH, Klinik für Kinder- und Jugendmedizin, D-Braunschweig SCHÄFER, CH., Frau Klinik für Kinder- und Jugendmedizin, Abt. Hämatologie/Onkologie, D-Homburg

List of Participants

XXV

SCHARRER, I., Frau Prof. Dr. Universitätsklinikum Frankfurt, Hämophilieambulanz, D-Frankfurt/Main SCHEEL, H., Dr. D-Leipzig SCHELLE, G. Interessengemeinschaft Hämophiler e. V., D-Bonn SCHELLE, F. Interessengemeinschaft Hämophiler e. V., D-Bonn SCHIRDEWAHN, B., Frau Dipl. Biologin DRK, Blutspendedienst Baden-Württemberg-Hessen gGmbH, D-Frankfurt/Main SCHLOMM, A., Frau Klinik f. Kinder und Jugendmedizin, Kinderaufnahme, D-Braunschweig SCHMIDT, O., Dr. Praxis für Gefäßkrankheiten, D-Magdeburg SCHNEIDER, M., Dr. Medizinische Klinik Innenstadt der Ludwig-Maximilians-Universität München, D-München SCHNEPPENHEIM, R., Prof. Dr. Universitätsklinikum Hamburg-Eppendorf, Abt. für Hämatologie/Onkologie, D-Hamburg SCHOLZ, U., Frau Dr. Universitätsklinikum Leipzig AöR, Med. Klinik und Poliklinik, Arbeitsbereich Klinische Hämostaseologie, D-Leipzig SCHRAMM, W., Prof. Dr. Medizinische Klinik Innenstadt der Ludwig-Maximilians-Universität München, Abt. Hämostaseologie, D-München SCHRÖDER, W., Frau Dr. Institut für Humangenetik, D-Greifswald SCHUBERT, CH., Frau Dr. Helios Klinikum Erfurt, Klinik für Innere Medizin, D-Erfurt SCHULTE OVERBERG, U., Frau Dr. Charité Campus Virchow-Klinikum, Universitätsmedizin Berlin, D-Berlin

XXVI

List of Participants

SCHULZE, S., Frau Otto-von-Guericke-Universität, Zentrum für Kinderheilkunde, Klinik für Päd. Hämatologie/Onkologie, D-Magdeburg SCHUMACHER, R., Dipl.-Mediziner D-Schwerin SCHÜTT, I., Frau AOK Hessen – Die Gesundheitskasse, D-Eschborn SEDLAK, W., Dr. A-Linz SEIFRIED, E., Prof. Dr. Blutspendedienst Baden-Württemberg-Hessen gGmbH, D-Frankfurt/Main SELLENG, K., Frau Dr. Ernst-Moritz-Arndt-Universität, Abt. Transfusionsmedizin, D-Greifswald S¸ERBAN, M., Frau Prof. Dr. University of Medicine, RO-Timisoara SEUSER, A., Dr. Kaiser-Karl-Klinik, Fachklinik für Orthopädie, D-Bonn SIEGEMUND, TH., Dipl. Biochemiker Universität Leipzig, Medizinische Klinik I, Intensivtherapiestation, D-Leipzig SITTINGER, K., Frau cand. med. Blutspendedienst Baden-Württemberg-Hessen gGmbH, D-Frankfurt/Main SPANNAGL, M., Dr. Medizinische Klinik Innenstadt der Ludwig-Maximilians-Universität, D-München STECOVA, N., Frau MU Dr. FNsP Trieda SNP1, SLO-Kosice STEIN, G. AOK Baden-Württemberg, Pharmazeutische Beratungsstelle, D-Lahr STOCKSCHLÄDER, M., Dr. Institut für Blutgerinnung und Transfusionswesen, D-Düsseldorf STOLL, H., Frau Universitätsklinikum Frankfurt, Kinderklinik, D-Frankfurt/Main

List of Participants

XXVII

STREIF, W., Univ.-Prof. Dr. Universitätsklinik Innsbruck, A-Innsbruck STUMPE, CH., Frau Krankenhaus im Friedrichshain, Klinische Hämostaseologie, D-Berlin STUMPF, U., Frau Dr. Orthopädische Uniklinik, Friedrichsheim, D-Frankfurt/Main STURM, P., Dr. A-Mittersill SYRBE, G., PD Dr. Asklepios-Fachklinikum, D-Stadtroda THEDIECK, S., Frau Universitätsklinik, Pädiatrische Hämatologie und Onkologie, D-Münster TIEDE, A., Dr. Medizinische Hochschule Hannover, Medizinische Klinik, Abt. Hämatologie-Onkologie, D-Hannover TIMELTHALER, H., Dr. A-Uttendorf TREGEL, M., Dr. Klinikum Frankfurt/Oder, D-Frankfurt/Oder TRUMMER, A., Dr. Medizinische Hochschule Hannover, Abt. Hämatologie-Onkologie, D-Hannover TÜRK-KRAETZER, B., Frau Dr. D-Oldenburg UDVARDY, M., Prof. Debreceni Tudományegyetem AOK, HU-Debrecen VERBRUGGEN, B., Dr. University Medical Center Nijmegen, NL-Nijmegen VIDOVIC, N., Frau Dr. Universitätskliniken Bonn, Institut für Experimentelle Hämatologie und Transfusionsmedizin, D-Bonn VIGH, TH., Dipl. Biochemiker J.W. Goethe Universität, Hämostaseologie, D-Frankfurt/Main

XXVIII

List of Participants

VOERKEL, W., Dr. Labor Dr. Reising-Ackermann und Partner, D-Leipzig VOGEL, G., Prof. Dr. D-Erfurt VOGT, B., Frau Universitätsklinikum Leipzig AöR Universitätsklinik u. Poliklinik für Kinder und Jugendliche, D-Leipzig VON AUER, CH., Frau

Dr. Universitätsklinik Frankfurt, Hämostaseologie, D-Frankfurt/Main

DEPKA PRONDZINSKI, M., PD Dr. Medizinische Hochschule Hannover, Abt. f. Hämatologie-Onkologie, D-Hannover

VON

WEID, N., Dr. CHUV, CH-Lausanne

VON DER

VON PAPE, K.-W.

Klinikum Fulda gAG, Institut für Laboratoriumsmedizin, D-Fulda ROENNE, B., Frau D-Bonn

VON

WANK, H., Dr. St. Anna Kinderspital, A-Wien WARMBIER, H., Frau Techniker Krankenkasse, D-Hamburg WEIDENHAMMER, A., Frau Prof.-Hess-Kinderklinik, Gerinnungsambulanz, D-Bremen WEINSPACH, S. Universitätskinderklinik Düsseldorf, Abt. für Hämatologie-OnkologieImmunologie, D-Düsseldorf WEINSTABEL, M., Dr. Tiroler Gebietskrankenkasse, A-Innsbruck WEINSTOCK, N., Dr. Labor Wagner + Partner, D-Göttingen

List of Participants

WEISS, J. Österr. Hämophiliegesellschaft, A-Wien WEISSER, J., Dr. Fachkrankenhaus gGmbH, D-Neckargemünd WENDISCH, E., Frau Dr. D-Dresden WERMES, C., Frau Dr. Medizinische Hochschule Hannover, Kinderklinik, D-Hannover WIEDING, J.U., Dr. D-Göttingen WIELAND, I., Frau Dr. Medizinische Hochschule Hannover, Kinderklinik, D-Hannover WILKEN, G., Frau Klinikum Bremen Mitte gGmbH, Prof.-Hess-Kinderklinik, Ambulanz Dr. Auerswald, D-Bremen WINDYGA, J., Dr. Institute of Hematology and Blood Transfusion, PL-Warsaw WINKELMANN, TH., Dipl. med. Städt. Klinikum Heinrich-Braun, Klinik für Kinder- und Jugendmedizin, D-Zwickau WULFF, K., Dr. Universität Greifswald, Institut für Humangenetik, D-Greifswald WUNDERLICH, S. Deutsche Hämophilie-Gesellschaft e.V., D-Hamburg WÜSTNER, J., Dr. A-Schoppernau ZACH, D., Frau Dr. Universitätsklinik Graz, A-Graz ZANIER, U., Frau Dr. Krankenhaus Dornbirn, A-Dornbirn

XXIX

XXX

List of Participants

ZELLHOFER, J. Österr. Hämophiliegesellschaft, A-Wien ZIEGER, B., Frau PD Dr. Universitätskliniken Freiburg, Universitäts-Kinderklinik, Abt. für Hämatologie, D-Freiburg ZIMMERMANN, R., Prof. Dr. Kurpfalz-Krankenhaus Heidelberg gGmbH, D-Heidelberg ZOGHLAMI, C., Frau Univ.-Prof. Dr. A-Wien ZOTZ, R., PD Dr. Universitätsklinik Düsseldorf, Institut für Transfusionsmedizin, D-Düsseldorf ZUPANCIC-SALEC, S., Frau Dr. University Hospital Rebro, CRO-Zagreb ZWIAUER, K., Univ.-Prof. Dr. Krankenhaus St. Pölten, A-St. Pölten

Johann Lukas Schoenlein Prize 2004 I. Scharrer

The Johann Lukas Schoenlein Prize was donated in 1977 by the company Immuno, today known as Baxter, in memory of Johann Lukas Schoenlein, who gave hemophilia its name. The aims of the prize are laid down in the statutes of the foundation: The foundation serves to promote clinical research in the area of chronic bleeding diseases, in particular, hemophilia and related congenital clotting disorders. It follows exclusively and directly charitable purposes and awards the prize for outstanding scientific research. The board of trustees responsible for awarding the prize is made up of seven independent scientists. This year there were four applicants for the prize. The prize committee had an extremely difficult time choosing the prizewinner, as the research work that was submitted was of a very high standard. The criterion for awarding the prize were and are as follows: ● Scientific value ● Clinical relevance ● Innovation ● Originality ● Correct Methodology ● Form and Style After extensive evaluation of all the submitted research, the 16th Johann Lukas Schoenlein Prize is awarded to PD Dr. Barbara Zieger for her publications on »Investigations into the significance of the septins 4, 5 and 8 for thrombocyte secretion.« The results are new and show a delightful originality. The scientific value of the publications has been rounded off to obtain an impact factor of 23.

XXXII

I. Scharrer

Dr. Zieger submitted four publications for consideration by the prize committee:

Zieger, B., Tran, H., Hainmann, I., Wunderle, D., Zgaga-Griesz, A., Bläser, S., Ware, J. Characterization and expression analysis of two human septin genes, PNUTL1 and PNUTL2. GENE 261 (2000) 197–203

Bläser, S., Jersch, K., Hainmann, I., Wunderle, D., Zgaga-Griesz, A., Busse, A., Zieger, B. Human septin-septin interaction: CDCrel-1 partner with KIAA0202. FEBS Letters 519 (2002) 169–172

Bläser, S., Jersch, K., Hainmann, I., Zieger, W., Wunderle, D., Busse, A., Zieger, B. Isolation of new splice isoforms, characterization and expression analysis of the human septin SEPT8 (KIAA0202). GENE 312 (2003) 313–320

Bläser, S., Horn, J., Würmell, P., Bauer, H., Strümpell, S., Nurden, P., Pagenstecher, A., Busse, A., Wunderle, D., Hainmann, I., Zieger, B. The novel human platelet septin SEPT8 is an interaction partner of SEPT4. Thromb Haemost (2004) 91: 959–966 The manifold difficult and elaborate methods which were used to obtain the results include cloning and expression studies, as well as Northern and Western analyses. Septins play an important role in platelet secretion. Dr. Zieger was able to show that the septins SEPT4, SEPT5 und SEPT8 are produced in the thrombocytes. Together with her work group, she was able to determine the cDNA of the three new human septins referred to, and to analysis the exons and introns. Her workgroup was also able to investigate and explain the expression patterns of these newly discovered genes and proteins by means of Northern and Western analyses. It was also possible to establish the necessary antibodies by producing the respective recombinant proteins. Furthermore, Dr. Zieger has identified the interaction partners for the human septins 4 and 5. This was the first description of the interactions between the human septins SEPT4 and SEPT8, and between SEPT5 and SEPT8. The septins SEPT4, 5 und 8 are localized around the alpha-granules in the thrombocytes. A translocation of septins SEPT4 and SEPT8 on the surface of the platelets followed stimulation of the platelets. This shows that the septins are involved in the secretion of thrombocytes. Furthermore, it is suspected that septins also regulate the release of the vonWillebrand factors. The investigations of Dr. Zieger and her workgroup have contributed, and will continue to contribute, to a better understanding of the activation and secretion in and from the thrombocytes.

Johann Lukas Schoenlein Prize 2004

XXXIII

These studies can be considered fundamental research. The clinical relevance of Dr. Ziegers results will still need to be seen, however, it is likely that they will involve the explanation of thrombocytopathia, and will eventually also have relevance towards the significance of the stimulated thrombocytes. In summary: SEPT4, 5 and 8 You have won the prize


SEPT4, 5 und 8 (acht) ihr habt den Preis gemacht

You emerged onto the surface Out of Freiburgs brook


Ihr tauchtet auf zur Oberfläche heraus aus Freiburgs Bächle

No longer do you remain hidden To cause problems for the Freiburgers


Nicht länger seid ihr verborgen und macht den Freiburgern Sorgen

But please reveal to us What is your true function?


Doch verratet uns nun schon Was ist eure Funktion?

Just which diseases do you cause? Because that interests us greatly


Mit welcher Krankheit treibt ihr euer Spiel? denn das interessiert uns doch recht viel

So we wish much joy and many blessings
Dazu wünschen wir viel Glück und Segen For all »Ziegers« further journeys in research auf »Ziegers« weiteren Forschungswegen

Dr. Zieger was born in 1960. She is a pediatrician, and has worked at the University Pediatric Clinic in Freiburg since April, 1997. She worked at the laboratory of Prof. Ruggeri and Prof. Ware in the Scripps Clinic in La Jolla in the USA from 1994–1997, and it was there that the foundations of the published award-winning studies were laid. The J. L. Schoenlein is not the only prize that Dr. Zieger has won. She was also awarded the Prof. Landbeck Prize at the GTH 2004, among other awards. In real life, we often find that joy and suffering lie close together. Thus, we remember the death of our honored friend, Professor Anton Sutor, at this time. Prof. Sutor would certainly have been very glad to join us and celebrate the fact that his successor Dr. Zieger has been awarded the J. L. Schoenlein Prize today.

I. Epidemiology Chairmen: W. Schramm (Munich) L. Gürtler (Greifswald)

HIV Infection and Causes of Death in Patients with Hemophilia in Germany (Year 2003/2004 Survey) W. Schramm, and H. Krebs on behalf of the participating German hemophilia centers

Introduction The annual survey »HIV Infection and Causes of Death in Patients with Hemophilia in Germany« already goes along with a fine tradition. Already in the late 1970s Professor Landbeck began to survey annually hemophiliacs living at that time in West Gemany for causes of death and the prevalence of diseases. This was carried on till today, so that our actual insights rest upon a broad database. However data quality could be much more improved in future.

Patients and Methods Questionnaires called »Todesursachenstatistik 2003/2004« were sent to all established hemophilia centers in Germany. Prompted was information about patients with hemophilia A, B and von Willebrand disease. In particular anonymous data concerning the last 12 months about number of treated patients, type and severity of illness, HIV-status and causes of death was inquired. This data was merged with existing data returning to 1982 and analyzed statistically. In the 2003/2004 survey, a total number of 8445 patients (including possible double registrations) have been reported from the participating centers.

Results Participating centers Since the first survey the number of participating centers has increased every year with a particularly rise in 1991 when the hemophilia treatment centers of the former East Germany joined in. Today these centers contribute a significant portion of the overall data (Fig. 1). In the last years’ surveys the number of reporting centers persisted relatively constant. In this year 70 centers contributed data (Table 1). Thereby the total number of patients (including patients with von Willebrand disease) reported from all centers added up to 8445 patients compared to 8070 patients in the last years’ survey (Table 2).

I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer-Verlag Berlin Heidelberg 2006

4

W. Schramm, H. Krebs

Fig. 1. Numbers of participating hemophilia centers and regions Table 1. Numbers of participating hemophilia centers 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 East West

47 18

62 18

79 24

Totals

65

80

103

111

119

119

71

75

93

87

72

75

71

70

Patients The distribution of patients with hemophilia A (45,9%), B (8,1%) and patients with von Willebrand disease (46,0%) is given in Table 2. Compared to the data of the previous surveys these are relative consistent findings. When severity of disease is analyzed with a cut-off of 2% factor activity, the distribution between the two subgroups, i.e. below 2% and above 2%, is similar in patients with hemophilia A (51,7/48,3%) and B (46,4/53,6%) as shown in Table 2. 22,6% of patients with von Willebrand disease showed Ristocetin co-factor levels below 30% as demonstrated in Table 2.

Inhibitors In 3,5% (134) of the patients with hemophilia A and in 2,4% (16) of the patients with hemophilia B an inhibitor was found (see Table 2). These findings correspond to international large-scale prevalence studies and registry data indicating that the prevalence of inhibitors in the hemophiliac A population overall is up to 5% [6].

HIV Infection and Causes of Death in Patients with Hemophilia in Germany

5

Table 2. Cumulative data from 71 centers as of 2003/2004 Hemophilia B

N

%*

N

%

Total

3877

45,9 (–)

685

8,1 (–)

3883

46,0 (–)

8445

Factor activity ≤ 2%

2005

318

–

2323

1872

3,8 (46,4) 4,3 (53,6)

–

Factor activity > 2%

23,7 (51,7) 22,2 (48,3)

–

–

2239

Ristocetin Co-factor ≤ 30%

–

–

–

–

879

Ristocetin Co-factor > 30%

–

–

–

–

2954

10,4 (22,6) 35,0 (76,1)

Inhibitor (low responders)

35

8

–

43

99

0,1 (1,2) 0,1 (1,2)

–

Inhibitor (high responders)

0,4 (0,9) 1,2 (2,6)

–

–

107

HIV negative

3325

3875

HIV positive

552

7,1 (88,0) 1,0 (12,0)

45,9 (99,8) 0,1 (0,2)

39,4 (85,8) 6,5 (14,2)

367

8 603 82

von Willebrand disease N %

S

Hemophilia A

8

N

879 2954

7803 642

* percentage of all patients; in brackets: percentage of row

HIV-Status Of all reported patients a total of 642 (45,2%) were infected with HIV. Analyzed for HIV distribution in subgroups 14,2% of all patients with hemophilia A, 12,0% of all patients with hemophilia B, and 0,2% of all patients with von Willebrand disease were HIV-infected (Table 3).A total of 33 patients (5,1% of all HIV positive patients) has reached the stage of full-blown AIDS, compared to 290 patients (45,2% of all HIV positive patients) that have up to now not shown severe symptoms of the immune disease (Table 3). 227 HIV positive patients with no further details concerning stadium were reported.

Mortality from all cases In the 2003/2004 period a total of 22 patients (10 HIV–, 12 HIV+) were reported dead with the distribution of causes of death given in Table 4. Since the beginning of the survey in 1982 a total of nearly 800 patients have been reported dead (Fig. 6). The development of mortality and causes of death since 82/83 are depicted in Figures 4 and 5. In this years’ survey liver disease (22,7%) again has been one of the main causes of death while AIDS (4,5%) loses ground anymore (see Fig. 5a, b). Up to

6

W. Schramm, H. Krebs

Table 3. HIV-status von Willebrand disease N %

S

Hemophilia A

Hemophilia B

N

%*

N

%

HIV positive, no AIDS

243

37,8 (44,0)

42

6,5 (51,2)

5

0,8 ()

290 45,2

HIV positive, CD4<200 cell/µl

72

11,2 (13,0)

17

2,6 (20,7)

3

0,5 (37,5)

92

14,3

HIV positive, full blown AIDS

29

4,5 (5,3)

4

0,6 (4,9)

0

0,0 (0,0)

33

5,1

HIV positive, no comment

208

32,4 (37,7)

19

3,0 (23,2)

0

0,0 (0,0)

227 35,4

S

552

86,0

82

12,8

8

1,2

642 100

N

%

* percentage of all patients; in brackets: percentage of row

Table 4. Distribution of death causes Cause of death

HIV– N

%*

HIV+ no AIDS n %

HIV+ AIDS CD4+ < 200 n % n %

Died of AIDS

—

—

0

0,0 (0,0)

0

0,0 (0,0)

1

Died of liver disease

0

0 (0,0)

4

18,2 (44,5)

0

0,0 (0,0)

Died of bleeding

3

13,6 (30,0)

0

0,0 (0,0)

0

Died of cancer

1

4,5 (10,0)

1

11,1 (4,5)

Died of other diseases 3

13,6 (30,0)

4

Died of accident, 1 suicide, drugs, murder

4,5 (10,0)

Died, no comment

2

S

10

S n

%

4,5 (50,0)

1

4,5

1

4,5 (50,0)

5

22,7

0,0 (0,0)

0

0,0 (0,0)

3

13,6

0

0,0 (0,0)

0

0,0 (0,0)

2

9,1

18,1 (44,4)

0

0,0 (0,0)

0

0,0 (0,0)

7

31,8

0

0,0 (0,0)

0

0,0 (0,0)

0

0,0 (0,0)

1

4,5

9,1 (20,0)

0

0,0 (0,0)

1

4,5 (100)

0

0,0 (0,0)

3

13,6

45,5

9

41,0

1

4,5

2

9,1

22

100

* percentage of all patients; in brackets: percentage of row

total number of deaths per year (cancer)

Liver disease

c)

Cancer

e) total number of deaths per year (HIV)

a)

total number of deaths per year (liver disease)

total number of deaths per year (HIV)

HIV

total number of deaths per year (cancer)

total number of deaths per year (liver disease)

HIV Infection and Causes of Death in Patients with Hemophilia in Germany

p < 0,001

b)

p < 0,025

d)

p < 0,001

f)

Fig. 2a–f. Comparison total number of deaths of HIV, liver disease and cancer

7

8

W. Schramm, H. Krebs

% of total number of deaths per year

% of total number of deaths per year

1995 the number of AIDS-related deaths increased continuously with decline taking place since. As at present more than half of the primary HIV-infected patients with hemophilia are still alive (642), the main reason for this development can probably be attributed to improved antiretroviral therapies as described by many authors [1–3]. In contrast liver disease (22,7%) and cancer (9,1%) as a cause of death remained relatively constant (see Fig. 5) still staying on an high level. 3 patients (13,6%) died of a bleeding. Overall annual mortality in patients with bleeding disorders in the 2003/2004 survey adds up to 0,26% per year. 7 patients (31,8%) died of other diseases not distinguished in this survey. No indications for Creutzfeldt-Jakob disease in our patient collective has been reported since 1978. Once again mentionable is the low portion of only 2 reported deaths with no comment (13,6%), improving data quality clearly. Arranging data for greater periods of time one can see these changes obviously (see Fig. 2b, d, f). Clustered data for the years 1982 to 1994 and 1994 to 2004 gives us a statistical significant difference between these periods concerning all important causes of death as HIV (p < 0,001), liver disease (p < 0,025) and cancer (p < 0,001).

a)

c)

HIV

b)

Liver disease

Cancer

Fig. 3a–c. Comparison % of total number of deaths of HIV, liver disease and cancer

Total number of deaths per year

HIV Infection and Causes of Death in Patients with Hemophilia in Germany

9

other diseases bleeding HIV liver disease cancer no comment accident, suicide, drugs, murder

Fig. 4. Causes of death since the beginning of the survey

The same numerical picture shows the HIV/liver disease/cancer deaths expressed as percentage of all deaths per year (see Fig. 3a–c).

Mortality from liver disease Therewith in this year’s survey the increase of liver disease as a causes of death has reached statistical significance the second time, suggesting a further increase in future (see Fig. 2d). The obvious reason for this probably can be attributed to the increasing number of deaths induced by liver cirrhosis and hepatocellular carcinoma due to chronic HCV [4]. As we did not discriminate type of cancer in our surveys up to now there might be a relevant portion of patients in this group having died of primary hepatocellular carcinoma induced by chronic HCV intensifying the impact of liver disease on causes of death in patients with hemophilia even more.

Conclusion Comparing actual data to those of the previous surveys we got consistent findings indicating good data quality. In addition our data is comparable to that of international large-scale prevalence studies and registry data. Despite mortality from HIV

10

W. Schramm, H. Krebs

total number of deaths per year

a)

% of total number of deaths per year

b)

other diseases bleeding HIV liver disease cancer no comment accident, suicide, drugs, murder

other diseases bleeding HIV liver disease cancer no comment accident, suicide, drugs, murder

Fig. 5. Chart of deceased patients per year, separated for causes of death

Total number of deaths per year (cummulated)

HIV Infection and Causes of Death in Patients with Hemophilia in Germany

11

other diseases bleeding HIV liver disease cancer no comment accident, suicide, drugs, murder

Fig. 6. Cumulative chart of deceased patients, separated for causes of death

in patients with hemophilia is keeping on decreasing, HIV still remains an important factor as an HIV/HCV coinfection seems to increase risk of progression of liver disease to cirrhosis and hepatocellular carcinoma [5–7]. A relevant portion of patients reported dead of cancer might have died of primary hepatocellular carcinoma induced by chronic HCV. This hypothesis has to be proved in future surveys by discriminating type of cancer. Moreover we will have to investigate the cohort of chronic HCV-infected patients in order to be able to calculate cumulative risks of death from liver disease in patients with hemophilia infected between 1969 and 1985 with HCV-contaminated blood products.

References 1. Chorba TL, Holman RC, Clarke MJ, Evatt BL: Effects of HIV infection on age and cause of death for persons with hemophilia A in the United States. Am J Hematol. 2001 Apr;66(4):229–40 2. Darby SC, Kan SW, Spooner RJ, Giangrande PL, Lee CA, Makris M, Sabin CA, Watson HG, Wilde JT, Winter M; UK Haemophilia Centre Doctors’ Organisation: The impact of HIV on mortality rates in the complete UK haemophilia population. AIDS. 2004 Feb 20;18(3): 525–33 3. Quintana M, del Amo J, Barrasa A, Perez-Hoyos S, Ferreros I, Hernandez F, Villar A, Jimenez V, Bolumar F: Progression of HIV infection and mortality by hepatitis C infection in patients with haemophilia over 20 years. Haemophilia. 2003 Sep;9(5):605–12

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4. Ragni MV, Belle SH: Impact of human immunodeficiency virus infection on progression to end-stage liver disease in individuals with hemophilia and hepatitis C virus infection. J Infect Dis. 2001 Apr 1;183(7):1112–5. Epub 2001 Mar 01 5. Santagostino E, Colombo M, Rivi M, Rumi MG, Rocino A, Linari S, Mannucci PM; Study Group of the Association of Italian Hemophilia Centers: A 6-month versus a 12-month surveillance for hepatocellular carcinoma in 559 hemophiliacs infected with the hepatitis C virus. Blood. 2003 Jul 1;102(1):78–82. Epub 2003 Mar 20 6. Wight J, Paisley S: The epidemiology of inhibitors in haemophilia A: a systematic review. Haemophilia. 2003 Jul;9(4):418–35 7. Yee TT, Griffioen A, Sabin CA, Dusheiko G, Lee CA: The natural history of HCV in a cohort of haemophilic patients infected between 1961 and 1985. Gut. 2000 Dec;47(6):845–51

Acknowledgment Anstadt, M. Auerswald, G. Aumann, V. Balleisen, L. Beck, C. Berthold, B. Beutel, G. Böhmann, J. Brackmann, H.-H. Bruhn, H. D. Clemens, P. von Depka Prondzinski, M. Dockter, G. Döhner, K. Eberl, W. Edelmann, H. Effenberger, W. Eifrig, B. Erler, T. Franke, D. Freund, W. Geib, R. Gerhardt, A. Gildberg, P. Gnad, M.

Göbel, U. Gortner, L. Greinacher, A. Griesshammer, M. Großmann, R. Güldenring, H. Günther, G. Haberland, H. Hähling, D. Henze, G. Hesse, V. Hofmann, K. Holfeld, E. Holzhüter, H. Horneff, S. Horst, H. A. Hossfeld, K. Kabus, M. Kemkes-Matthes, B. Kentouche, K. Kiesewetter, H. Kirchmaier, M. Klamroth, R. Klare, M. Klarmann, D. Klinge, J.

Knöfler, R. Koscielny, J. Krebs, H. Kreibich, U. Kretschmer, V. Kreuz, W. Kurnik, K. Kyank, U. Laws, H. J. Lenk, H. Loreth, R. Maak, B. Mayer, B. Mittler, U. Möller, J. Niekrens, C. Nimtz, A. Notheis, G. Pasold, R. Pindur, G. Pralle, H. Reiter, W. W. Richter, P. Ries, M. Scharf, E. Scharrer, I.

Schmeltzer, B. Schneppenheim, R. Schobeß, R. Schramm, W. Schubert, C. Schulte-Overberg, U. Schulz, A. Schumacher, R. Seyfert, T. Steiner, B. Stuckert, T. Suttorp, M. Syrbe, G. Walter, U. Wedemeyer, U. Weigel, S. Weinspach, S. Weippert, M. Weisser, J. Wendisch, E. Winkelmann, T Winterstein, E. M. Zieger, B. Zimmermann, R. Zintl, F.

Hemophilia Registry of the Medical Committee of the Swiss Hemophilia Association – Update and Annual Survey 2004 S. Hartmann, and K. Locher

Database transformation to online version of internet communication The software of the registry was transformed to be retrievable from an extranet system. At the same time the registration of the hemophiliac patients has been changed: the former register-domain with anonymous coded names (BGA code) was generally opened to full name personal data (former confidential safe-domain), provided the informed consent of the patients could be obtained. Only about 6% denied permission; these patients will remain in the registry with anonymous data. With the transformation of the software administration has become much easier to handle. The new authorized version is fully compatible with the Swiss law of data protection. Only Swiss specialists of hemophilia will have access to the registry, if they are members of the medical committee and are selectively authorized by the administration board. In addition to the patients’ data the registry provides the address of each treatment center (actually 18) and the name of its responsible medical chief. The rights of the individual users vary according to their status and affiliation to the treatment center, the individual users have different rights. The head of the center (and its authorized collaborators) may change the database of the individual patients of their own treatment center (new registration, mutation, changes of parameters etc.), however, they will only have insight into the data of patients of other treatment centers without authorization for changes. Crucial data of the master file, i.e. name and surname and date of birth, are blocked for regular users, for security reasons these data can only be modified by selected administrators. Security planning was given first priority. The access to the registry is shielded by multiple coding and multi-stage passwords. All the logins are registered, every mutation in the file is completely documented. Thus it is possible to reconstruct the history of data at any time and as far back as is desired. A daily backup of the database is provided automatically. The hemophilia registry has a modular conception. It is already planned to extend the software with a module for factor substitutes which will include different parameters of application. We are planning as well to further differentiate the statistics of deaths. The date of the last clinical control has recently been included to get a better survey of the medical care of the patients. The new online-registry has shown to be a handy tool, expeditious and convenient for the user, a fact which will make the hemophiliac patient the main beneficiary. I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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S. Hartmann, K. Locher

Registration The inclusion criteria were slightly modified; the definition is given as follows: 1. Factor VIII/IX level < 40% (before <30%). 2. Clinically relevant von-Willebrand disease, Ristocetin-Cofactor < 10% 3. All other forms of severe plasmatic coagulopathies (factor deficiencies) The online concept makes it possible to retrieve data for statistical evaluation whenever desired. The latest records of 26th October 2004 are presented as follows:

Patients A total number of 743 patients are included in the registry. 46 patients are not affiliated to a treatment center at the moment. As the data cannot be actualized they are excluded from evaluation. 697 patients (674 in year 2002) remain for statistical analysis. Double registration is not a problem any more as the patients may choose to register at two different treatment centers simultaneously. About 75 patients were newly registered; on the other hand approximately 50 cases had to be definitely excluded from the registry. Registration by full personal data revealed a remarkable number of various mistakes; many inconsistencies might to be hidden behind the BGA-code. The age distribution is presented according to the classification of WFH: children (0–4 y.) 25, youths (5–14) 86, adults (15–24) 90, (25–34) 81, (35–44) 105, (45–54) 75, (55–64) 57, and seniors (>65) 46. The mean age for hemophilia A is 37, for hemophilia B 34, and for von-Willebrand disease 38 years.

Classification of hemophilia The distribution of hemophilia type is given in Figure 1: hemophilia A 464 (67%), hemophilia B 103 (15%), other coagulopathies 44 (6%), including deficiencies for factor VII 17, factor XIII 12 and afibrinogenemia 10. Von Willebrand disease 86 (12%), differentiated into type I 59%, type II 35%, type III 6% The severity of disease is classified following ISTH standard. The grading is defined as severe FVIII/IX < 1%, moderate 1–5% and mild 6–40%. The corresponding results are shown in Figure 2: severe type A 175 (38%), type B 30 (29%), moderate type A 94 (20%), type B 39 (38%) and mild type A 186 (40%), type B 32 (31%). In 10 patients the results are missing, we know that these are mainly mild cases that do not request regular medical control. Additionally we register the mode of substitution, the type of therapy shown in Figure 3. It is evident by far, that in Switzerland treatment on demand exceeds the prophylactic treatment. More than 2/3 (71%) of the patients are substituting on demand, type A 330, type B 73. For a total of 63 (11%) patients no data are available. The differentiation of factor substitutes (Fig. 4) shows a relevant shift in favor of the recombinant factor VIII products, 262 (56%) patients as compared to 48% in year

Hemophilia Registry of the Medical Committee of the Swiss Hemophilia Association

86

44

103 Hemo A Hemo B Other coagulopathies

464

von Willebrand

Fig. 1. Distribution of hemophilia type and other coagulopathies 200 186 180

severe 1% severe< <1%

175

moderate moderate1–5% 1-5% light light6–40% 6-40%

160

unknown unknown 140

120

100

94

80

60

39 40 30

32

20 9 2 0

Hemophilia A (total 464) Hemophilia B (total 103) Fig. 2. Severity grading of hemophilia A and B following ISTH standards

15

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S. Hartmann, K. Locher

350

330

Hemophilia A Hemophilia B

300 250 200 150 100

84

73 56

50

11

5

1

7

0 on demand

prophylaxis

no therapy

unknown

Fig. 3. Mode of substitution, on demand versus prophylaxis in hemophilia A and B

2002. For type B the use of BeneFIX did not increase; there remain only 7 (7%) patients. 17 hemophilia A and 3 hemophilia B are reported to need no substitutes. In 47 resp. 6 patients no data could be obtained. The product NovoSeven is generally available but is only rarely applied in selected patients with inhibitors. From the registry we can retrieve a list of all the products (5 manufacturers) chosen by the patients, but the numbers do not correlate with the amount of substitutes clinically applied. 100% 90% 80% 70%

DDAVP NovoSeven recombinant plasmatic nihil

60% 50% 40% 30% 20% 10% 0%

Hemophilia A

Hemophilia B

Fig. 4. Range of substitutes, recombinant versus plasma-derived factor VIII/IX

Hemophilia Registry of the Medical Committee of the Swiss Hemophilia Association

17

Hemophilia B

Hemophilia A

1%

4% 20%

30%

actually negative actually unknown actually positive 69% 76%

low responder 17 responder2 high low responder

low responder 1 low responder high responder1 1

Fig. 5. Inhibitor status of hemophilia A and B

There is a new separate recording for the total use of factor concentrate per year. However, the data are not yet complete, since this registration was started in April 2004 only. The date of the last clinical control was included in the registry as additional parameter, there again the analysis will not yield reasonable results before the next year. The inhibitor status is registered in different steps. The results are shown in Figure 5: the quota is very low, inhibitor actually positive for hemophilia A 19 (4%), for hemophilia B 2 (1%). The results are not representative for the cohort, as there are still 20% respectively 30% patients with no corresponding data. Indeed we have some doubts regarding the results for low responders (max. < 5 BU) in 18 of 21 inhibitor patients. Eight patients with immune tolerance therapy are reported, in 7 cases the inhibitor is actually negative.

Mortality statistics Registration of numbers and causes of death is still a very small and incomplete file. In the period 1996–2003 a total number of 37 patients have been reported dead (compared to 24 up to year 2002). The increase is mainly due to improved registration technique. The cumulative causes of death are shown in Figure 6. The small numbers in each category hardly admit interpretation, all the less with 7 deaths of unknown origin. Some tendencies seem to become apparent: 1. HIV associated deaths are not dominant (we have to recall that registration started in 1996), 2. the most imminent causes of death are bleedings (7 patients) – of all localizations and independent of the inhibitor status – and chronic liver disease (7 patients), including 3 liver cirrhosis and 3 liver cancer, the latter increasing over the last 4 years. The mean age at death for all patients was 58.1, for HIV positive patients 41.7 years.

18

S. Hartmann, K. Locher

40 35 30 Unknown Others

25

Cardiovascular disease

20

Malignant Neoplasia

Liver disease 15

Bleeding HIV-associated

10 5 0 1996 1997 1998 1999

2000 2001 2002 2003

Fig. 6. Cumulative causes of death registered in the period 1996–2003

By extending the modular concept further differentiation in death registration would be achieved.

Conclusion We think that the online version of the Swiss Hemophilia Registry can be considered a good step on the road to progress. Registration of a remarkable number of additional hemophiliacs has now become possible. Verification of the files by personal data registration helped to identify double settings, and in particular to eliminate definite outright errors. Security of data is given priority in all fields. In future we would like our users to login frequently in the registry, thus providing the best update of hemophiliac data obtained in their daily consultation.

II. Risk of Infections and Inhibitors in Hemophilia Chairmen: W. Schramm (Munich) R. Zimmermann (Heidelberg)

Update on the Safety of Clotting Factors, Mainly Regarding the Risk of Transmission of vCJD (Variant Creutzfeldt Jakob Disease) L.G. Gürtler

Infectious agents may be divided according to their conventional classification starting with virus, bacteria, fungi, protozoa and worms. Of these agents only virus has been important for transmission by clotting product and due to donor selection, NAT (nucleic acid testing) and antibody screening, inactivation and removal, the transmission rate of transfusion relevant virus, such as HIV (human immunodeficiency virus), HTLV-I (human T leukemia virus type 1), HBV (hepatitis B virus) and HCV (hepatitis C virus) has been reduced to figures below 107. In 2004 newly arisen has the risk of transmission of prion by blood transfusion and consecutively by plasma components as well, since two cases of transmission of vCJD by blood transfusion had been reported in UK [13, 17]. There are two new viruses and the vCJD agent which are giving concern to consider the safety of the administration of plasma component once more.

Virus West Nile Fever Virus (WNV) When epidemiological data available from both Americas are taken spread of this virus has continued and WNV is present on the whole North-American continent where mosquitoes are found [9]. It seems so that human cases in the East, where the epidemic started in 1999, are declining. WNV has spread to Mexico and Cuba and will further be spread to the southern parts of Latin America. In USA and Canada screening for WNV by NAT and IgM antibody ELISA (enzyme linked immunosorbent assay) led to identification of infected donors and donations and elimination of these blood units. Since the beginning in 1999 there has been no transmission of WNV reported by inactivated plasma components, despite the transmission of WNV by blood and organ transplant. The explanation is quite simple since WNV behaves as a normal flavivirus and is easily inactivated by all procedures active against enveloped virus [20]. In Europe there are still very few cases of WNV infected people either residing or traveling in the delta-mouth of big rivers with warm climate or returning after several weeks of travel in high prevalence areas in USA. There has been no transmission of WNV by blood transfusion nor by plasma component in Europe, despite the reports of few cases of encephalitis and even deaths during endemics in I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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L. G. Gürtler

Southern Europe. The very low prevalence of WNV and its lacking transmission is most probably related to a certain degree of immunity in European birds and people by the exposure to WNV related flavivirus spread by birds of passage and mosquitoes since millennia. Future aspect will be that WNV is not an important infectious agent concerning blood transfusion and infusion of plasma components as long as the safety conditions employed presently are unimpaired. When immunity in both American continents in birds and people has established WNV will be of minor importance. There is a high chance to get a vaccine protective against the pathogenic action of WNV, as is established for other flavivirus as tick borne encephalitis virus (TBEV) and yellow fever virus (YFV) and Japanese encephalitis virus (JEV). From the virological point of view a virus can change very simple properties by recombination when two different viruses of one group infect one cell. Recombinational events within other flavivirus, such as Dengue fever virus or yellow fever virus have not been reported. But recombination within the genes for envelope proteins which could lead to changes in the envelope proteins of WNV and alter its cell tropism cannot be predicted.

Rabies virus In autumn 2004 the transplantation of contaminated organs in Germany infected 4 recipients from whom 3 died. The recipient of the liver transplant who survived was most probably vaccinated as child. The organs were taken from a young women who had returned a few months ago from India and who suffered from severe encephalopathy with untypical signs of rabies. Retrospectively it was reported that the women was scratched by a dog in India and had a very high viremia when the organs were taken. In the aspect of safety of clotting factors rabies virus will not be a problem. First argument in this case is donor selection: she had traveled in a malaria endemic area and should have been excluded from donation by the quarantine for travelers from malaria endemic areas which presently is 6 months. Second she was suffering from unknown encephalopathy – and prominent neurological signs are a reason for blood donor exclusion. Both, the travel and the clinical symptoms, would have been strong arguments for her referral as a blood donor and elimination of her donation. Secondly rabies virus is an enveloped virus [21] which is easily removed by the fractionation and purification process preparing plasma components and is inactivated using the obligatory inactivation procedures. A final aspect to this recent transmission, with always sad outcome for the recipient, for the safety of blood is that rabies prevalence in middle Europe is very low and regional recruitment of donors remains a huge and safe advantage. Finally whenever rabies should be a problem there is still a very safe and efficiently protecting vaccine available.

Update on the Safety of Clotting Factors, Mainly Regarding the risk of Transmission

23

Variant Creutzfeldt Jakob Disease (vCJD) agent The infectious nature of this spongiform encephalopathy is a prion (proteinaceous infectious agent) causing mad cow disease in cattle, which was transferred to humans around 1985 first in England followed by further European countries, especially France. Pathogenic action of the prion is seen predominantly on nerve cells, which since birth have no capacity to replicate. Prion attached to the cell surface is incorporated in the neuronal cell by a clathrin receptor mediated process and is cleaved by proteases in the proteasome [4].

Prpc (prion protein) Cellular Prpc has a three-dimensional structure that allows its intracellular cleavage by proteases linked to the endoplasmatic reticulum in the proteasome. Prpc has a predominant alpha helical structure of the N-terminal parts of the protein. Only a part of the function of Prpc is known: this is its involvement in protein synthesis close to the ribosome. Prpc knockout mice survive well, thus this function of prion cannot be indispensable.

Prpsc (scrapie prion protein) Scrapie is a disease of mainly sheep which was reported being present in European countries 300 years ago. Most probably the scrapie prion was transmitted from sheep to cattle, causing in cattle BSE (bovine spongiform encephalopathy) and in a further step from cattle to man, causing vCJD. Prpsc differs from the Prpc in the formation of beta-sheets especially in the N-terminal part of the protein, that convert the three-dimensional structure of Prpc in such a manner that it cannot be cleaved by cellular proteases. There are different strains of Prpsc causing disease [2] hinting to a long evolution of spongiform encephalopathy. When the misfolded Prpsc has entered a cell it will initiate a harmful process, by converting the majority of newly synthesized cellular Prpc from its alpha helical to the Prpsc beta sheet structure [4].

Pathogenic action In nerve cells Prpsc after entering through the cell surface will be permanently produced from Prpc, accumulates first in lysosomes, finally in plasma and leads to cell death [4]. The liberated Prpsc cannot be digested by proteases, thus it will stick to neighbouring nerve cells and finally lead to destruction of huge areas in the brain. Destruction of a whole set of nerve cells leads to impaired function and finally clinical symptoms. The time course of the manifestation of CJD is 20 to 30, perhaps up to 45 years, while the time course of manifestation of vCJD when the Prpsc is taken up by the oral route is 10–12 years and by the blood route is 5–6 years, as

24

L. G. Gürtler

far as this can be judged from the 2 cases known infected by blood transfusion [13, 17].

Manifestation of vCJD according to the human genotype Within the prion protein in position 129 there is either methionine (M) or valine (V). The information of the synthesis of this protein is drawn from the gene on chromosome 20 which might be homozygous for M or V or heterozygous for M/V. Until now only people with the M/M codon have been affected by vCJD [17].

Presence of PrPsc in blood

prion titer in mouse U/ml

Prpsc diagnostics is hampered by the abnormal configuration of this infectious protein. Since the protein only hypothesis of S Prusiner holds true [19], there is neither a special antigenic structure on this protein, nor is there any nucleic acid which might be detected by methods like the polymerase chain reaction. Since the structures between Prpc and Prpsc are similar there is no antibody formed in defense of the Prpsc of the immune system and there are no signs of inflammation. According to the mouse model established by P Brown [3] it might well be that initially when Prpsc is taken up from the gastrointestinal system there is a period of prion-aemia be it as a free molecule be it by the transport in macrophages and dentritic cells that finally lead to the deposition of Prpsc in lymphoid organs as tonsils, appendix, lymph node and/or spleen [10, 11, 1, 18]. A hypothetical model for man is given in Figure 1. When the mouse gets clinically symptomatic Prpsc content in blood increases 100-fold and there is no reason to assume that prion-aemia is absent in humans. Only half of the Prpsc is removed from blood by leukocyte depletion in hamster [8]. Cynomolgus macaques after oral and intracerebral uptake of Prpsc show an equivalent tissue distribution as mice [12].

120 110 100 90 80 70 60 50 40 30 20 10 0 0

2

4

6

year

8

10

12

Fig. 1. Putative prion-aemia in man adapted from Brown [3] from the mouse model. Since the way of transportation of prion after oral ingestion in an organism seems roughly clear a time period of 5 to 10 years might be assumed. Unclear is the titer of the prion in plasma and in leukocytes

Update on the Safety of Clotting Factors, Mainly Regarding the risk of Transmission

25

Elimination of PrPsc by the fractionation process Data published on the removal of Prpsc has been summarized by Foster [7]. Data published are not absolutely comparable since for spiking of plasma the infectious prion from mice, hamster, sheep and man had been used. As well as assay for the detection of remaining infectious prion western blot or an bioassay using mouse or hamster had been used. Nevertheless it has been shown that by cryoprecipitation 1 log 10 was removed (is equivalent with 90%) and by the further Cohn fractionation additionally between 1.3 to 5.3 log 10. In successive steps the main amount of infectious prion was removed by fraction step III with up to 6.2 log 10, and finally depth filtration of albumin and IgG removed between 2.8 and 4.9 log 10. Adsorption chromatography removed between 2.7 and 4.6 log 10.

Removal of prion by nanofiltration The ambiguities in results of data for the removal of Prpsc arise from the material used for spiking. Prpsc or vCJD prion cannot be grown in tissue culture nor be prepared from body fluids from laboratory animals, except from brain of infected and clinically symptomatic mouse, hamster or sheep. Initially homogenized whole brain was used, later microsomal fractions and finally highly purified prion protein concentrates [14]. In 2004 for the first time blood of hamster was used [8]. There are several reports, that infectivity of vCJD can be removed by use of filters with a pore size of 15 nm. Prion protein has a molecular weight of 19.000 to 30.000 related to the degree of glycosylation. As mentioned prion has the high tendency to aggregate and all high aggregates will be removed by filtration. The hitherto unresolved problem is what happens with monomers, with prion associated to other proteins as plasmin [6] and finally what is really the infectious dose administered intravenously which will lead to transport and attachment of the prion to brain cells. In conclusion it can be stated that a considerable part of the prion – between 1 to 3 log 10 – can be removed by nanofiltration for example for a FIX preparation. Due to the high molecular weight this process will fail with FVIII preparation.

Stability of Prpsc Some infectious agents can be easily inactivated as bacteria and HIV, some other need higher temperature or time or concentrations of disintegrating chemical substances to be inactivated as poliovirus and parovirus B19. Prpsc withstands all procedures developed for virus inactivation. Chemical inactivation with 1 N sodium hydroxide, sodium hypochloride or autoclavation at 134°C, which is effective [5] cannot be used for plasma or plasma components and thus inactivation of Prpsc is presently impossible for blood, blood product and plasma component.

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Risk evaluation for the transmission of Prpsc by blood in Germany in 2005 As long as plasma from German donations is used the risk of transmission of the vCJD agent is presently utmost low since no case of vCJD has occurred. The occurrence of the first case of vCJD in Germany is only a question of time, as has to be assumed looking at the cases reported from England [11], France, the Netherlands and Italy. That the putative risk of contamination is real has been demonstrated recently by the possible contamination of a Haemate HS preparation with one donation originating from France and a further FVIII preparation from Biotest. Looking at England and the two cases of transmission of vCJD by blood transfusion a very rough calculation can be made. Since 1984 when the BSE agent was introduced in the human population 20 years have elapsed. The annual number of blood transfusions drawn in England are 2 million (2 x 106), which are given to 106 recipients. 106 x 20 gives 2 x 107 and since 2 infected recipients have been found the risk of transmitting vCJD in England by blood transfusion is 107. The risk in Germany is 10 or more probably 100 times lower (108) or even lower. What is a calculable risk for a human being that is at the end of the scale of probability to occur but utmost rare. This risk is that somebody is hit on his head by a meterorite. There are 1010 people living on earth. When one of the 1010 is hit per year by meteorite the risk will be 1010, or when every 10 years one is hit by meteorite the risk will be 1011. This general risk of head vulneration is easily accepted by anyone. When the risk of transmitting vCJD by blood transfusion in Germany is 108 or lower, the risk of transmitting vCJD by clotting factor preparation is as cited above [7] even by a factor of 3 to 5 log 10 lower, that means it is lower than 1011 or 1013. And this is a risk which is easily acceptable or has without any further remarks being accepted.

Short term aspect for the prion disease Presently, as described above, the risk of transmitting the vCJD prion by blood in Germany is extremely low. The risk of acquiring vCJD by the oral route is even low but magnitudes higher compared to blood, since still in Germany every year by the cattle brain stem slaughterhouse screening more than 20 cows are found to be infected with the prion agent. Despite the ban and further measurements to stop the transmission in England and on the British island every year more than 1000 infected cattle are identified. The Prpsc is very stable also under environmental conditions, and time periods of 5 to 30 years have been assumed from the spread of scrapie in sheep on the island of Iceland by the oral route. The prion agent is as shown in mule deer in Colorado horizontally transmitted [15]. Thus for the next 30 years the risk of contamination with prion is existent for sheep and cattle by natural feeding and thus as well the transmission to man by brain and nerve material remains possible. The second way of transmission of the vCJD agent is from man to man. To block this route transfusion recipients are no longer allowed to donate blood in France,

Update on the Safety of Clotting Factors, Mainly Regarding the risk of Transmission

27

England [22], Canada and Switzerland. According to the risk evaluation it remains to be decided whether a similar step is appropriate in Germany as well today. Much more desirable is the establishment of a sensitive test for the detection of the prion agent which would solve, as with other infectious agents, the majority of the problems we face today.

References 1. Andreoletti AO, Simon S, Lacroux C, Morel N, Tabouret G, Chabert A, Lugan S, Corbiere F, Ferre P, Foucras G, Laude H, Echyenne F, Grassi J, Schelcher F. Prpsc accumulation in myocytes from sheep incubating natural scrapie. Nature Med 2004; 10: 591–593 2. Arima K, Nishida N, Sakaguchi S, Shigematsu K, Atarashi R, Yamaguchi N, Yoshikawa D, Yoon J, Watanabe K, Kobayashi N, Mouillet-Richard S, Lehmann S, Katamine S. Biological and biochemical characteristics of prion strains conserved in persistently infected cell cultures. J Virol 2005; 79: 7104–7112 3. Brown P, Cervenakova L, McShane LM, Barber P, Rubenstein R, Drohan WN. Further studies of blood infectivity in an experimental model of transmissible spongiform encephalopathy, with an explanation of why blood components do not transmit Creutzfeldt-Jakob disease in humans. Transfusion 1999; 39: 1169–1178 4. Collins SJ, Lawson VA, Masters CL. Transmissible spongiform encephalopathies. Lancet 2004; 363: 51–61 5. Fichet G, Comoy E, Duval C, Antloga K, Dehen C, Charbonnier A, McDonnel G, Brown P, Lasmezas CI, Deslys JP. Novel methods for disinfection of prion contaminated medical devices. Lancet 2004; 264: 521–526 6. Fischer MB, Roecki C, Parizek P, Schwarz HP, Aguzzi A. Binding of disease-associated prion protein to plasminogen. Nature 2000; 408: 479–483 7. Foster PR. Removal of TSE agents from blood products. Vox sang 2004; 87 Suppl 2: S7–S10 8. Gregori L, Mc Combie N, Palmer D, Birch P, Coker SOS, Giulivi A, Rohwer RG. Effectiveness of leukoreduction for removal of infectivity of transmissible spongiform encephalopathies from blood. Lancet 2004; 364: 529–531 9. Harrington T, Kuehnert MJ, Kamel H, Lanciotti RS, Hand S, Currier M, Chamberland ME, Petersen LR, Marfin AA. West Nile virus infection transmitted by blood transfusion. Transfusion 2003; 43: 1018–1022 10. Herzog C, Sales N, Etchegaray N, Charbonnier A, Freire S, Dormont D, Deslys JP, Lasmezas I. Tissue distribution of bovine spongiform encephalopathy agent in primates after intravenous or oral infection. Lancet 2004; 363: 422–428 11. Hilton DA, Ghani AC, Conyers L, Edwards P, McCardle L, Ritchie D, Penney M, Hegazy D, Ironside JW. Prevalence of lymphoreticular prion protein accumulation in UK tissue samples. J Pathol 2004; 203: 733–739 12. Lasmezas CI, Comoy E, Hawkins S, Herzog C, Mouthon F, Konold T, Auvré F, Correia E, Etchegaray NL, Sales N, Wells G, Brown P, Deslys JP. Risk of oral infection with bovine spongiform encephalopathy agent in primates. Lancet 2005; 365: 781–783 13. Llewelyn CA, Hewitt PE, Knight RSG, Amar K, Cousens S, Mackenzie J, Will RG. Possible transmission of Creutzfeldt-Jakob disease by blood transfusion. Lancet 2004; 363: 417–421 14. Marsh RF, Dees C, Castle BE, Wade WF, German TL. Purification of the scrapie agent by density gradient centrifugation. J Gen Virol 1984; 65: 415–421 15. Miller MW, Williams ES. Horizontal prion transmission in mule deer. Nature 2003; 425: 35–36 16. Mohan J, Bruce ME, Mabbott NA. Neuroinvasion by scrapie following inoculation via the skin is independent of migratory Langerhans cells. J Virol 2005; 79: 1888–1897 17. Peden AH, Head MW, Ritchie DL, Bell JE, Ironside JW. Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient. Lancet 2004; 364: 527–529

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18. Prinz M, Heikenwalder M, Junt T, Schwarz P, Glatzel M, Heppner FL, Fu YX, Lipp M, Aguzzi A. Positioning of follicular dentritic cells within the spleen controls prion neuroinvasion. Nature 2003; 425: 957–959 19. Prusiner SB, Scott MR, DeArmond SJ, Cohen FE. Prion protein biology. Cell 1998; 93: 337–348 20. Remington KM, Trejo SR, Buczynski G, Li H, Osherhoff WP, Brown JP, Renfrow H, Reynolds R, Pifat DY. Inactivation of West Nile virus, vaccinia virus and viral surrogates for relevant and emergent viral pathogens in plasma derived products. Vox sang 2004; 87: 10–18 21. Warrell MJ, Warrell DA. Rabies and other lyssavirus diseases. Lancet 2004; 363: 959–969 22. Wilson K, Ricketts MN. Transfusion transmission of vCJD: a crisis avoided ? Lancet 2004; 364: 477–479

nvCJD and Blood Products in the UK P. L. F. Giangrande

A risk assessment has recently been carried out by the department of health in the UK in order to determine the risk of transmission of vCJD through transfusion of blood and plasma-derived products. The principal conclusion was that »all patients with bleeding disorders … who have received clotting factor concentrates derived from UK-sourced plasma between 1998–2001 should be considered at risk of vCJD for public health purposes«. This has generated some alarm in the hemophilia community but also reflects an abrupt about-turn in opinion and policy at government level. The first case of BSE in cattle in the UK was identified in April 1985 [1]. The initial official response was to try and contain the epidemic in cattle by culling of affected herds. Legislation was rapidly introduced to ban the use of animal feeds for ruminants containing bone meal and to require removal of spinal and cerebral material from carcasses during processing. The official view was that such measures would suffice to render British beef safe for human consumption. However, the human equivalent of BSE was first diagnosed a decade later in 1996 [2]. Physicians in hemophilia centers in the UK were among the first to recognize the potential for transmission through blood products of a pathogen which presumably reached the nervous system via the bloodstream after ingestion in food. Recombinant factor VIII had already been launched in the UK in 1994 and the unanimous opinion of UKHCDO (United Kingdom Hemophilia Center Doctors’ Organization) that »recombinant factor VIII is the treatment of choice for all patients« was published in early 1997 [3]. Approaches for funding to permit such a switch were firmly rejected by the government at the time in view of the cost implications. In response to the initial refusal of funding for recombinant products, UKHCDO published a letter in the Lancet to express concern about the continued use of products derived from British plasma and concluded that »it is likely that any risk of transmission would be reduced by the use of concentrates prepared from plasma collected in other countries, such as the USA, where there are no recorded cases of nvCJD or BSE [4].« Shortly afterwards, the British Government took the decision to abandon fractionation of plasma derived from British volunteer donors and resort to importation of imported plasma derived from paid donors in the USA. This remains the current position and other precautions introduced in subsequent years include leukodepletion, issuing guidelines on optimal use of blood products in order to limit blood transfusion and the banning of recipients of transfused blood as blood donors. It was only in early 2004 that funding was eventually I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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P. L. F. Giangrande

granted which will permit the target of recombinant for all to be reached by April 2005. It is a fact that physicians in hemophilia centers were notified in 1997 and again in 2000 by the Department of Health in the UK that patients diagnosed with vCJD had been blood donors and contributed to pools used to manufacture a number of specified batches of coagulation factor concentrates. However, the official advice at the time was not to inform patients and no special precautions were advocated [5]. What has happened to change the government’s perception with regard to safety of plasma-derived products? It must be emphasized that not a single person with hemophilia has been diagnosed as having vCJD or is even currently suspected of having contracted the condition. Furthermore, a limited pathological study of the brains of deceased patients with hemophilia who had received large quantities of British plasma-derived plasma products showed no evidence of prion disease [9]. A national study involving histological examination of surgical and post mortem samples from patients with hemophilia in the UK has also been established and will continue to gather vital evidence. The number of subjects in the general population who have been diagnosed with vCJD in the UK remains relatively small at 151 (November 2004) and published annual data do not suggest any upward trend [10]. However, the potential for transmission of BSE/vCJD through blood transfusion has now been convincingly demonstrated. This was initially demonstrated in an animal model using susceptible sheep [6]. Two case reports of presumptive transmission of vCJD in humans were subsequently published in 2004 [7, 8], the second case report also being particularly important as this described the first case of vCJD in a subject heterozygous for methionine/valine at codon 129 in the prion gene and implying a wider susceptibility in the general population than previously reported. Of course, the fact that vCJD can be transmitted by a transfusion of whole blood does not automatically imply that fractionated plasma-derived products carry a similar risk. There are several other examples of pathogens which can be transmitted by whole blood but which are not transmitted by fractionated plasma-derived products (e.g. malaria, cytomegalovirus and HTLV-1). Individual physicians in hemophilia centers were informed by e-mail of the outcome of the risk assessment on September 9th. The details of specific batches to which a donor who subsequently developed vCJD were also circulated at the same time (16 batches of factor VIII, 8 batches of factor IX and a further 77 in which only the albumin added as an excipient was potentially contaminated). However, the risk assessment acknowledged that further batches of UK-sourced plasma will be identified in the future as more cases of vCJD are diagnosed and for this reason the decision was taken to consider all recipients of British plasma products at risk for public health purposes, and not just the recipients of the specified batches. Center staff had to identify all patients who had received pooled plasma products derived from UK donors in the period 1980–2001 and individuals identify recipients of the specific implicated batches. Templates of letters were provided for treatment centers send out to individuals registered with each center on September 20th, so that they would be received to coincide with a planned conference scheduled for September 21st. The patient notification exercise involved a considerable amount of work in a short timeframe and has clearly demonstrated the value of having a reliable database. In

nvCJD and Blood Products in the UK

31

the case of a large hemophilia center such as Oxford, 781 out of 1064 patients were identified as having received pooled plasma products of British origin during the relevant period. 114 of these had received batches known to be potentially contaminated and 234 had left the region since their treatment. In total, 527 patients were known to be still alive and were sent personalized letters. Furthermore, details of any surgical procedures and endoscopies undertaken in recipients of all British plasma-derived products (and not just the specific implicated batches) had to be provided to the local infection control officer. All patients were given the opportunity to know whether they had received batches of concentrate known to be potentially contaminated by virtue of the fact that a donor who contributed to the pool had subsequently developed vCJD. Patients were also notified that their »exposure will be recorded … in hospital and general practitioner notes and a national database.« It will be important to ensure that these patients do not become stigmatized, as happened with HIV in the 1980s, and that their treatment does not suffer. This could happen in the setting of surgery or other invasive procedures, where the current guidelines mandate disposal of instruments used in certain types of procedure involving exposure to lymphoid or neural tissue: this includes intraocular surgery and neurosurgery as well as rectal surgery, tonsillectomy, appendectomy and splenectomy [11]. This requirement may lead to subtle discrimination on the grounds of cost. Even though the official policy is that no special precautions are required for most procedures including dentistry and endoscopy, there are already anecdotal reports that people with hemophilia are being singled out. This can only serve to exacerbate the grievance felt by patients, particularly those exposed to batches of implicated product which were released in 1996 and 1997, that funding for recombinant products was not granted much earlier. However, the repercussions of this notification exercise will also be felt well outside the national borders of the UK. Some British patients treated with these products are now living abroad and a significant number of foreign patients have been treated with British plasma products whilst visiting the country in the two decades 1980–2001. Furthermore, plasma derived products (including albumin and immunoglobulin as well as factor VIII, IX and XI) have been exported to several countries during this period. However, no information whatsoever was provided in response to specific enquiries from the World Federation of Hemophilia (WFH) by the manufacturers themselves or government agencies. Some limited information of product usage around the world is held by the WFH, which now collects data on treatment around the world on an annual basis, and this proved helpful in identifying some of the countries which had used British plasma-derived products. Although patients and physicians in the UK had been provided with detailed information, many in other countries felt they had not been treated on an equal basis. There is also considerable potential for confusion around the world as regulatory authorities in other countries may well perceive the level of threat associated with the use of plasma-derived concentrates of British origin differently. Health Canada was the first agency to respond to the issue, focusing on imported factor XI and issued a statement on October 18th that »the grounds for action related to potential secondary transmission are theoretical. The Steering Committee of Infection Control

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P. L. F. Giangrande

Guidelines recommends that no extra infection control precautions need to be taken with surgical instruments used in surgeries on individuals who have received a transfusion of factor XI theoretically contaminated with vCJD at the rate described in the above Risk Assessment.« Of course, the United Kingdom has been the principal country affected by BSE but it is by no means the only affected country. BSE has been reported in a significant number of cattle in several other European countries and furthermore potentially contaminated food product (bone meal) for cattle was exported to many countries around the world from the UK at the height of the BSE epidemic. Contrary to the impression of many, BSE in cattle has not yet been completely eliminated although the number of affected animals is now very small. The threat of vCJD is therefore a real and persistent one which is not just confined to the UK. This is emphasized by the recent reports of the very first case of indigenous vCJD in the Republic of Ireland and the latest (eighth) case in France in a person who was a regular blood donor.

References 1. Ridley RM, Baker HF. Fatal protein: the story of CJD, BSE and other prion diseases. Oxford University Press (1998) [page 150: early history of BSE in UK] 2. Will RG, Ironside JW, Zeidler M, Cousens SN, Estibeiro K, Alperovitch A: A new variant of Creutzfeldt-Jakob disease in the UK. Lancet 347: 921–925 (1996) 3. Guidelines on therapeutic products to treat hemophilia and other hereditary coagulation disorders. UK Hemophilia centre Directors’ Organisation Executive Committee. Hemophilia 3: 63–77 (1997) 4. New-variant Creutzfeldt-Jakob disease and treatment of hemophilia. Executive Committee of the UKHCDO. United Kingdom Hemophilia Centre Directors’ Organisation.Lancet 350: 1704 (1997) 5. Giangrande PLF, Taylor S. Communicating risk: the example of vCJD. Proceedings of the World Federation of Hemophilia Global Forum on the Safety and Supply of Hemophilia Treatment Products (2002); 37–39 6. Hunter N, Foster J, Chong A, McCutcheon S, Parnham D, Eaton S, MacKenzie C, Houston F. Transmission of prion diseases by blood transfusion. Journal General Virology 83: 2897– 905 (2002) 7. Llewelyn CA, Hewitt PE, Knight RS, Amar K, Cousens S, Mackenzie J, Will RG. Possible transmission of variant Creutzfeldt-Jakob disease by blood transfusion. Lancet 363: 417–421 (2004) 8. Peden AH, Head MW, Ritchie DL, Bell JE, Ironside JW: Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient. Lancet 364: 527–529 (2004) 9. Lee CA, Ironside JW, Bell JE, Giangrande PLF, Ludlam CA, Esiri MM, McLaughlin JE: Retrospective neuropathological review of prion disease in UK hemophilic patients. Thrombosis and Hemostasis 80: 909–911 (1998) 10. Regular updates on cases of vCJD in the UK as well as copies of the latest annual report of the UK CJD Surveillance Unit are available at : www.cjd.ed.ac.uk 11. www.advisorybodies.doh.gov.uk/acdp/tseguidance/Index.htm

Mutation Type Dependent Inhibitor Risk – a Single Center Study on 432 Patients with Severe Hemophilia A J. Oldenburg, J. Schröder, R. Schwaab, C. Müller-Reible, E. Seifried, P. Hanfland, and H.-H. Brackmann

Alloantibodies (inhibitors) against factor VIII (FVIII) in Hemophilia A represent a major complication in patient care because they render classical substitution therapy ineffected. Inhibitors occur at a frequency of 20-30 %. Herein we report on 432 patients with severe hemophilia A, known mutation with the FVIII gene and known inhibitor status representing the largest cohort ever been reported from a single center (Bonn). The underlying mutation was correlated to the inhibitor prevalence as well as to the inhibitor titer. From the 432 patients, 205 (47,45 %) hemophiliacs showed the prevalent intron22-inversion, 8 (1.85 %) the intron-1-inversion, 61 (14.12 %) a nonsense mutation, 65 (15.05%) a small deletion/insertion, 61 (14.12%) a missense mutation, 17 (3.94%) a large deletion and 15 (3.47 %) a splice site mutation. 79 (18.29 %) of the 432 patients developed an inhibitor of which 46 (58 %) showed a high titer and 33 (42 %) a low titer. Inhibitor prevalences for the main mutation types were 35.29 % in large deletions, 25 % in intron-1-inversion, 21.46 % in intron-22-inversion, 21.31 % in nonsense mutations, 13.85 % in small deletions/insertions, 13.33 % in splice site mutations and 4.92 % in missense mutations. However, for mutations subtypes inhibitor prevalences could be as high as 100 % (3 out of 3) in multi domain large deletions and as low as 0 % (0 our of 16) in small deletions/insertion affecting an adenine series. Although for some mutation types the numbers are still small the results corresponding well to those reported earlier on merged patient samples and underline the significant influence of the mutation type to the risk of inhibitor formation.

I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

Inhibitor Incidence in Previously Untreated Patients (PUPs) with Hemophilia A and B. A Prospective Multi-Center Study of the Pediatric Committee of the German, Swiss and Austrian Society for Thrombosis and Hemostasis Research (GTH) W. Kreuz, G. Auerswald, U. Budde, H.J. Klose, H. Lenk and the GTH-PUP-Study-Group

One of the most serious complications in the treatment of the current generation of hemophiliacs is the development of inhibitors occurring in about 30% of patients with severe hemophilia A and to a lesser extent in those with moderate and mild disease [4]. The development of inhibitors in hemophilia B is rare (0–9%) and affects predominantly severe hemophiliacs with major factor (F) IX gene defects. In order to observe inhibitor development in previously untreated patients (PUPs) with hemophilia A and B a prospective multi-center study was started in 1993 by the Pediatric Committee of the GTH (German, Swiss and Austrian Society for Thrombosis and Hemostasis Research). PUPs with hemophilia A and B are recruited prospectively all over Germany, Austria and Switzerland (32 hemophilia centers) since 1993 (observation period: 11 years/update June 2004) in this ongoing study (recruitment period 15 years). The patients are restricted to one single plasma derived (pd) or recombinant (r) concentrate available on the market throughout the whole study period. The study protocol provides rigorous inhibitor testing: – prior to the first exposure – every 3rd to 4th exposure day (ED) for the first 20 EDs – until the 200th ED every 10th ED – after the 200th ED every 3 months – additional testing in any situation if inhibitor development is suspected – rarely exposed patients should be controlled every 3 months Inhibitor assays are performed at a central lab (Professor Budde, Laborgemeinschaft Keeser, Arndt und Partner; Lademannbogen 61-63; 22339 Hamburg) by modified Bethesda method out of 1.3–3 ml citrated blood. Additional inhibitor testing is done against porcine and recombinant FVIII. The following parameters are investigated and correlated with the development of inhibitors: – type and severity of hemophilia – age at first exposure and at inhibitor development – number of EDs until inhibitor development – therapy regimen (prophylaxis, on-demand, continuous infusion) – type and dosage of factor concentrate administered I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

Inhibitor Incidence in Previously Untreated Patients with Hemophilia A and B

35

– analysis of mutation type and HLA-type – epitope mapping in inhibitor patients – neutralizing and non-neutralizing FVIII-antibodies Patients are classified as inhibitor patients if they exceed inhibitor titers of 0.6 Bethesda Units (BU) at least at two consecutive times. Low responder (> 0.6–5 BU), high responder (> 5 BU).

Patients Until June 2004, 324 PUPs (321 Caucasians, 3 of other ethnic origin) have been enrolled (278 hemophilia A, 46 hemophilia B). 213 out of those 324 have been exposed at least once to FVIII or to FIX concentrate respectively (Table 1). Age at first exposure was 0.8 years in median (range 0.1–24.9). During a median observation period of 4.6 years per patient (median; range 0.1-10.3) the patients had 47 EDs in median (range 1–> 1000). Table 1. Treated patients (n=213) – update June 2004 Hemophilia A

Hemophilia B

Severe (≤1%) Moderate (>1-5%) Mild (>5%)

102 52 28

15 12 3

Total

183

30

Type of concentrate Out of 30 hemophilia B patients 28 received pd FIX concentrate. 2 patients were treated with a rFIX concentrate. In the hemophilia A population 95 patients received rFVIII and 88 patients pd FVIII (Table 2). Table 2. Type of concentrate in patients with hemophilia A (n=183) rFVIII

pd FVIII

Severe (≤1%) Moderate (>1-5%) Mild (>5%)

47 30 18

57 20 11

Total

95

88

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W. Kreuz et al.

Results Inhibitor development in hemophilia B Out of 30 patients 2 developed an inhibitor (1 high titer > 5 Bethesda Units/BU, 1 low titer). The preliminary incidence for severe hemophilia B (FIX ≤ 1%) is 13% (2/15 patients).

Inhibitor development in hemophilia A Out of 183 patients 34 developed an inhibitor (15 high titer, 17 low titer [>0.6-5 BU], 2 transient inhibitors) at the age of 0.9 years in median after 12 EDs in median (156). Inhibitor incidence for severe hemophilia A patients was 28.4% and 7.7% for moderately affected ones (Table 3). Table 3. Frequency of inhibitor development in hemophilia A dependent on severity Inhibitor patients

Frequency

Severe (=1%) Moderate (>1-5%) Mild (>5%)

29/102 4/52 1/29

28.4% 7.7% 3.5%

Total

34/183

18.6%

The preliminary inhibitor incidence as well as the number of EDs until inhibitor development (12 EDs in median) confirm the results from former prospective PUP studies [4]. Further known risk factors, in particular the underlying gene mutation has also been corroborated by our data [1]. 70% of patients with Intron-22 inversion developed FVIII antibodies. Inhibitor development was rare in the low risk mutations (e.g. in patients with missense mutations). Up to now, inhibitor incidence in hemophilia A treated with pd FVIII is lower than in patients receiving rFVIII: The percentage of the inhibitor development for severely affected patients in the recombinant treated group was 36% and 21% in the plasma-derived group. However, the difference was not significant (p=0.08; Fisher´s exact test). More patients have to be included and followed up to evaluate further subgroups (e.g., full-size and B-domain-deleted rFVIII; the role of VWF etc). Recently a positive correlation between age at first exposure and inhibitor development in hemophilia A has been described [2, 3]. This aspect was not fully confirmed by the GTH-PUP-Study data. No correlation between age at first exposure and inhibitor development was found. However, patients firstly exposed at the age <0.5 years developed predominantly high titer inhibitors whereas the larger proportion of the older patients (>0.5 years at 1st ED) developed low titer inhibitors.

Inhibitor Incidence in Previously Untreated Patients with Hemophilia A and B

37

A correlation between the therapy regimen and the inhibitor development was found in the GTH-PUP-Study hemophilia A patient cohort: Patients receiving primary prophylaxis developed significantly less inhibitors (10%) than those receiving on-demand treatment (45%) (p=0.003; Fisher’s exact test). In order to validate the preliminary results and to evaluate further subgroups further patients have to be included in this still ongoing prospective PUP study. For study protocol and CRFs please contact: PD Dr. Wolfhart Kreuz/Ulrike Hauber Klinikum der Johann Wolfgang Goethe Universität ZKI, 31-1 Theodor-Stern-Kai 7 60596 Frankfurt Tel. 069/6301-83030

GTH-PUP-Study-Group Kreuz W, Auerswald G, Budde U, Lenk H, Klose HJ† (Steering committee) Aumann V, Becker S, v Beutel, Bergmann F, Bodmer N, Brackmann HH, Brockhaus W, Buschmann, vDepka M, Eberl W, Edelmann, Eckhof-Donovan, Effenberger W, Eifrig B, Eisert S, Funk M, Escuriola Ettingshausen C, Göbel U, Güldenring H, Gutsche S, Halimeh S, Hassenpflug W, Heller Ch, Hess, Herrmann FH, Holfeld E, Kentouche K, Kern A, Kirchmaier CM, Klarmann D, Klien U, Klinge J, Knöfler R, Kobelt R, Kolb R, Kosch, Kotitschke S, Kruck, Kurme A, Kurnik K, Kurnik P, Kyank U, Lenz E, Maak B, Martinez Saguer MI, Mauz-Körholz Ch, Mayr WR, Mößeler J, Müller S, Niekrens C, Nowak-Göttl U, Oldenburg J, Rister M, Röhrenbach, Rusicke E, Schimpf K, Schneppenheim R, Schulte Overberg-Schmidt U, Schumacher R, Schwaab R, Siemens HJ, Sigl-Kretzig M, Streif W, Sutor AH, Weippert, Weissbach G, Wendisch E, Wendisch J, Wermes C, Wieland, Zeitler P, Zieger B, Zimmermann R. References 1. Oldenburg J, Schröder J, Brackmann HH, Müller-Reible C, Schwaab R, Tuddenham E. Environmental and genetic factors influencing inhibitor development. Semin Hematol 2004; 41 (Suppl 1): 82 2. Lorenzo JI, Lopez A, Altisent C, Aznar JA. Incidence of factor VIII inhibitors in severe hemophilia: the importance of patient age. Br J Haematol 2001; 113(3): 600-3 3. Van der Bom JG, Mauser-Bunschoten EP, Fischer K, Van den Berg HM. Age at first treatment and immune tolerance to factor VIII in severe hemophilia. Thromb Haemostas 2003; 89: 475-79 4. Kreuz W, Ettingshausen CE, Zyschka A, Oldenburg J, Saguer IM, Ehrenforth S, Klingebiel T. Inhibitor development in previously untreated patients with hemophilia A: A prospective long-term follow-up comparing plasma-derived and recombinant products. Semin Thromb Hemost 2002; 28 (3): 285-90

ADVATE Inhibitor Risk Profile: 18 Months post-Licensure E. Gomperts

By intention, there are similarities between ADVATE (ADVATE Antihemophilic Factor (Recombinant), Plasma/Albumin-Free Method (r-AHF-PFM) and its predecessor Recombinate (Recombinate Antihemophilic Factor [Recombinant][rAHF]). Both are manufactured utilizing a CHO-cell clone, with the ADVATE master cell bank being derived from a protein/albumin free culture medium-adapted Recombinate cell line. Consequently, both cell lines include the human FVIII and human von Willebrand Factor genes. In addition, the development of ADVATE for licensure showed that both recombinant FVIII proteins are very similar in structure and function. Thus it was not a surprise that the clinical research carried out on ADVATE demonstrated an almost identical pharmacokinetic (PK) profile to that of Recombinate. The Recombinate-ADVATE PK study showed a Recombinate mean +/–SD adjusted recovery of 2.59 +/– 0.52 with that of ADVATE being 2.41 +/– 0.50. Also the half-life in hours for Recombinate was 11.24 +/– 2.53 versus 11.98 +/– 4.28 for ADVATE [1]. Given the importance of inhibitor development to the person with hemophilia, it is appropriate to ask the question as to whether there are differences in the inhibitor profile between the two products. Here the comparison between them are not as straight forward, because a clinical research study to answer this question was not carried out. Nevertheless there is data on inhibitor incidence that has been generated by clinical research study on both products in previously treated patients (PTPs). There is data on PUPS for Recombinate, but the ADVATE PUP study is still ongoing and it is too early to make any comparison. However, there is data generated by pharmacovigilance reporting post-licensure with both products that can provide some insight as to inhibitor prevalence in clinical practice across all age groups. The information provided in Table 1 shows the inhibitor incidence that has been generated by the clinical research projects on ADVATE through January 2005. The study subject number in the Pivotal, Pediatric and Japan PTP studies amounted to 176 study subjects, with one non-persistent low titer inhibitor being documented in a single study subject. In addition, no study subjects who underwent surgery using ADVATE for hemostasis and no study subject entered into the Pivotal study who continued to receive ADVATE until licensure developed an inhibitor. This compares favorably with the situation in Recombinate with one low titer transient inhibitor being identified in 69 previously treated children and adult research subjects. I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

ADVATE Inhibitor Risk Profile: 18 Months post-Licensure

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Table 1. Inhibitor incidence in clinical research studies on ADVATE and Recombinate Eligibility

Age (yr)

Number

Total Days of exposure (ED)

Inhibitor Reports

ADVATE Pivotal

> 150 ED

> 10

108

12,636

1 nonpersistent 2.0 BU

ADVATE Continued

Ex Pivotal

> 10

82

1528

0

ADVATE Pediatric

> 50 ED

< 6

53

3237

0

ADVATE Surgery

> 150 ED

> 5

42

839

0

ADVATE Japan

> 150 ED

> 10

15

829

0

Recombinate Phase II/III

›PTP‹

Children 23 Adults 46

69

16,615

Study

1 nonpersistent 0.8 BU

The pharmacovigilance experience for Recombinate has been assessed from January 1, 1993 to December 31, 2002. The information has been published [2]. In summary, a total of 89 adverse event (AE) inhibitor reports were received during this period. The inhibitor incidence rate was estimated by a mixed-effects Poisson regression analysis. This was expressed as the percentage of patients treated assuming that PTPs used 148,700 units and PUPs used 48,600 units per annum. This data was generated by the respective Recombinate clinical research studies. Over the 10 years of observation, the total inhibitor incidence rate was 0.317% (CI 0.148–0.677). The total high titer (> 5BU) incidence was 0.158% (CI 0.086–0.286). That of the PTPs was 0.123% (high titer 0.0554%) and on the PUPs 11.9% (CI 5.05–28.0%), with high titer incidence as 5.96% (CI 3.00–11.8%). The ages of the group separated into 50% < 2 years, 30% 2 to 6 years and 20% greater than 6 years of age. Almost 80% of the group were reported having severe hemophilia, with almost 10% having mild disease. Almost 80% had less than 50 exposure days to Recombinate, with 17 % with data indicating >150 exposure days. The inhibitor titer in the reported patients was > 5BU in 52.4% of reports and < 1BU in 15.5 %. ADVATE was first licensed in the USA in late July 2003. Over the course of the first 18-month post-launch period (through January 2005), over 402 million units of ADVATE were distributed worldwide, with a total of 16 inhibitor cases reported. Of these 16 cases, 14 were deemed at least possibly related, while 2 were judged unlikely related to ADVATE due to determination of inhibitor development prior to exposure to the product. Of the 14 inhibitor cases considered at least possibly rela-

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E. Gomperts

ted to ADVATE exposure, 11 involved previously untreated patients (PUPs) or patients with a history of less than 50 FVIII exposure days (ED) at time of inhibitor detection. One of the 14 cases involved a 13 year-old PTP with an estimated history of greater than 50 prior ED and a history of high-titer inhibitor development (182 BU) on previous FVIII therapy. In this case, the patient demonstrated symptoms of inhibitor re-development at the time of initial Advate exposure; an inhibitor with maximum titer of 0.6 Bethesda Unit (BU) was detected shortly thereafter with FVIII recovery levels since being reported as within expected range. For the two remaining ADVATE-related inhibitor cases, the extent of factor VIII exposure prior to ADVATE administration was unclear. The first case involved a 46year-old male with baseline factor VIII of 15% who underwent an unspecified surgery. An inhibitor (unknown titer) developed after 16 ED to the product and became undetectable within 2 months without immune tolerance therapy. The second case involved a 3-year-old boy with severe hemophilia A who received ADVATE following a surgical procedure for phimosis. An inhibitor titer of 0.9 BU was detected after the second ADVATE exposure day. The inhibitor was judged to be transitory with a maximum titer of 2.2 BU being documented. With 14 at least possibly related inhibitor cases reported for 402 million units distributed, the reporting rate of inhibitor development for ADVATE may be expressed as 0.035 reports per million units distributed. While this is not a head-to-head study, and no claims as to superiority can be made by a comparison using these numbers, information about the experience with Recombinate during its first 18 months of market availability may offer context. During the respective first 18month post-launch period, 143 million units of Recombinate were distributed. Over this time period 14 inhibitor cases were reported. Of the 14 inhibitor cases, 11 were judged at least possibly related to this agent. Thus, the reporting rate of at least possibly related inhibitor development for Recombinate was 0.077 reports per million units distributed, which is more than twice that currently reported for ADVATE. These numbers are summarized in Table 2. In summary, both the clinical research-generated information and the postapproval clinical experience of a newly licensed product is critical to better understand its safety profile. The adverse event data actively collected during the comprehensive clinical trials of ADVATE that permitted its licensure, as well as the passively collected pharmacovigilance data post-licensure, clearly indicates that the safety and efficacy profile of ADVATE poses no problematic issues. Table 2. Summary of the pharmacovigilance experience of both ADVATE and Recombinate during the first 18 months post licensure Units distributed (million units) during first 18 months post-licensure ADVATE Recombinate

402 143

Number of reported inhibitor events (at least possibly related) 14 11

Inhibitor rate per million units distributed

0.035 0.077

ADVATE Inhibitor Risk Profile: 18 Months post-Licensure

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References 1. Ewenstein BM, Collins P, Tarantino MD, Negrier C, Blanchette V, Shapiro AD, Baker D, Spotts G, Sensel M, Yi SE, Gomperts ED. Hemophilia Therapy Innovation: Development of an advanced category recombinant factor VIII by a plasma/albumin-free method. Seminars in Hematology, 41, Suppl 2: 1–18, 2004 2. Ewenstein B, Gomperts E, Pearson S, O’Banion, M: Inhibitor Development in Patients Receiving Recombinant Factor VIII (Recombinate rAHF/Bioclate): A Prospective Pharmacovigilance Study. Haemophilia, 10: 1–8, 2004

EACH-Registry: An European Registry for Acquired Hemophilia A. Huth-Kühne, P. Lages, R. Fischer, and R. Zimmermann

Acquired hemophilia (AH) is a rare bleeding disorder with an incidence of approximately 1.5 cases/million/year. It is caused by the development of autoantibodies against clotting factor VIII (FVIII). This autoimmune disorder is characterized by spontaneous bleeds in patients with no previous history of a bleeding diathesis. In about 50% of cases an underlying medical condition is found, including autoimmune diseases, solid tumors and lymphoproliferative malignancies. Due to the highly variable phenotypic presentation, the severity of the hemostatic defect varies between life-threatening bleeds, which may be fatal and mild bleeding episodes requiring no treatment with hemostatic agents in about 30% of patients. In contrast to alloantibodies, which may arise in patients with congenital hemophilia following replacement therapy, autoantibodies to FVIII display a non linear type II kinetic. Therefore we see no correlation between residual FVIII activity, inhibitor titer and bleeding tendency and therapeutic decisions concerning the management of acute bleeding have to be based mainly on the clinical presentation. Inhibitor elimination is usually attempted with steroids alone or in combination with cytotoxic agents. The literature on AH consists only of small, referral center case collections and retrospective surveys of referral centers. To collect and evaluate data on all patients with AH in Europe, a group of European experts has developed an online registry, the European Registry for Acquired Hemophilia, called EACH Registry. Members of the steering committee are: A. Huth-Kühne, P. Knöbel, F. Baudo, L. Nemes, P. Marco, P. Collins, L. Tengborn. The database started with patients treated for AH since 01.01.2003. To date 256 patients have been enrolled from all over Europe. The EACH registry contains the largest pool of data on this rare condition collected to date. The first interim analysis will be presented in August 2005 during the ISTH Congress in Sydney and the registry will remain open for patient enrollment at least through years 2006 and 2007. Every treating physician of patients with AH is most welcome to participate in this unique European registry and enter data including the diagnostic procedures, treatment regimens and response to treatment. Final data analysis will hopefully provide an improvement in the management of patients by patient tailored treatment algorhythms. I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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The EACH registry is owned by the members of the steering committee, who are specialists in the field of AH. Only the members of the steering committee will have access to the database. Each participant of the registry has only access to his own data. No third party has any access to the database. Data security is given by fully anonymized patient data and secure limited access to the database. Data analysis will be performed on a national and European level. The pan-European evaluation will be done by the EACH steering committee members and for Germany and Austria by the local steering committee members (A. Huth-Kühne, P. Knöbl). All participating centers will be mentioned in the publication. The registry is accessible in the internet by www.each-registry.org.

Elective Orthopedic Surgery for Hemophilia Patients with Inhibitors P. L. F. Giangrande

Only ten years have passed since the distinguished orthopedic surgeon Robert Duthie wrote that »elective surgery is absolutely contraindicated in the presence of significant levels of factor VIII antibodies [1].« This is clearly no longer the case and a recent paper reports the pooled experience on the outcome in 108 hemophilic patients with inhibitors who have undergone surgery in nine centers around the world in more recent years [2]. The average age of patients was 22.5 years (range 5–40 years), and the average follow-up time was 2 years (range 1–5 years). However, it must be pointed out that radiosynoviorthesis accounted for 88 (81%) of these cases: this is hardly major surgery, and typically involves a single injection of isotope into a joint. In fact, this series only includes six cases of elective knee arthroplasty and two cases of hip arthroplasty. In the synoviorthesis group (41 patients, 88 synoviortheses) the average age was 14.3 years (range 5–40 years) and the average follow-up was 6.5 years (range 1–10 years). Few centers, even large hemophilia centers, have accumulated experience of more than a handful of cases of patients with inhibitors undergoing surgery. This point was recognized when these data were presented at 8th congress of the Musculoskeletal Committee of the World Federation of Hemophilia (WFH) in Bonn in May 2003, when it was resolved to systematically collate case reports on a worldwide basis. It would also be desirable to establish consensus on protocols, particularly with regard to choice of products and dosage regimes. Recombinant activated factor VII (rVIIa) was used in 38 cases, including all the cases of arthroplasty. FEIBA was used in 50 cases (including 47 cases of radiosynoviorthesis). It would appear that rVIIa is becoming increasingly considered the agent of choice for cover of surgery in patients with hemophilia and inhibitors because of a perceived increased risk of thromboembolism with activated prothrombin complex concentrates such as FEIBA. However, data accumulated over several years indicate this risk, even in the setting of surgery, is actually very small and patients who develop this complication usually have additional risk factors [3, 4]. In this context, consensus on the need for thromboprophylaxis in patients with hemophilia undergoing surgery is also highly desirable [5]. The maximum dose of rVIIa used in this series was 90 mg/kg every 2 hours initially for 24–48 hours and significant postoperative bleeding was reported in three cases (15%), including 2/6 cases of total knee arthroplasty. It would be fair to say that nowadays the view is that higher doses of rVIIa should be used in the setting of surgery and a standard regimen of 150–200 mg/kg body weight every 2 hours for the first 48 hours has been advocated [6]. I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

Elective Orthopedic Surgery for Hemophilia Patients with Inhibitors

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Laboratory monitoring of treatment in patients with inhibitors is a particular problem. Methods being developed which could offer a solution include thromboelastography, thrombin generation and wave form analysis using automated coagulometers [7]. A thrombin generation assay has recently been specifically developed for monitoring FEIBA therapy but this is not one which is easy to establish in most clinical laboratories and more data on clinical efficacy in relation to thrombin generation are needed before we can judge the feasibility of using this assay as a therapeutic guide for dosing [8]. An assay for rVIIa has also been developed and is now available as a commercial kit (Staclot VIIa-rTF assay, Diagnostica Stago). This employs a truncated recombinant soluble thromboplastin that does not form complexes with factor VIII zymogen, thus ensuring measurement of rVIIa exclusively [9, 10]. However, it is also not widely used in clinical practice currently. There is no consensus on what plasma levels need to be achieved in the setting of surgery anyway. The recent demonstration of an increased clearance of rVIIa in children has implications for pediatric surgical cases [11]. No details of cost are given in the recent published series. From both personal experience as well as published case reports, it is clear that the final bill for the use of blood products to support major surgery in patients with hemophilia and inhibitors can be very high and is typically at least € 1,000,000. Limited clinical trial data do not suggest that significant savings can be made by giving rVIIa by continuous infusion [12]. However, the costs incurred by surgery may be significantly offset by savings in subsequent years through reduction in bleeding episodes in the affected joint and thus consumption of product [13]. It is particularly important that clinical outcome is assessed in such patients. Of the 20 major orthopedic procedures performed in this series, outcome was assessed as »good« in 16, »fair« in one and »poor« in three cases. Validated quality of life questionnaires have already been used to obtain data on patients with hemophilia and inhibitors and justify the high cost of treatment through demonstrating a satisfactory quality of life [14]. Data on quality of life should also be routinely collated as part of the routine assessment of outcome of surgery.

References 1. Duthie RB, Dodd CAF. Reconstructive surgery and joint replacement. In: The management of musculoskeletal problems in the haemophilias. Duthie RBD, Rizza CR, Giangrande PLF, Dodd CAF. Oxford University Press, 1994 (page 193) 2. Rodriguez-Merchan EC, Wiedel JD, Wallny T, Caviglia H, Hvid I, Berntorp E, Rivard GE, Goddard NJ, Querol F. Elective orthopedic surgery for hemophilia patients with inhibitors: new opportunities. Seminars in Hematology 41: 109–116 (2004) 3. Negrier C, Goudemand J, Sultan Y Bertrand M, Rothschild C, Lauroua P and the members of the French FEIBA study group. Multicenter retrospective study on the utilization of FEIBA in France in patients with factor VIII and IX inhibitors. Thromb Haemost 1997; 77: 1113–1139 4. Ehrlich HJ, Henzl MJ, Gomperts ED. Safety of factor VIII inhibitor bypass activity (FEIBA): 10-year safety compilation of thrombotic adverse events. Haemophilia 2002; 8: 83–90 5. Franchini M, Tagliaferri A, Rossetti G, Pattacini C, Pozzoli D, Lorenz C, Gandini G. Absence of thromboembolic complications in patients with hereditary bleeding disorders under-

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6. 7. 8. 9. 10. 11. 12.

13. 14.

P. L. F. Giangrande going major orthopaedic surgery without antithrombotic prophylaxis. Thromb Haemost 2004; 91: 1053–1055 Rodriguez-Merchan EC, Wiedel JD, Wallny T, Hvid I, Berntorp E, Rivard G-E, Goddard NJ, Querol F, Caviglia H. Elective orthopaedic surgery for inhibitor patients. Haemophilia 2003; 9: 625–631 Barrowcliffe TW. Monitoring haemophilia severity and treatment –new or old laboratory tests? 2004 Haemophilia (in press) Varadi K, Negrier C, Berntorp E, Astermark J, Bordet JC, Morfini M, Linari S, Schwarz HP, Turecek PL. Monitoring the bioavailability of FEIBA with a thrombin generation assay. J Thromb Haemost 2003; 1: 2374–2380 Morrissey JH, Macik BG, Neuenschwander PF, Comp PC. Quantification of activated FVII levels in plasma using a tissue factor mutant selectively deficient in promoting factor VII activation. Blood 1993; 81: 734–44 Cid AR, Lorenzo JI, Haya S, Montoro JM, Casana P, Aznar JA. A comparison of FVII:C and FVIIa assays for monitoring of recombinant factor FVIIa treatment. Haemophilia 2001; 7: 39–41 Villar A, Aronis S, Morfini M, Santagostino E, Aurswald G, Thomsen HF, Erhardtsen, Giangrande PLF. Pharmacokinetics of activated recombinant coagulation factor VII (NovoSeven) in children vs. adults with haemophilia A. Haemophilia 2004; 10: 1–8 Ludlam CA, Smith MP, Morfini M, Gringeri A, Santagostino E, Savidge GF.A prospective study of recombinant activated factor VII administered by continuous infusion to inhibitor patients undergoing elective major orthopaedic surgery: a pharmacokinetic and efficacy evaluation. British Journal of Haematology 2003;120:808–813 Lacey L. Economic impact of treating inhibitor patients. Pathophysiology of Haemostasis and Thrombosis 2002; 32 (Suppl. 1): 29–32 Gringeri A, Mantovani LG, Scalone L, Mannucci PM; COCIS Study Group. Cost of care and quality of life for patients with hemophilia complicated by inhibitors: the COCIS Study Group. Blood 2003 ;102: 2358–63

III. Chronic Synovitis and Long-term Results of Orthopedic Treatment Chairmen: I. Scharrer (Frankfurt/Main) A. Kurth (Frankfurt/Main)

Results after Total Knee and Hip Replacement in Patients with Hemophilia A – A Single Center Experience R. Klamroth, Ch. Heinrichs, S. Gottstein, and R. Koch

Introduction Severe bleeding disorders like hemophilia A are complicated by spontaneous joint bleeding. The joint cartilage is damaged by the free intra-articular blood. The hemophilic arthropathy results in severe secondary arthrosis and is characterized by cartilage destruction, deformities and fibrous contracture. There are different options to avoid hemophilic arthropathy. First choice is the prophylactic substitution of clotting factor concentrates to completely avoid any joint bleeds, second choice is to treat bleeds very early by self injection and home treatment. After the beginning of joint destruction there are various possibilities to treat synovitis and the possibility of total joint replacement [1, 2, 3].

Patients From 1996 to 2004 we performed in 14 patients (13 severe hemophilia A and 1 moderate hemophilia A) 18 total knee replacements and 6 total hip replacements. In all patients surgery was performed by the same orthopedic team. Perioperative hemostasis management was provided by the hemophilia treatment center. The mean age at the time of surgery was 44.4 years (range 38–54). No patient was HIV-infected, 6 patients had chronic hepatitis C. In 2 patients total knee and hip replacement was performed in one operation. The indication for joint replacement therapy was an anatomically and functionally altered knee or hip. In all patients the main symptom was pain. 11 patients had a flexion contracture in 17 knees and 2 patients showed increased bleeding frequency.

Surgery and replacement therapy All patients got a non-constrained bicondylar total knee arthroplasty (MillerGalante type or Nexgen/Zimmer) with cementation. The total hip arthroplasty was cement free. Surgery was done under routine single shot antibiotic prophylaxis. Replacement therapy with factor VIII concentrates was started at the day before surgery by intravenous bolus injection and was continued every 6–8 hours to I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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R. Klamroth et al.

provide factor VIII levels above 70%. Bolus injection was continued every 8–12 hours during hospital stay depending on factor VIII levels. Desired factor VIII levels were > 70% in the first week and > 50% in the second week after operation. Every patient received a thromboembolic prophylaxis with low molecular weight heparin. In the first week no non-steroidal analgetics were used. The patients stayed a mean time of 17 days (range 10–30) in hospital. The average coagulation requirement was 81,500 IU factor VIII (44,000 to 123,000 IU, bodyweight 65 to 95 kg) during the hospital stay. The patients used different plasma-derived and recombinant factor VIII concentrates. Replacement therapy was continued after discharge and the dose was adapted to intensity of rehabilitation measures.

Postoperative Outcome No infection, no inhibitor development or venous thromboembolism occurred. Two patients had a postoperative hemorrhage after knee replacement at the second postoperative day for surgical reasons requiring an open revision. Three patients required transfusion of two units packed red cells after total hip replacement. Two patients developed fibrosis of suprapatellar recessus with flexion deficit after total knee replacement which required an open arthrolysis.

Postoperative rehabilitation Mobilization and physiotherapy started at the first day after surgery and were continued up to three months. All patients went for at least three weeks in a special rehabilitation center, where intensive practice and training was performed.

Results after total knee replacement Results are based on the clinical and radiological follow-up. The mean observation time for total knee replacement was 4.6 years, range 0.5–8.6 years. All 18 joints were infection-, pain- and bleeding free until now. 17/18 patients had full extension and flexion of at least 90°. One patient had an extension deficit which existed already before operation. Despite of an intraoperatively good result it was not possible to achieve full extension with physiotherapy. All patients were content with the results. Walking distance remained decreased in 4 patients, due to impairment of other joints.

Results after Total Knee and Hip Replacement in Patients with Hemophilia A

51

Results after total hip replacement Results are based on the clinical and radiological follow-up. The mean observation time for total knee replacement was 4.5 years, range 0.5–9.5 years. All 6 joints were infection-, pain- and bleeding free until now. One patient had atrophy of the musculus glutaeus medius one year after operation with recurrent pain in the hip. All patients were content with the results. Walking distance remained decreased in 4 patients, due to impairment of other joints.

Conclusions Surgery for total knee and hip replacement in hemophilic patients requires a close interdisciplinary cooperation between orthopedic surgeons and hemophilia treaters. Total joint replacement therapy in severe hemophilic arthropathy is possible without major bleeding complications and results in better quality of life due to pain reduction and reduction of bleeding frequency. In comparison to patients without hemophilia the results after total joint replacement are equally successful until now.

References 1. Beeton K, Rodriguez-Merchan EC, Aloltree J. Total joint arthroplasty in hemophilia. Haemophila 2000 (6), 474–481 2. Cohen I, Heim M, Martinowitz U, Chechick A. Orthopaedic outcome of total knee replacement in haemophilia A. Haemophilia 2000 (6), 104–109 3. Löfqvist T, Sanzen L, Pertersson C, Nilsson IM. Total hip replacement in patients with hemophilia. Acta Orthop Scand 1996; 67, 321–324

Motion Analysis Epidemiology in Hemophilic Children A. Seuser, G. Schumpe, T. Wallny, and H.-H. Brackmann

Motion analysis was performed in 203 children with hemophilia in a prospective multicenter trial involving German hemophilia centers in Hamburg, Bremen, Hanover, Brunswick, Bonn, Frankfurt, Würzburg, Ulm, Berlin, Potsdam, Magdeburg, Halle, Erfurt, Erlangen and Munich. 406 knees were investigated in total. Full data sets are available for 172 children to date. The children were aged 3 to 18 and the average age was 9.75 years. Both knees were analyzed during treadmill walking and during knee bends. Motion analysis was conducted using an original ultrasound topometer. Two transmitters were attached above and below the knee joint for this purpose. Threedimensional spatial data were determined accurate to the millimeter and converted mathematically to angle, angular velocity, angular acceleration, and the roll-andglide pattern during knee bends. In addition, a hematology questionnaire, sport and activity questionnaire, and pain questionnaire were completed and an orthopedic examination was performed. The motion analysis data were rated according to a score which factored in regularity, rhythm and sinus curve pattern. Analysis was continued on the basis of these score data.

Correlations between epidemiology data and function score Gait pattern displayed a relative dependence on age, with a regression coefficient of 0.56. Knee bends display an even closer correlation with age, with a regression coefficient of 0.59. In contrast, roll-and-glide pattern in the knee joint displays no correlation with age, with a coefficient of 0.04. Both height and weight display a similar dependency as age but are less robust as a basis for drawing conclusions. Interesting results were obtained in terms of a correlation between extent of sporting activity and the respective biomechanical score. Nine children practiced no sports, 73 children practiced sports for up to one hour per week, 65 children for up to 2 hours, and 33 children for more than 2 hours per week. The extent of sporting activity had no effect on the gait score. Knee bend analysis showed, however, that sporting activity was associated with a higher knee bend score. This is merely a trend which was identified. Statistical significance has not been achieved. In particular, the 9 children who did not practice I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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any sports produced better score ratings than children who engaged in up to one hour of sporting activity, and produced similar ratings to children who practiced up to 2 hours of sports or more. However, as the number of children who practiced no sports was markedly lower than the number of active children, this observation is not statistically significant. A positive correlation is evident in terms of the roll-and-glide pattern. In this aspect, children who do not practice sports score significantly below children who practice sports. Moderate sporting activity of up to 2 hours per week seems to be best. Sporting activity in excess of 2 hours does not improve the roll-and-glide score in children. There was no correlation between demeanor and gait score, knee bend score or roll-and-glide score. The evaluation disclosed 18 children with a quiet demeanor, 47 average, 64 children who were described as active, and 54 highly active children. Quiet, average and active children tended to do better in roll-and-glide analysis and in knee bend tests than highly active children. Sporting activity can also be seen as a function of the children‘s demeanor. Highly active children had by far the highest rates of sporting activity and sporting frequency. Quiet children engage in sports once or twice a week for 1.1 hours, those with an average demeanor 1.7 hours once a week, active children 1.7 hours twice a week, and highly active children 1.9 hours 2.8 times per week. Fatigue (as interviewed) does not have a major influence on the total motion analysis score for knee bends and roll-and-glide pattern. Nor is parental motivation/attitude - whether inhibitory, average or encouraging – associated with a biomechanical correlate. When asked about pain, the children replied as follows: No pain in 158 children, mild pain in 13 children, moderate pain in 10 children, severe pain in 2 children. Interestingly, the average total score increases slightly from no pain to severe pain. This might be a signal that these children did more physiotherapy exercises.

Inter-site comparison The possibility of a comparison between all sites in terms of biomechanical outcome might prove the suitability of this method as a quality criterion for the musculoskeletal apparatus. At the very least, each center can be given an overall summary indicating the weaknesses of the individual children in terms of coordination and strength or, pathologically speaking, with regard to the roll-and-glide pattern.

Retest The first retest produced a surprising outcome. The average score in site 1 at age 11.5 was 233.2 points. Retesting at 12.7 years showed an overall decline in the number of points to 213.8. A significant deterioration was evident in terms of the rolland-glide pattern. Scores for gait and knee bends rose slightly. An interview revea-

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led that no physiotherapy had been performed. This had been recommended to all at the first visit and had been written down in each individual child’s report. The average biomechanical score in site 2 for the first examination at 10.7 years was 164.1 points. The average score per child was 239.7 per child at the retest at age 12.1. Gait and roll-and-glide pattern improved significantly while knee bends remained approximately unchanged. Physiotherapy had been performed regularly in all children as recommended in the biomechanical report, in some cases at the child’s home.

Discussion Both coordination and strength are related to age, as shown by the correlation between gait score and age and between knee bends and age. There was no correlation between the roll-and-glide pattern and age. This suggests that the roll-and-glide pattern is more pathognomonic than age-related in terms of being a specific marker of the internal kinematics of the knee joint. Weight and height on their own are not correlated with biomechanical score to the same extent as age, but weight has more of an effect than height. This might be due to the fact that a heavier weight is more inclined to induce the muscles of the body to work, and thus has an effect similar to training with weights. Interestingly, as was also the case for age, the roll-and-glide pattern does not depend on the child’s height and weight. Hemophilic children practice sports (including physical education at school). Only 9 out of the 173 children did not engage in sporting activity! A correlation with gait score was not evident, but a certain connection with the roll-and-glide pattern was detected. Assuming that the roll-and-glide pattern is disease-specific, it would be plausible to assume that sporting activity has a positive effect on internal knee joint kinematics. A certain dependency is also evident with regard to knee bends. The 9 children who did not engage in sporting activity tended to get good scores. This was a non-significant trend. Differences in demeanor also had an effect on the extent and frequency of sporting activity. Active children express their activity. There is no conclusive evidence at present for an impact of demeanor on gait, knee bend performance or roll-and-glide pattern. Parental motivation, and whether or not the children tired easily, likewise seemed to have no effect on the overall motion analysis score. The few children who reported pain tended to score slightly above average in terms of biomechanical score. Possible explanations are more frequent physiotherapy in these children or the fact that these children are slightly above the average age. However, since only 25 children reported mild, moderate or severe pain, this outcome is of no statistical significance. Again, re-testing showed no more than a trend. There was quite a marked improvement in the scores from site 2, especially the roll-and-glide score. All the children at this site received regular physiotherapy! In contrast, the pathognomonic roll-and-glide pattern deteriorated markedly in site 1 where not a single child underwent physiotherapy.

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Summary Motion analysis as introduced 2 years ago on a national basis effectively identifies individual functional disorders and provides a means for describing them on the basis of a score. Individual therapy planning is possible. More treatment tends to produce better function scores and thus helps to lower the strain on the joint. Further study is required in order to determine whether this reduces the incidence of bleeding. Knee function is age-dependent. Height, weight and sporting activity seem to be influencing factors. Demeanor, pain, fatigue and parental motivation do not seem to have an impact. The roll-and-glide pattern is not age-dependent and probably shows functional abnormalities of the knee. Functional benchmarking of the sites is possible but difficult because each site selects the children differently. Age differences also render an overall assessment difficult. Some sites performed negative screening so as to only test children with more severe problems, while other sites performed no such selection. In other sites, the only children to show up for motion analysis were those with well informed parents and who are always involved in all the other activities on offer too. However, motion analysis has shown itself to be useful as an instrument for measuring quality. Being motivated enough to adhere to individual treatment recommendations seems to produce a functional benefit for each patient concerned. Studies in other areas such as intervertebral disk surgery, total hip and knee arthroplasty, and in patients with refractory pain have shown that optimization of therapy is possible and that an improvement in function is associated with improvement of subjective well-being and objective quality control and treatment monitoring.

References 1. Seuser A., G. Schumpe, H.-H. Eickhoff, H.-H. Brackmann, J. Oldenburg: Analyse der Kniekinematik bei Patienten mit Hämarthopathie beim Leg Press Training, in: 24. Hämophilie-Symposium Hamburg 1993 (I. Scharrer und W. Schramm, Hrsg.), Springer Verlag Berlin Heidelberg, S. 150–157 2. Seuser A., G. Schumpe, H.-H. Eickhoff, H.-H. Brackmann, J. Oldenburg: Das Ultraschalltopometer: Eine neue Meßmethode zur 3D-Bewegungsanalyse, in: 24. HämophilieSymposium Hamburg 1993 (I. Scharrer und W. Schramm, Hrsg.) Springer Verlag Berlin Heidelberg, S. 165–172 3. Seuser A., G. Schumpe, H. Gäbel: Quantifizierung von rehabilitativen Therapiemaßnahmen und Qualitätssicherung durch die Verlaufskontrolle mittels Ultraschalltopometrie, Wien. med. Wschr. (1994), Nr. 110, S. 15 4. Seuser A., G. Schumpe, H. Eickhoff, W. Effenberger, H.-H. Brackmann: Gait analysis of the hemophilic knee with different shoes and inlays, in Abstracts of the XXI International Congress of the World Federation of Hemophilia, Mexico City 1994 (R. Ambriz, Hrsg.), S. 144 5. Seuser A., T. Wallny, G. Schumpe, H.-H. Brackmann, W.J. Ribbans: Biomechanical Research in Haemophilia in: Musculoskeletal Aspects of Haemophilia, Edited By E. C: RodriguezMerchan, N. J. Goddard & C. a. Lee, Blackwell Science 2000, Seite 27–36

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6. Seuser A., G. Schumpe, H.-H. Brackmann, T. Wallny. Functional Disorders and Treatment Modalities in Hemophilic Children. Seite 125–134. 34th Hemophilia Symposium Hamburg 2003, Seite 232–237. Springer Medizin Verlag Heidelberg 2004 7. Seuser A., T. Wallny, H.-H. Brackmann, G. Schumpe. The physics of the young haemophilic knee. Results of a multi centre motion analysis study of over 200 knee joints. Haemophilia, Volume 10, Supplement 3, October 2004. (79-87) Session M4–7

Radiosynoviorthesis in Hemophilic Arthropathy – A Single Center Experience S. Gottstein, and R. Klamroth

We have been using radiosynoviorthesis (RSO) in hemophilia patients in two years, since we got the possibility of collaborating with Dr. E. Stelling, who is a specialist in nuclear medicine and has great experience with this method in patients with rheumatoid arthritis. Until now we have treated 10 patients with severe hemophilia A. None of them ever had an inhibitor. Their age at the time of treatment was between 20 and 56 years. They are all HIV negative. Two are positive for HCV-RNA, two others have been successfully treated for hepatitis C. The number of joints treated was 14, their stage of arthropathy was between III and V (according to the classification of Arnold and Hilgartner). 6 ankle joints, 5 elbows, 1 interphalangeal joint, 1 wrist and 1 pedal digital joint were affected. 3 of the joints had been operated earlier: There had been done an arthrodesis on one ankle and 2 surgical synovectomies on one ankle and one elbow, respectively. Before the actual planning of the RSO, there were done conventional x-ray-studies of the joints as well as MRT scans. Ultrasound imaging showed exsudation and synovial thickening indicating chronic inflammation. In half of the cases a blood pool scintigram with technecium was performed additionally. The RSO procedure was performed under sterile conditions as follows: After local skin anesthesia a test injection with contrast fluid was performed to assure the optimal position of the needle. Then, eventually after aspiration of exsudate from the joint, the injection of the radioactive material followed. Depending on the size of the joint, Dr. Stelling used 1,5 mCi Rhenium (for joints and elbows) or 0,3 mCi Erbium (for digital joints). To reassure the correct intraarticular localization, a distribution scintigram was performed. Finally, the joint was immobilized for 24 hours with a splint. Concerning the substitution of factor VIII, the procedure was quite economical, compared to surgical synovectomies: The patient was advised to inject 2000 IU of factor VIII concentrate prior to the treatment and 1000 to 2000 IU on the following day. Table 1 shows the patients opinion about the effect of the RSO. The very biggest part of the patients had a lower bleeding frequency after the treatment, 3 even were free from bleedings into the treated joint. Though one patient had no effect on the bleeding pattern at all. Similarly, there was a positive effect on pain: Two joints even became pain free, but the same single patient still had the same amount of pain in his ankle joint. I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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Table 1. Treatment results- the patients view Patients Stage joint age of *sooner arthro- op. pathy

Effect on Bleeding 1–5

Effect on pain 1–5

Time to start of positive effect after treatment

Duration of positive effect

22 26 30 33 26 58

3 4 3–4 4 3 4–5

5 2 3 3 0 2–3

3 2 3 3 0 2–3

0 1 Month 2 Months 0

since 20 Months 3 Months 3 Months ? (OP after RSO)

4–6 Weeks

4 Months

20 32 50 56

3 4 4–5 5

5 5 3 2

5 5 2–4 3

1 Week 0 None/2–3 Months 2 Weeks

4 Months since 18 Months since 21 Months 6 Months

Ankle Ankle* Ankle* Ankle Ankle PIP III wrist Elbow Ankle Toe Elbow* Elbow bilat. Elbow

There was remarkable variation in the time, until the positive effect of the RSO became evident. 3 patients reported, they had relief immediately after the treatment, some felt the benefit not before 3 months after. The duration of pain relief and reduced bleeding was between 3 months an more than 20 months until now. Interviewed months after the RSO, all patients said, they would accept the offer of a second treatment and they would recommend it to other patients. This positive judging is also due to the lack of adverse events. We saw one mild inflammatory reaction in an ankle one the day after RSO, which resolved spontaneously. There were no infections and no bleeding complications. Only one procedure was planned in in-patient regime, because the patient lives far from our treatment center. Moreover, he was the first and the youngest patient treated, and he was not very experienced with self-i.v.-injection. None of the patients had to stay from work longer than 2 days because of the RSO. After our experience, the RSO appears to be a save and relatively uncomplicated method to reduce bleeding frequency and pain in »target joints«. We think, that it should be offered to the patient, if there is no surgical option or if the patient does not or not yet wish an operation. RSO in hemophiliacs should be in the hands of an experienced specialist and there should be good communication between him or her and the hemostaseologist. Pre-examinations including ultrasound, MRI-imaging and scintigram help to identify those patients who can most probably benefit from RSO. Still, it is important to have the patient informed about the individual variability concerning the effect of the RSO on pain and bleeding.

IV. Laboratory Diagnostics: Coagulation Factor, Inhibitors, Monitoring Chairmen: U. Budde (Hamburg) R. Schneppenheim (Hamburg)

Individual Therapy of Hemophilia – New Laboratory Methods Considering Platelets Th. Siegemund, A. Siegemund, S. Bassus, W. Wegert, S. Petros, U. Scholz, and L. Engelmann

Introduction FVIII/IX replacement therapy based on body weigth and concentration of the corresponding coagulation factors and in case of operations in dependence of the kind of operation procedure. But the hemostatic balance in hemophiliacs is influenced also by other coagulation factors like von-Willebrand factor, platelets, activation state of platelets and also thrombotic risk factors like factor V-Leiden or prothrombinvariant 20210 GA. To measure the overall capacity of the coagulation system to generate thrombin we measured the thrombin generation assay (TG) in platelet rich plasma in patients with hemophilia A and B and inhibitor patients. We used a fluorogenic substrate and determined several parameters of the TG like the endogenous thrombin potential (ETP) and the maximum reaction velocity (Vmax). We monitored the substitution therapy in order to find the optimal amount of the coagulation factor concentrate and an individual substitution concept with is effective to avoid bleeding and in the same time to be cost effective. Additionally, we determined coagulation parameters such as concentration of the coagulation factors II, VIII, IX and XI, the von-Willebrand factor, prothrombinvariant 20210 GA and factor V-Leiden.

Materials and Methods We included 9 patients with hemophilia A und 7 patients with hemophilia B visiting the adult hemophilia center of the university of Leipzig after informed consent. Blood was drawn directly before and 30 minutes after substitution therapy. We measured in platelet-poor plasma (PPP) and also in platelet-rich plasma (PRP) a defined number of platelets obtained by mixing samples of PRP with autologous PPP. The fluorescence measurements were performed with Z-Gly-Gly-Arg-AMC (Bachem, Heidelberg, Germany) as substrate using the Fluoroscan Ascent 2.2 (Labsystems Helsinki, Finland) at wavelengths of 340 nm (excitation) and 440 nm (emission). The measurement lasted 120 minutes and ETP was given as arbitrary units of fluorescence intensity (FU). The calculation was done with an own mathematical procedure published in 2003. I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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Results Patients with comparable levels of coagulation factor VIII/IX show differences in thrombin generation. These differences we obtain in PPP and PRP. The effect of substitution of a definite amount of factor VIII increases the ETP and Vmax, but the effect is also different in each patient. 500

before substitution after substitution

Endogenous Thrombin Potential (ETP) [FU] 400

300

200

100

0 A1

A2

A3

A4

A5

A6

A7

A8

A9

Fig. 1. The effect of factor VIII substitution on thrombin generation in platelet rich plasma

600

Endogenous Thrombin Potential (ET P) [FU]

before substitution after substitution

500

400

300

200

100

0 B1

B2

B3

B4

B5

B6

B7

Fig. 2. The effect of factor IX substitution on thrombin generation in platelet rich plasma

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The same relations we obtain in hemophilia B patients and not only in the classic endogenous thrombin potential, but also in all related parameter like height of the thrombin peak (Vmax). Factor IX has a stronger effect on the ETP than factor VIII. Another finding are the differences of the substitution effects if we compare the measurement results in platelet rich and platelet poor plasma. The platelet rich plasma shows higher interindividual differences at the base level of ETP and lower effects compared with platelet poor plasma. 500

before substitution after substitution

Endogenous Thrombin Potential (ETP) [FU] 400

300

200

100

0 B1

B2

B3

B4

B5

B6

B7

Fig. 3. The effect of factor IX substitution on thrombin generation in platelet poor plasma

Fig. 4. ETP in hemophilia A patients after substituation of 1000 U FVIII with (prothrombin mutation 20210 GA, upper) and without (lower) thrombotic risk factors

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Figure 4 shows the increase in thrombin generation in two patients with hemophilia A. Corresponding to the initial level of FVIII we obtain an increase in ETP as effect of the substitution on 1000 U of a concentrate but at different levels caused by simultaneous occuring of hemophilia and thrombogenic risk factors like prothrombin variant 20210 GA. In this case the prothrombin mutation gives a higher effect on thrombin generation than the substitution of factor VIII.

200%

FVIII

180%

RCoF

160%

vWF:Ag

140% 120% 100% 80% 60% 40% 20%

a) 0% 06.10.2003

09.10.2003

12.10.2003

15.10.2003

18.10.2003

21.10.2003

250

0,35 Thrombinpeak [FU/s]

F VIII [%] ETP [FU]

FVIII

200

0,28

ETP

Peak_h 150

0,21

100

0,14

50

0,07

b) 0,00

06 .1 0. 20 03 07 .1 0. 20 03 08 .1 0. 20 03 09 .1 0. 20 03 10 .1 0. 20 03 11 .1 0. 20 03 12 .1 0. 20 03 13 .1 0. 20 03 14 .1 0. 20 03 15 .1 0. 20 03 16 .1 0. 20 03 17 .1 0. 20 03 18 .1 0. 20 03 19 .1 0. 20 03 20 .1 0. 20 03 21 .1 0. 20 03

0

Fig. 5a, b. FVIII, von-Willebrand factor and thrombin generation in a patient with vonWillebrand syndrome type III underwent orthopedic surgery

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The interaction between hemophilia and thrombogenic risk factors plays also an important role when hemophilia patients underwent surgical procedures, for instance knee replacement therapy, especially to discuss the question of heparin prophylaxis in cases of high factor levels immediately postoperative in elderly hemophilic patients. The ETP as global hemostatic parameter includes pro- and anticoagulant effects and describes the overall capacity of the coagulation system. In the postoperative phase with high levels of fibrinogen, coagulation activation because of wound healing, elevated d-dimer levels we measure higher ETP-levels despite of lower FVIII-levels.

Conclusions The ETP is a global parameter that reflects the overall capacity of the coagulation system to generate thrombin. The clotting factors VIII and IX and platelets have a key influence on thrombin generation. Substitution with clotting factor concentrates enhance thrombin generation and this elevated thrombin generation can be measured by ETP. We can determine the point on which the ETP reaches an optimal level and further substitution does not improve the hemostatic potential. The problem is that this optimal level differs from patient to patient. But with our measurements we can identify this level and optimize the treatment of hemophilia patients. Because of the interaction between platelets and coagulation system we measure in platelet rich plasma. This method is also suitable to monitor alternative therapies in inhibitor patients like FEIBA or NovoSeven. For understanding the physiology of clotting not only the ETP but also other kinetic data such as the maximal thrombin peak or the begin of the thrombin generation should be considered.

Epitope Mapping during FVIII Inhibitor Elimination with Rituximab Reveals Conformational Epitopes on FVIII and Identifies Small Molecules Blocking Inhibitor and Targeting B Cells C. Kessel*, C. Königs*, R. Linde, C. Escuriola-Ettingshausen, J. Roland, H. Stoll, T. Klingebiel, U. Dietrich, and W. Kreuz

Introduction Hemophilia A is a X-chromosome linked bleeding disorder. Mutations in the factor VIII (FVIII) gene result in reduced or non-functional expression of FVIII [1], which is an essential regulatory protein cofactor [2] to the serine protease FIX in the blood coagulation cascade [3]. FVIII increases the activity of FIX by 200 000 [4]. The FVIII molecule is a large plasmatic glycoprotein consisting of 2332 amino acids [5]. The plasmatic form of FVIII is a heterodimer consisting of a heavy chain (comprising domains A1-A2-B) and a light chain (A3-C1-C2). The heterodimer is bound to the von-Willebrand Factor (VWF) via C2 [6] and thus protected from proteolysis. In its active form FVIIIa dissociates from VWF and is subsequently processed into a heterotrimer consisting of the A1 subunit, the light chain (A3-C1-C2) and the A2 subunit [7]. FVIIIa binds to negatively charged phospholipids on the surface of activated thrombocytes via amino acids in C2. Essential amino acids for binding of FIXa were found within A2 (484-509 and 558–565) [8] and A3 (1811–1818) [9]. FVIIIa binds to negative phospholipids on the surface of activated thrombocytes via amino acids in C2. Hemophilia A patients are commonly treated by substitution of recombinant (rFVIII) or plasma derived (pdFVIII) FVIII preparations. However, a significant number of patients develop an antibody response (inhibitors) to substituted FVIII. Inhibitors usually belong to IgG1 and IgG4 [10] and neutralize FVIII by various mechanisms. So far inhibitors hydrolyzing FVIII [11, 12] or binding to non-functional domains of the molecule have been described [13]. Best characterized are inhibitors which bind to functional domains of FVIII and thereby sterically or competitively block its interaction with FIX, FX or phospholipids [14–18]. Inhibitor epitopes, especially in A2 and C2, have been characterized in the past more or less precisely by a variety of different methods [14, 16, 19, 20, 21]. Phage display is a very convenient method to exactly identify amino acids involved in an antibody binding, even if arranged in a conformational epitope [22]. The phage display technology has as well been employed for the identification of FVIII inhibitor epitopes [23, 24] but so far no conformational epitope could be identified and mapped to the FVIII molecule.

* Both authors contributed equally to this work I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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In our study, phage displayed random peptide libraries were screened with plasma from a patient before, under and after rituximab treatment who has been described earlier [25]. We set out to identify relevant epitopes at different time points of elimination approaches and to determine whether a change in epitope specificity occurs and accounts for observed clinical changes.

Materials and Methods Patient The patient with hemophilia A has been described earlier. In brief, he was diagnosed at the age of 1 year. Inhibitors appeared 20 days after initial substitution of FVIII. Multiple attempts to induce immunotolerance using known protocols failed. In the light of persisting bleedings, several rounds of rituximab infusions were given from 10/00 to 01/02 to eliminate the inhibitor [25].

Sampling Citratblood was sampled as part of routine checks. Plasma was stored at –20°C. From blood cells PBMCs were prepared by ficol and stored at –80°C.

Phage Display Patient’s plasma was bound to paramagnetic beads coated with goat anti-human mAb. For negative selections plasma from healthy donors mixed in a ration of 1:1 with commercial ivIg was immobilized on beads. Linear 7mer and 12mer and a constrained 7mer M13 phage displayed peptide library (New England Biolabs) were used for screening. From each library 1x1011 pfu (plaque forming units) were mixed with beads coated with patient’s IgG. After thorough washing steps, bound phages were either eluted by pH-shift with glycine-HCl (pH 2,0) or competition with FVIII (Monoclate). Subsequently eluted phages were used in a negative selection on beads coated with normal IgG to remove irrelevant binders. Supernatants of the negative selected phage pool were amplified in E. coli. Three selection rounds of biopanning were performed. The phage pool was titered in a blue/white screen and single clones were amplified in 96well plates. Supernatants were used for capture ELISA and ssDNA isolation for sequencing.

Capture ELISA ELISAs to confirm specificity were done as described earlier [22]. In brief, plates were coated with patient’s plasma or control plasma, washed and blocked with 5% skim milk for 2h at 37°C. Phage clones were added and left at rt for 90 minutes.

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Bound phages were detected by HRP conjugated mouse anti-M13 IgG. Strongly positive binders from the capture ELISA were used for ssDNA extraction and sequencing.

Sequencing ssDNA was extracted from phage clones that were positive by ELISA. DNA sequencing was performed according to the manufacturer’s instruction. Sequences were analyzed by using DNAStar software.

Epitope Mapping Mapping of identified peptide sequences to the primary amino acid sequence was performed using Provar software. Homologies to the surface of a three dimensional FVIII model [26] was performed using 3D-Epitope-Explorer software (3DEX) [27]. Identified amino acids on the FVIII molecule were the visualized by PyMol software.

Depletion and Inhibition ELISA To confirm specificity of the identified amino acid motive for FVIII inhibitors the strongest binding phage clone (clone 82) was used in an inhibitor depletion and an inhibition assay. For inhibitor depletion ELISA plates were coated with clone 82, wild-type phage or no phage as controls. Plates were washed, blocked and washed again. Patient’s plasma was applied to the first plate, incubated for 2h and then transferred to the next phage coated plate. After each round of depletion, an aliquot was transferred to an ELISA plate coated with FVIII (Advate, Baxter) to monitor depletion of inhibitors. A maximum of 5 rounds was performed. Binding of inhibitors to FVIII was detected with a HRP conjugated goat anti-human IgG (Dianova). Percent depletion of inhibitors was calculated in comparison to depletion of inhibitors by wild-type phage. For direct inhibition of inhibitor binding to FVIII, different concentrations of clone 82 were mixed with patient’s plasma and the mixtures were then applied to a FVIII coated ELISA plate and incubated for 2h at rt. Bound inhibitor was detected as above. Percent inhibition was calculated in comparison to controls without phage.

Synthetic Peptides and ELISAs Synthetic peptides with sequences corresponding to phage inserts of clone 82 and two irrelevant peptides (HIV epitopes) were generated and conjugated to biotin (Thermohybaid). ELISA plates were coated with peptides diluted in carbonate buffer, pH 8.9, or with patient’s plasma for direct or sandwich ELISA respectively. For direct ELISA, plates were incubated with patent’s plasma 2h at 37°C and bound IgG

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was detected by a goat anti-human IgG conjugated to HRP. For the sandwich ELISA, plasma coated plates were incubated with a range of peptide dilutions. Bound peptides were detected by HRP-conjugated streptavidin. Inhibition of inhibitor binding to FVIII was performed as described for inhibition with phages. In brief, the mixtures of peptide dilutions and plasma were pre-incubated for 1h at 37°C, transferred to FVIII coated plates and incubated for another 1h at 37°C. Percent inhibition was calculated in comparison to inhibition by control peptides.

Staining of Patient’s B Cells Thawed patient’s PBMCs were washed with RPMI/10% FCS and resuspended at a density of 1x107 cells/ml. Approximately 1x105 cells were incubated with 1x1011 pfu of clone 82 or wild-type phage (wt) as control for 2h at 4°C. Cells were washed three times with ice-cold RPMI/3%FCS (washing buffer). Thereafter cells were incubated with a mouse anti-M13-IgG (Amersham Pharmacia), which was previously labeled to FITC (Sigma, according to instructions of Fluorotag FITC conjugation kit) (1:10 dilution) and with a PE-conjugated anti CD19 or anti CD138 (1:10 dilutions) for 30 minutes at 4°C. Cells were washed three times, fixed in formaldehyde and 1x104 cells of each staining were analyzed on a FACSCAlibur flow cytometer using Cellquest Pro software (Becton Dickinson).

Results Screening Plasma samples before, under and after rituximab treatment were chosen for screening of three different phage display peptide libraries. A total of nine independent biopannings was performed. The libraries have an approximate diversity of 109 different clones and 100 copies of each clone are present in the library. After three rounds of screening, single phage clones were tested for their avidity to autologous plasma by ELISA. Clones with a significantly higher optical density compared to the negative control (wild-type phage) were regarded positive and chosen for ssDNA extraction and sequencing. Approximately 300 clones per sample were analyzed by ELISA. 116 (38%), 36 (12%) and 50 (16%) phage clones obtained from screening with patient’s plasma before, under and after rituximab treatment specifically bound to inhibitor by ELISA. Sequencing of these clones revealed a very frequently appearing amino acid motive within the random peptide inserts of the linear 7mer and 12mer library exclusively. The EV/IPN-motive appeared among 77 (66%), 36 (94%) and 16 (32%) sequenced ELISA positive phage clones from screening patient’s plasma before, under and after therapy, respectively (Fig. 1). This amino acid motive was mapped to the surface of the FVIII molecule. The 12mer phage clone (clone 82) with highest avidity for inhibitor positive plasma was chosen for further assays.

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a Positive Selection

Negative Selection Inhibitor negative plasma Elution by pH-shift or competition with FVIII

Amplification

Inhibitor positive plasma

HRP

Repeated selections and amplifications 50 5560 65 70 75 80 85 90 95 100105110115 Bases

b

Sequencing

Specificity

Fig. 1a–c. Screening of phage display libraries with patient’s plasma before, under and after rituximab treatment. Patient’s inhibitor titers in BDU and CD20+ cell numbers are depicted in a. The inhibitor titre declines shortly after the first infusions. After final rituximab infusion CD20 cells increase in peripheral blood, 18 months after rituximab treatment the inhibitor reappears. Both number of CD20+ cells and inhibitor titre have reached starting level by now (data not shown). Time points of plasma samples taken for screening are indicated by arrows. A scheme of screening procedure with positive and negative selection is seen in b.

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Fig. 1c. Graph c shows stabilities of the analysis of all phage clones obtained

Epitope Mapping The EV/IPN amino acid motive obtained from the phage display screenings was used to search for homologies in the primary amino acid sequence of FVIII. The search revealed no significant homologies. The search for sequence homologies on the surface of a B-domain depleted three dimensional model of the FVIII molecule was carried out with 3DEX software. The surface of FVIII was scanned for an EV/IPN motif within a range of 6 to 8 Å. Several motifs were found, which were all visualized on the surface of the FVIII molecular model by PyMol software to confirm their position and relevance as possible epitopes. One cluster involving the amino acids E589 I591 P598 N597 was mapped to the surface of the A2 subunit (Fig. 2). Additional amino acids next to the EV/IPN motive in the sequenced peptide inserts of positive phage clones cluster next to the EIPN motive on the surface of A2. The motive is in close proximity of a known binding site of FIX (amino acids 484–509).

FVIII Inhibitor Depletion and Inhibition For further confirmation of specificity of the selected peptide ligands for FVIII inhibitors, a phage clone (clone 82) with strongest avidity to patient’s plasma was chosen for additional assays. In a depletion assay, inhibitors subsequently depleted from plasma by several rounds of exposure to clone 82 coated ELISA plates. Clone 82 could deplete up to 70% of inhibitor in comparison to depletion with wild-type phage (Fig. 3a).

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Fig. 2a–c. Epitope mapping. a) shows the EIPN motive in A2 in context with the whole B-domain depleted FVIII molecule. The enlarged EIPN motive with additional amino acids from obtained phage clones is shown in detail in b). The proximity of the identified epitope to a known FIX binding site is depicted in c)

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Fig. 3a, b. FVIII inhibitor depletion and inhibition. Figure a shows a sequential depletion of FVIII inhibitors by several rounds of absorption on plates coated with clone 82. Residual binding is measured by binding to immobilized FVIII. Direct inhibition of inhibitor binding to immobilized FVIII by phage particles (pp) of clone 82 is depicted in b. Clone 100 is another isolated clone, WT is the wild type phage without peptide insert

In a direct inhibition assay the addition of different concentrations of clone 82 to patient’s plasma could inhibit binding of inhibitors to immobilized FVIII in a concentration dependent manner (Fig. 3b).

Synthetic Peptides In order to analyze the identified peptide sequences without the scaffold of M13, synthetic peptide ligands were generated and used similarly to phage in binding

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and inhibition assays. In direct and sandwich peptide ELISAs either plasma binding to coated peptide or peptide binding to immobilized plasma was observed. Both assays revealed peptide binding in a concentration dependent manner in contrast to control peptides or plasma of healthy donors (Fig. 4a–c). Inhibitors in patient’s plasma after rituximab treatment (251103) reveal a slightly weaker binding to Pep82 in comparison to patient’s plasma before therapy start (100400). This finding was observed with clone 82 as well (Fig. 7). The significance of this finding is not clear.

Fig. 4a

Fig. 4b

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Inhibition of inhibitor binding in patient’s plasma by synthetic peptides to immobilized FVIII was shown. A pre-incubated mix of patient’s plasma and different concentrations of Pep82 revealed reduced inhibitor binding to FVIII in a peptide concentration dependent manner. As seen with the binding assays inhibitors in plasma after therapy reveal a weaker binding to Pep82 and therefore inhibitor inhibition by Pep82 is reduced (Fig. 4d).

Fig. 4a–d. Reactivity of synthetic peptides with FVIII inhibitors. Figure a and b demonstrate binding of FVIII inhibitor in patient’s plasma before (a) and after (b) rituximab elimination therapy to immobilized Pep82 in a concentration dependent manner. No concentration dependent reactivity with negative control peptides (irrelevant sequences) was observed. Figure c specific binding of immobilized patient’s plasma to Pep82 compared to inhibitor negative plasma. Figure d demonstrates inhibition of inhibitor binding to FVIII in patient’s plasma samples before (100400) and after (251103) rituximab Therapy

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EVPN

Fig. 5. Pep82 tertiary structure prediction

The peptide sequence of clone 82 was used to predict a possible 3D structure of the peptide mimotopes in a hydrophilic environment by using the Raghava Peptide Tertiary Structure Prediction Server. The obtained pdb-data were visualized by PyMol (Fig. 5).

Targeting of Patient’s B Cells The use of specific peptide ligands presented on phage has been shown to be a useful tool for targeting of B cells with surface immunoglobulin (sIg) [28]. Patient’s PBMCs or PBMCs of a healthy donor were incubated with clone 82 or wild-type phage (wt) as control. Phage binding to B cells was assessed by staining with anti M13-FITC and subsequent FACS analysis. Either anti-CD19 or anti-CD138 was chosen for double staining of B or plasma cell (PC) surface marker. Clone 82 revealed a significantly higher binding to patient’s CD138 and CD19 positive cells. Nearly no binding of clone 82 was observed to CD138 and CD19 positive cells of healthy donor (Fig. 6).

Discussion Phage display is a very convenient method to identify essential amino acids in ligand binding sites or antibody epitopes. Phage displayed peptide libraries were used to map epitopes of FVIII inhibitors from patient plasma at different time points of rituximab treatment. This approach identified a possible epitope in the A2 domain of FVIII. Despite complete depletion of B cells by rituximab and improved clinical outcome, we could identify the same amino acid motive. This suggests an identical inhibitor epitope before and after therapy. The specificity of isolated phagotopes as well as synthetic peptide ligands for inhibitors has been shown by various approaches. Targeting of patient’s B cells with peptides presented on the surface of phages was shown by FACS and might lead to a novel immune therapy in the treatment of inhibitors in hemophilia A.

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Fig. 6a, b. Targeting of patient’s B cells with phage. Exemplary dot plots of FACS results are depicted in a. Clone 82 reveals significantly stronger binding to patient’s CD19+ cells in contrast to wt or CD19+ cells of healthy donor. Percent of phage binding to whole lymphocytes of patient’s PBMCs or whole CD19/CD138+ cells is shown in b

Specificity of the selected peptide ligands with the EI/VPN on the surface of the phage or as part of a synthetic peptide was confirmed by inhibition of inhibitor binding to immobilized FVIII. More than about 50% inhibition was not achieved with clone 82 or the corresponding Pep82. However, inhibition close to 100% was not expected as FVIII inhibitors are known to be a polyclonal Ig-population directed to various epitopes on FVIII. In our assays we found evidence for the presence of at least one additional inhibitor. In inhibition assays performed with FVIII preparations without VWF no inhibition was observed (data not shown). Without VWF, a possible inhibitor specific for the C2 domain binds to this subunit and cannot be blocked with clone 82, which represents an A2 epitope. Consequently, the following inhibition assays with clone 82 or Pep82 were performed with VWF containing FVIII preparations. The relevance of the identified epitope was confirmed by showing its cross-reactivity with a panel of heterologous inhibitor positive plasma (Fig. 7).

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Fig. 7. Cross reactivity of clone 82 with various hemophilia A patients` plasma. Different patients’ plasma (grey) did reveal binding to immobilized clone 82. Binding of plasma of the index patient is shown in black for samples before (100400) and after (251103) rituximab treatment

Weaker binding of plasma from patient 1, 2 and 4 compared to plasma of the index patient corresponds to lower inhibitor titers as determined by Bethesda assay. Despite having a high inhibitor titer, the plasma of patient 3 shows no binding to clone 82. Thus, FVIII inhibitors of patient 3 do not share the same epitope. The successful targeting of antigen specific B cells by peptides selected from a phage library has been shown before [28]. In our approach, selected phagotopes were used to target inhibitor specific B cells by binding to surface immunoglobulin (sIg). B cell populations were stained with CD19 and CD138. Both cell types express sIg, although CD138 only show a low expression of sIg [29]. For patient’s B cells, a subset of CD19 or CD138 cells was stained with phage clone 82, but no subset of CD19 or CD138 cells of a healthy control was stained. The control phage (wild type) showed a significantly lower binding to the patient’s B cells. This strongly argues that an inhibitor specific subset of B cells was stained by clone 82. Further analysis is being performed to proof this hypothesis. Another approach, staining PBMCs in addition to CD19 or CD138 with biotinylated peptide Pep82 failed. The used label streptavidin produced a very strong background. Thus no detection was possible. The same amino acid motif was isolated before and after rituximab treatment strongly suggesting that the same epitope is recognized despite therapy. This finding raises the question whether this is due to a persisting immunological memory or a newly established immune response to FVIII. Although a close monitoring of peripheral blood samples revealed no CD20 positive cells, there is the possibility for a lasting immunological memory, as resident plasma cells can migrate to survival niches in the bone marrow [30, 31]. They provide stable antibody titers for years [32]. However, in this case it is unlikely, that the relapsing inhibitor was cased by resident plasma cells. The patient was continuously treated with the antigen (FVIII). Even after the final rituximab infusion, no inhibitor appeared for months. It was shown that presence of antigen (tetanus-toxin) leads to a release of large numbers

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of toxin-specific, antibody-secreting plasma cells from the bone marrow within 7 days [33]. In our case inhibitors were first detected months after CD20+ cells reappeared in peripheral blood. Inhibitor titers rose slowly and were delayed to increasing CD20+ cell numbers. We believe that the relapse of patient’s inhibitors is not due to immunological memory, but to a newly established B cell response. Therefore we argue that the identified amino acids are part of an immunodominant epitope, which is repeatedly recognized by the patient’s immune system. The data shown here demonstrates a successful mapping of a conformational inhibitor epitope from patient plasma at various time points of a novel inhibitor elimination therapy. The technique chosen yielded a small peptide molecule that inhibits binding of inhibitor to FVIII. Additionally, the phage bearing the peptide stained a subset of the patient’s B cells that are suspected to be inhibitor specific. This approach might lead to novel immune therapies, which specifically eliminate inhibitors and inhibitor specific B cells in patients that fail established immune tolerance regiments. References 1. Bowen, D. J. Haemophilia A and haemophilia B: molecular insights. Mol Pathol 55, 1–18 (2002) 2. Lawson, J. H., Kalafatis, M., Stram, S. & Mann, K. G. A model for the tissue factor pathway to thrombin. I. An empirical study. J Biol Chem 269, 23357–66 (1994) 3. Mann, K. G. Biochemistry and physiology of blood coagulation. Thromb Haemost 82, 165–74 (1999) 4. van Dieijen, G., Tans, G., Rosing, J. & Hemker, H. C. The role of phospholipid and factor VIIIa in the activation of bovine factor X. J Biol Chem 256, 3433–42 (1981) 5. Kane, W. H. & Davie, E. W. Blood coagulation factors V and VIII: structural and functional similarities and their relationship to hemorrhagic and thrombotic disorders. Blood 71, 539–55 (1988) 6. Saenko, E. L. & Scandella, D. The acidic region of the factor VIII light chain and the C2 domain together form the high affinity binding site for von willebrand factor. J Biol Chem 272, 18007–14 (1997) 7. Pipe, S. W. & Kaufman, R. J. Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa. Proc Natl Acad Sci U S A 94, 11851–6 (1997) 8. Fay, P. J. & Scandella, D. Human inhibitor antibodies specific for the factor VIII A2 domain disrupt the interaction between the subunit and factor IXa. J Biol Chem 274, 29826–30 (1999) 9. Lenting, P. J., van de Loo, J. W., Donath, M. J., van Mourik, J. A. & Mertens, K. The sequence Glu1811-Lys1818 of human blood coagulation factor VIII comprises a binding site for activated factor IX. J Biol Chem 271, 1935–40 (1996) 10. Fulcher, C. A., de Graaf Mahoney, S. & Zimmerman, T. S. FVIII inhibitor IgG subclass and FVIII polypeptide specificity determined by immunoblotting. Blood 69, 1475–80 (1987) 11. Lacroix-Desmazes, S. et al. Catalytic activity of antibodies against factor VIII in patients with hemophilia A. Nat Med 5, 1044–7 (1999) 12. Lacroix-Desmazes, S. et al. Antibodies with hydrolytic activity towards factor VIII in patients with hemophilia A. J Immunol Methods 269, 251–6 (2002) 13. Vermylen, J. & Briet, E. Factor VIII preparations: need for prospective pharmacovigilance. Lancet 342, 693–4 (1993) 14. Scandella, D. et al. Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipidbinding site. Blood 86, 1811–9 (1995)

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15. Scandella, D. H. et al. In hemophilia A and autoantibody inhibitor patients: the factor VIII A2 domain and light chain are most immunogenic. Thromb Res 101, 377–85 (2001) 16. Healey, J. F. et al. Residues 484–508 contain a major determinant of the inhibitory epitope in the A2 domain of human factor VIII. J Biol Chem 270, 14505–9 (1995) 17. Shima, M., Yoshioka, A., Nakajima, M., Nakai, H. & Fukui, H. A monoclonal antibody (NMC-VIII/10) to factor VIII light chain recognizing Glu1675-Glu1684 inhibits factor VIII binding to endogenous von Willebrand factor in human umbilical vein endothelial cells. Br J Haematol 81, 533–8 (1992) 18. Zhong, D., Saenko, E. L., Shima, M., Felch, M. & Scandella, D. Some human inhibitor antibodies interfere with factor VIII binding to factor IX. Blood 92, 136–42 (1998) 19. Scandella, D. et al. Epitope mapping of human factor VIII inhibitor antibodies by deletion analysis of factor VIII fragments expressed in Escherichia coli. Proc Natl Acad Sci U S A 85, 6152–6 (1988) 20. Fulcher, C. A., de Graaf Mahoney, S., Roberts, J. R., Kasper, C. K. & Zimmerman, T. S. Localization of human factor FVIII inhibitor epitopes to two polypeptide fragments. Proc Natl Acad Sci U S A 82, 7728–32 (1985) 21. Ware, J., MacDonald, M. J., Lo, M., de Graaf, S. & Fulcher, C. A. Epitope mapping of human factor VIII inhibitor antibodies by site-directed mutagenesis of a factor VIII polypeptide. Blood Coagul Fibrinolysis 3, 703–16 (1992) 22. Konigs, C. et al. Monoclonal antibody screening of a phage-displayed random peptide library reveals mimotopes of chemokine receptor CCR5: implications for the tertiary structure of the receptor and for an N-terminal binding site for HIV-1 gp120. Eur J Immunol 30, 1162–71 (2000) 23. Villard, S. et al. Peptide decoys selected by phage display block in vitro and in vivo activity of a human anti-FVIII inhibitor. Blood 102, 949–52 (2003) 24. Muhle, C. et al. Epitope mapping of polyclonal clotting factor VIII-inhibitory antibodies using phage display. Thromb Haemost 91, 619–25 (2004) 25. Linde, R., Escuriola-Ettingshausen, C., Voigt, B., Klingebiel T., Kreuz, W. First successful inhibitor elimination with a new protocol in a high responder hemophilia A patient after failure of various immune tolerance induction regimes. Blood 98, 11 (2001) 26. Stoilova-McPhie, S., Villoutreix, B. O., Mertens, K., Kemball-Cook, G. & Holzenburg, A. 3Dimensional structure of membrane-bound coagulation factor VIII: modeling of the factor VIII heterodimer within a 3-dimensional density map derived by electron crystallography. Blood 99, 1215–23 (2002) 27. Schreiber, A., Humbert, M., Benz, A. & Dietrich,U. 3D-Epitope-Explorer (3DEX): Localization of conformational epitopes within threedimensional structures of proteins. JCC, accepted for publication 28. Buhl, L., Szecsi, P. B., Gisselo, G. G. & Schafer-Nielsen, C. Surface immunoglobulin on B lymphocytes as a potential target for specific peptide ligands in chronic lymphocytic leukaemia. Br J Haematol 116, 549–54 (2002) 29. Horst, A. et al. Detection and characterization of plasma cells in peripheral blood: correlation of IgE+ plasma cell frequency with IgE serum titre. Clin Exp Immunol 130, 370–8 (2002) 30. Manz, R. A., Thiel, A. & Radbruch, A. Lifetime of plasma cells in the bone marrow. Nature 388, 133–4 (1997) 31. Manz, R. A., Lohning, M., Cassese, G., Thiel, A. & Radbruch, A. Survival of long-lived plasma cells is independent of antigen. Int Immunol 10, 1703–11 (1998) 32. Ahmed, R. & Gray, D. Immunological memory and protective immunity: understanding their relation. Science 272, 54–60 (1996) 33. Odendahl, M. et al. Generation of migratory antigen-specific plasma blasts and mobilisation of resident plasma cells in a secondary immune response. Blood (2004)

Lack of Factor VIII Expression Represents a Novel Mechanism Leading to Hemophilia A O. El-Maarri, H. Singer, H.-H. Brackmann, J. Schröder, J. Graw, C.R. Müller, W. Schramm, R. Schwaab, P. Hanfland, and J. Oldenburg

Introduction Hemophilia A (HA) is caused by impaired F8 protein activity. Various point mutations leading to either altered or truncated FVIII protein and the two major inversion hot spots, intron 22 and intron 1 inversions, have been reported. Most interestingly, in about 2% of the HA cases we were unable to detect any mutation(s), even after sequencing the whole F8 exons and their flanking intronic sequences [1]. Therefore, to further investigate the reason for HA in these small number of patients we have recently analyzed the F8 mRNA, isolated from lymphocytes, without finding any rearrangements [2]. However in one severe HA patient, we were unable to detect a RT-PCR product from the F8 gene. We followed the segregation of this allele in the family members, mainly his mother and his sister; the same allele present in the patient was found to be transmitted by the mother to his sister. In all three individuals we were unable to get an RT-PCR product corresponding to this allele (Fig. 1). Our data strongly suggest that the cause of HA in this patient is either absence or rapid degradation of the F8 mRNA, thus pointing to a novel mechanism leading to hemophilia A.

Materials Subjects The index patient is a 12-year-old boy suffering from severe HA. He is on a prophylactic treatment regime from his second year of life. No inhibitor has been reported during more than 1000 exposure days. He is the first hemophiliac in this family. Both, the mother and the sister are suggested to be carriers because of significantly reduced FVIII:C of 17-49% and 42% respectively while the VWF:Ag levels were in the normal range (mother: 109%, sister: 139%).

Results and Discussion The two alleles in both, the mother and the sister, were distinguished by the analysis of two known polymorphisms in exon 14 of the F8 gene (Fig. 1, 2). RT-PCR I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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I.

1

2

D N A

at aa 1241

C

G

C

at aa 1269

C

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A

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at aa 1241

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at aa 1269

II.

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at aa 1241

G

C

G

at aa 1269

A

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A

R N A

at aa 1241 at aa 1269

X

C A

X

No Product

Fig. 1. A pedigree of the family presented in this study. Different alleles present in both DNA and RNA are indicated under each member

revealed that the same GA allele that was not detected in the patient was also absent from the RT-PCR products of both the mother and the sister in three different experiments. In a control RNA sample both, the CA and GA alleles were detected by RTPCR (Fig. 2). The absence of detectable expression of the GA allele in the lymphocyte derived RNA in both, the mother and the sister, could theoretically be the result of skewed X-chromosome inactivation that is leaving only the allele from the active X- chromosome to be actively expressed. However, analysis of the HUMARA locus proved a normal random X-chromosome inactivation in the mother and the sister (Fig. 3). Another possibility of our finding could be a local abnormal ›closed‹ chromatin structure, that would affect the expression of the F8 gene among other neighbouring genes. The F8B transcript, which is completely nested within the F8 locus, represents a good candidate to further elucidate this possibility. An RT-PCR product specific for the F8B transcript could be obtained from the patient RNA. Furthermore, the level of DNA methylation at a CpG rich region located about 5 kb upstream from the transcription start site was not different from male controls, thus further arguing against any global regional silencing due to epigenetic factors.

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D N A

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No RT-PCR

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CG

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Two alleles

Only one allele detected

Fig. 2. Discrimination of the presence of one or two alleles in both the mother (I.1), the sister (II.1) and a female control. For simplicity only the polymorphism at codon 1241 is shown. To distinguish the presence of one or two alleles a SNuPE reaction was performed on either a DNA derived PCR or RT-PCR products. The product of the SNuPE reaction was run on HPLC to allow the separation and quantitation of the two alleles Random Inactivation

Skewed Inactivation

Undigested Genomic DNA

Hpa II Digested

Patient

Mother

Sister

Fig. 3. Test for the status of X-Chromosome inactivation in the family members, DNA which was either digested with HpaII or undigested were amplified by primers specific for the HUMARA locus. The products were run on an ABI machine to separate the alleles. The undigested DNA serve as a control to show if the locus is informative with two different size alleles. In the later cases and after HpaII digestion (methyltion sensitive enzyme) the presence of two alleles represent a normal random X-chromosome inactivation

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Reasons that could explain the absence of RT-PCR product from a single allele could be a mutation lying either deep in one of the large F8 introns that affects the stability or induces the rapid degradation of the mRNA or in a still unknown control region that enhance the expression of the gene. In conclusion, we are presenting a unique case of HA, where we provided strong evidence that the absence of expression of the F8 and/or the rapid degradation of the F8 mRNA is the cause of HA. This finding points to a novel mechanism leading to HA; however the molecular basis behind this phenomenon is still to be elucidated.

References 1. Klopp N, Oldenburg J, Uen C, Schneppenheim R, Graw J. 11 haemophilia A patients without mutations in the factor VIII encoding gene. Thromb Haemost. 2002; 88: 357–60 2. El-Maarri O, Herbiniaux U, Graw J, Schröder J, Watzka M, Brackmann H.H, Schramm W, Hanfland P, Schwaab R, Müller CR, Oldenburg O. Detailed RNA analysis in haemophilia A patients with previously undetectable mutations, Journal of thrombosis and haemostasis. 2005; Feb;3(2):332–9

V. Pediatric Hemostaseology Chairmen: W. Kreuz (Frankfurt/Main) K. Kurnik (Munich)

Effects of the Factor V G1691A Mutation and the Factor II G20210A Variant on the Clinical Expression of Severe Hemophilia A (< 2%) in Children – Results of a Multicenter Study K. Kurnik, C. Escuriola-Ettingshausen, S. Horneff, C. Düring, R. Schobess, C. Bidlingmaier, S. Halimeh, H. Pollmann, N. Bogdanova, and U. Nowak-Göttl

Summary It has been recently shown that the first bleeding onset in children with severe HA carrying prothrombotic risk factors is significantly later in life than in non-carriers. The present multicenter study was performed to determine whether the factor (F) V G1691A or the F II G20210A are associated with decreased annual bleeding episodes (ABE) in 104 pediatric PUP patients with severe HA consecutively admitted to German pediatric hemophilia treatment centers. Treatment was initiated according to the frequency of bleedings, and most patients received on demand therapy with a switch over to prophylactic therapy 3x/week when more than two bleedings (range 2–6) had occurred into the same joint. Prospective median (range) patient follow-up was 14 (5–24) years. Heterozygosity of the FV mutation was found in 8 subjects, homozygosity in one, and 5 children carried the FII mutation once combined with protein C-deficiency. Carriers of the FV and FII mutations had significantly fewer ABE than non-carriers (p = 0.004). 65 of 104 PUP patients had developed at least one target joint. The Pettersson score (median/range: 1/0–12) available in 56 patients is clearly dependent on age (p = 0.039), and on ABE (r = 0.42; p = 0.007). The »Nuss« joint score available in 32 subjects highly correlated with the Pettersson score (r = 0.8; p = 0.0004). Data presented here give evidence that the clinical expression of severe HA in children is influenced by the co-expression of the FV and FII mutation.

Introduction Hemophilia A and B are X-linked genetic hemorrhagic disorders resulting from deficiencies of blood coagulation factor VIII or IX. Subjects suffering from plasma levels of factor VIII coagulant activity or factor IX below 1% of normal are classified as severe hemophiliacs [1]. While mild or moderate hemophilia is not always diagnosed during childhood, severe hemophilia is generally diagnosed at an early age [2–6]. Although bleeding symptoms correlate with the levels of the remaining factor activity, it is reported that some hemophilic subjects with factor VIII levels < 1% do not all bleed with the same severity [7, 8]. Besides the possibility that the mutation type within the factor VIII gene may influence the clinical severity of hemophilia [9, 10], it has been recently suggested that the clinical phenotype of severe hemophilia A is influenced by co-inheritance I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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with the factor V G1691A mutation [11]. In addition, we have recently demonstrated that the first symptomatic bleeding onset in children with severe HA carrying prothrombotic risk factors is significantly later in life than in non-carriers [12]. The present study was therefore conducted to unravel the role of the factor V (FV) G1691A mutation or the factor II (FII) G20210A variant, coinherited with severe hemophilia A with respect to the clinical expression of the disease, e.g. the bleeding frequency.

Methods Ethics The present survey on consecutively recruited pediatric patients with hemophilia was performed in accordance with the ethical standards laid down in a relevant version of the 1964 Declaration of Helsinki and approved by the medical ethics committee at the Westfälische Wilhelms-University, Münster, Germany. With special regards to the data presented here, the ethical committee has approved the investigation of established prothrombotic risk factors possibly coinherited in pediatric patients with hemophilia A.

Inclusion Criteria Untreated Caucasian infants and children (PUP) with previously undiagnosed severe hemophilia A (FVIII activity < 1%) aged neonate to 16 years admitted to the university pediatric hospitals in Frankfurt, Halle, Munich and Münster, Germany, on the first symptomatic and spontaneous onset of the disease. In the patients enrolled the classification of HA based on the FVIII activity was confirmed by using the same aPTT reagents and factor VIII-deficient plasma in the patients investigated.

Exclusion Criteria Pretreated pediatric patients, subjects in whom surgery- or major (birth-) traumainduced bleeding had occurred. In addition, patients with prenatal diagnosis of HA were excluded, as well as children with diagnosis of hemophilia before the first bleeding episode. Children with hemophilia A and inhibitor development were not included in the present survey.

Study Period From October 1985 to December 2001, 144 consecutive Caucasian pediatric PUP patients with a first symptomatic onset of hemophilia A were recruited from different geographic areas of Germany.

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Study Population Of the 144 consecutively recruited children, 104 were suffering from severe HA. Due to individual decisions of the participating centers the majority of patients were initially treated »on-demand« and were switched over to a »prophylactic« treatment regimen when more than two bleeding episodes had occurred into the same joint within a 12-month period [13, 14]. In children with joint bleedings imaging of the affected joints was performed according to Pettersson [15], and in addition, in patients with a Pettersson score of 0, as well as in cases with suspected synovitis a MRI was additionally requested. The MRI classification was performed according to Nuss [16, 17]. Due to ethical reasons in children without clinical hints of joint bleedings no roentgenograms of the joints were performed. In each participating child the annual frequency of bleeding events (ABE) was recorded at comprehensive clinical visits based on review of patient infusion logs or the family report. Joint hemorrhage was defined as an acutely painful swollen joint necessitating factor replacement therapy. A target joint was defined when more than two bleeding episodes had occurred into the same joint within a three-month period [18, 19].

Laboratory Analysis With informed parental consent the G1691A polymorphism in the FV gene and the G20210A variant in the factor II gene were detected in patients with severe hemophilia A by PCR amplification [20, 21]. Activities of protein C, antithrombin, free protein S antigen and protein C antigen were measured as previously described [22]. The plasma levels of factor VIII were measured by one-stage clotting assays purchased from Behringwerke/Marburg, Germany using standard laboratory methods. Mutation analysis for hemophilia A was performed as described earlier [23].

Statistics Calculations of medians, ranges and nonparametric statistics (Mann-Whitney, Spearman rank correlation) were performed with the Stat view 5.0 programs. The significance level was set at 0.05.

Results HA Patient Population Of the 144 consecutively recruited children, 104 were suffering from severe HA. Median (range) patient follow-up was 14 years (5–24). The HA mutation spectrum was no different between carriers and non-carriers of prothrombotic risk factors (p = 0.3: Table 1). In addition, no significant differences were found with respect to

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Table 1. Spectrum of mutations in children with HA with respect to coinherited prothrombotic risk factors – Thrombophilia number [%]

+ Thrombophilia number [%]

Inversion 22 Missense Nonsense Large deletion Splice Frameshift Chromosomal abnormalities not identified so far

35 17 6 2 1 1 1 2

6 1 0 1 0 0 0 2

Total

65 [72.2]

[53.8] [26.2] [9.2] [3.1] [1.5] [1.5] [1.5] [3.1]

[60.0] [10.0] [10.0]

[20.0]

10 [71.4]

P=0.3

treatment modalities performed in both patient groups (p = 0.24): without knowledge of the thrombophilia status treatment was initiated according to the frequency of bleedings, and most patients received initially »on-demand« therapy, e.g. 50 IU/kg bw, and were switched over to prophylactic therapy 3x/week with the substitution of a median(range) dose of 40 IU/kg bw (30–60) factor VIIII concentrate, when more than two bleedings (range 2–6) had occurred into the same joint within a 12-month period. »On-demand« therapy was applied to 58 children without prothrombotic risk factors (64.4%) compared to 7 patients in the thrombophilia group (50%), and prophylactic factor VIII administration was administered in 32 subjects without additional thrombophilias compared with 7 children in carriers of the FV or FII mutation (p = 0.24).

Prevalence of Prothrombotic risk Factors in German Children with Severe HA From the entire study group 14 children carried additionally prothrombotic risk factors. The prevalence of prothrombotic risk factors in children with severe HA was no different from previously reported data [12]: FV G1691A 7.7% (8/105), FV A1691A 1.0% (1/104), FII G20201A 4.8% (5/104), once combined with protein C type I deficiency. No deficiency states of antithrombin or protein S were found in this cohort of hemophilic patients.

Annual Bleeding Episodes Carriers of the FV and FII mutations had significantly fewer median (range) ABE than non-carriers (p = 0.004): 1.3 (0–7) versus 5.5 (0–36) respectively. 65 of 104 PUP patients developed at least one target joint: the distribution within the two patients groups, however, was no different (no thrombophilia: ankle n=27, knee n=19, elbow n = 6, hip n = 1, knee & ankle n = 4; with thrombophilia: ankle n = 5, knee, elbow or hip n = 1 each [p = 0.76]). The median (range) Pettersson score performed in 56

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patients at a median (range) age of 12 (4–24) years was 1(0–12) in children without thrombophilic risk factors compared with 1(0–4) in children carrying either the FV mutation or the FII variant. The Pettersson score was clearly dependent on age (p = 0.039) and on ABE (r = 0.42; p = 0.007). The »Nuss« joint score documented in 32 subjects highly correlated with the Pettersson score (r = 0.8; p = 0.0004).

Discussion The purpose of the present study was to investigate whether the presence of prothrombotic risk factors influences the bleeding frequency in children with severe hemophilia A. In addition, in HA patients suffering clinically from joint bleedings the association between the bleeding frequency and the joint status were investigated. In addition to our previously findings, that the coinheritance of a thrombophilic gene mutation is associated with a later onset of a first symptomatic bleeding in HA patients, data of this multicenter survey give evidence that the clinical phenotype of severe HA in children is influenced by the presence of the FV and FII mutation: our results clearly demonstrate that the annual bleeding frequency in children treated since 1980 by the same treating physicians over a median time of 14 years was significantly lower in carriers of prothrombotic risk factors compared to noncarriers, thereby supporting the hypothesis of Nichols et al. [11], that the hemophilia phenotype is influenced by coinheritance with prothrombotic risk factors. The latter is also supported also by the findings of Lindvist et al. showing that women carrying the FV G1691A mutation experienced less intrapartum blood loss [24]. Our data, however, are in contrast to data reported by Arbini et al. and data shown by Lee et al. in adult patients [25, 26], showing that the proportion of severe hemophiliacs whose mild clinical course could be attributed to coinheritance with the FV G1691A mutation tended to be small. Pediatric patients with severe HA typically experience frequent bleeding episodes into joints or soft tissues [2, 3, 5], necessitating on-demand or prophylactic treatment twice or thrice weekly with individual amounts of factor VIII concentrates. The frequency of bleeding and the outcome with respect to joint damage investigated with the Pettersson score [27] has been discussed to be not only dependent on the severity of the disease, but also on the corresponding factor F VIII gene mutation, or the development of inhibitors [8]. In addition, the course of bleeding episodes is also controversially discussed with respect to individual therapeutic regimen performed by each hemophilia center [28–33]. Patients enrolled from the different pediatric hemophilia treatment centers in Germany, and due to the higher bleeding tendency not including children with inhibitor development, were treated since 1980 by the same medical teams. In similarity to the Canadian hemophilic cohort recently reported [19], treatment protocols used in the German pediatric PUP patients enrolled here have not changed with respect to treatment indications, and the criteria chosen to treat a bleeding episode »on-demand« or »on-prophylaxis« with an uniform increase of prophylactic treatment regiments since the late eighties [13, 14]. In addition, in the children reported here the mutation spectrum in HA subjects were no different in carriers and non-carriers of prothrombotic risk factors. Thus,

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since the treatment regiments were administered in the PUP patients presented here without knowledge of the individual thrombophilia status, and since the treatment regimens were no different between carriers and non-carriers of prothrombotic risk factors, evidence is given, that the thrombophilic gene mutation itself is responsible for the lower bleeding frequency associated with the better joint outcome in the children reported. The different results obtained from adults [25, 26], however, may be mainly due to different treatment modalities used before 1980 [32], e.g. the non-availability of purified factor VIII concentrates leading to a higher rate of untreated bleeding episodes in older hemophiliacs, as well as a lower rate of prophylactic administration of non-purified factor VIII preparations. In the elderly hemophilic patient, however, aging has to be discussed as an additional cause of arthropathic joint damage, which is normally not present in the young: therefore it seems to be likely, that the interaction hemophilic joint damage and age-related arthropathy will be combined in the adult patients previously reported [25, 26]. The latter, however, makes a clear differentiation difficult, if the origin of a joint arthropathy diagnosed in adult hemophiliacs is mainly based on the number of bleeding episodes, the process of aging, the interaction with prothrombotic risk factors, or a combination of multiple contributing factors respectively. In conclusion, data of the cohort study presented here suggest that the hemophilic phenotype is influenced by the presence or absence of prothrombotic risk factors, e.g. the factor V or factor II variant. However, to obtain further insight into the possible putative effect of these prothrombotic risk factors on the severity of hemorrhagic disorders, especially of severe hemophilia A, prospective large-scale studies in previously untreated hemophilic children are required. Acknowledgements. The authors thank all technicians from the participating laboratories, in particular Ursula Schulze-Horsel, Sabine Thedieck, Annette Sander and Heike Ringkamp for excellent technical assistance. In addition, we thank Gwyneth Schulz for help in editing this manuscript. This study was supported by grants from Bayer Vital AG (Leverkusen, Germany), and ZLB Behring GmbH (Hattersheim, Germany)

References 1. Hoyer LW. Hemophilia A. N Engl J Med 1994; 330: 38–47 2. Baehner RL, Strauss HS. Hemophilia in the first year of life. N Engl J Med 1966; 275: 524–528 3. Conway JH, Hilgartner MW. Initial presentations of hemophiliacs. Arch Pediatr Adolesc Med 1994; 148: 589–594 4. Ljung R, Petrini P, Nilsson IM. Diagnostic symptoms of severe and moderate haemophilia A and B. Acta Pediatr Scand 1990; 79: 196–200 5. Thacker KE, Lim T, Drew JH. Cephalhaematoma: A 10-year review. Aust NZ J Obstet Gynaecol 1987; 27: 210–212 6. Wiswell KE, Lim T, Drew JH. Risk from circumcision during the first month of life compared with those for uncircumcised boys. Pediatrics 1989; 83: 1011–1015 7. Walsh PN, Rainsford SG, Biggs R. Platelet coagulant activities and clinical severity in hemophilia. Thromb Diath Haemorrh 1973; 29: 722–729

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8. Bauer KA, Mannucci PM, Gringeri A, Tradati F, Barzegar S, Kass BL, ten Cate H, Kestin AS, Brettler DB, Rosenberg RD. Factor IXa- factor VIIIa- cell surface complex does not contribute to the basal activation of the coagulation mechanism in vivo. Blood 1992; 79: 2039–47 9. Oldenburg J, Schröder J, Schmitt C, Brackmann HH, Schwab R. Small deletion/insertion mutations within poly-A runs of the factor VIII gene mitigate the severe haemophilia A phenotype. Thromb Haemost 1998; 79: 452–453 10. Lakich D, Kazazian HH, Antonarakis SE, Gitschier J. Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A. Nature Genet 1993; 5: 236–241 11. Nichols WC, Amano K, Cacheris PM, Figueiredo MS, Michaelides K, Schwaab R, Hoyer L, Kaufman RJ, Ginsburg D. Moderation of hemophilia A phenotype by the factor V R506Q mutation. Blood 1996; 88: 1183–1187 12. Escuriola Ettingshausen C, Halimeh S, Kurnik K, Schobess R, Wermes C, Kreuz W, Pollmann H, Nowak-Göttl U. Hemophilia phenotype is dependent on the presence of prothrombotic risk factors. A multicenter cohort study. Thromb Haemost 2001; 85: 218–220 13. Kreuz W, Escuriola Ettingshausen C, Funk M, Pons S, Schmidt H, Kornhuber B. Prevention of joint damage in hemophilic children with early prophylaxis. Orthopäde 1999;28:341–346 14. Ljung R. Paediatric care of the child with haemophilia. Haemophilia 2002;8:178–182 15. Pettersson H, Nilsson IM, Hedner U, Norehn K, Ahlberg A. Radiologic evaluation of prophylaxis in severe haemophilia. Acta Paed Scand 1981;70:565–570 16. Nuss R, Kilcoyne RF, Geraghty S, Shroyer ALW, Rosky JW, Mawhinney S, Wiedel J, MancoJohnson M. MRI findings in haemophilic joints treated with radiosynoviorthesis with development of an MRI scale of joint damage. Haemophilia 2000;6:162–169 17. Nuss R, Kilcoyne RF. The MRI atlas of hemophilic arthropathy. Professional publishing group, Ltd, New York, 2002 18. Blanchette VS, McCready M, Achonu C, Abdolell M, Rivard G, Manco-Johnson MJ. A survey of factor prophylaxis in boys with haemophilia followed in North American haemophilia treatment centres. Haemophilia 2003;9(suppl 1):19–26 19. Kern M, Blanchette V, Stain AM, Einarson TR, Feldman BM. Clinical and cost implications of target joints in Canadian boys with severe hemophilia. J Pediat 2004;145:628–634 20. Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velde PA, Reitsma PH. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64–67 21. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3‘untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996; 88: 3698–3703 22. Junker R, Koch HG, Auberger K, Münchow N, Ehrenforth S, Nowak-Göttl U. Prothrombin G20210A gene mutation and further prothrombotic risk factors in childhood thrombophilia. Arterioscler Thromb Vasc Biol 1999; 19: 2568–2572 23. Bogdanova N, Markoff A, Pollmann H, Nowak-Göttl U, Eisert R, Dworniczak B, Eigel A, Horst J. Prevalence of small rearrangements in the factor VIII gene F8C among patients with severe hemophilia A. Hum Mutat. 2002 ;20:236–237. 24. Lindvist PG, Svensson PJ, Dahlbäck B, Marsal K. Factor V Q506 mutation (activated protein C resistance) associated with reduced intrapartum blood loss – a possible evolutionary selection mechanism. Thromb Haemost 1998; 79: 69–73 25. Arbini AA, Mannucci PM, Bauer KA. Low prevalence of the factor V Leiden mutation among ”severe” hemophiliacs with a ”milder” bleeding diathesis. Thromb Haemost 1995; 74: 1255–1258 26. Lee DH, Walker IR, Teitel J, Poon MC, Ritchie B, Akabutu J, Sinclair GD, Pai M, Wu JWY, Reddy S, Carter C, Growe G, Lillicrap D, Lam M, Blajchman MA. Effect of the factor V Leiden mutation on the clinical expression of severe hemophilia A. Thromb Haemost 2000; 83: 387–389 27. Fischer K, van Hout BA, van der Bom JG, Grobbee DE, van den Berg HM. Association between joint bleeds and Pettersson scores in severe haemophilia. Acta Radiologica 2002;43:528–532

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28. Ludlam CA. Treatment of haemophilia. Br J Haematol 1998; 101: S13–S14 29. Van den Berg HM, Fischer K, Mauser-Bunschoten EP, Beek FJA, Roosendaal G, van der Bom JG, Nieuwenhuis HK. Long-term outcome in individualized prophylactic treatment of children with severe haemophilia. Br. J Haematol 2001;112:561–565 30. Fischer K, Astermark J, van der Bom JG, Ljung R, Berntorp E, Grobbee DE, van den Berg HM. Prophylactic treatment for severe haemophilia: comparison of an intermediate-dose to a high-dose regimen. Haemophilia 2002;8:753–760 31. Carlsson KS, Höjgard S, Glomstein A, Lethagen S, Schulman S, Tengborn L, Lindgren A, Berntorp E, Lindgren B. On-demand vs. prophylactic treatment for severe haemophilia in Norway and Sweden: differences in treatment characteristics and outcome. Haemophilia 2003;9:555–566 32. Fischer K, van der Bom JG, Mauser-Bunschoten EP, Roosendaal G, Prejs R, Grobbee DE, van den Berg HM. Changes in treatment strategies for severe haemophilia over the last 3 decades: effects on clotting factor consumption and arthropathy. Haemophilia 2001;7:446– 452 33. Van den Berg HM, Fischer K. Prophylaxis for severe hemophilia: experience from Europe and the United States. Seem Thromb Haemost 2003;29:49-54

Continuous Infusion of Factor Concentrates in Children with Hemophilia A in Comparison to Bolus Injections C. Bidlingmaier, M. Deml, and K. Kurnik

Background The method of continuous infusion of factor concentrates is well known since the 1950s [1], and numerous studies have evaluated the efficacy and safety of this form of factor concentrate administration. In 1975 Hermes calculated a possible reduction in factor usage of approximately 30% compared to bolus injections every 12 hours [5], which matched with the first clinical experiences published by Hathaway [3]. This is thought to be the result of the more stable factor VIII (FVIII) through levels without high peaks and a decrease in the clearance rate of factor VIII during the first three postoperative days [6]. In 1994, Schulman and coworkers showed the stability of resolved factor VIII over several days [9], and several studies found that factor VIII concentrates are not prone to bacterial contamination [10]. Side effects appear to be rare and the most often reported occurrence of thrombophlebitis can be avoided by parallel saline infusion [4]. Some studies suggest that continuous infusion (CI) might trigger the development of inhibitors especially in patients with mild hemophilia [7, 11], but the data are still inconclusive. However, prospective data in children are rare [8] and CI does not form the standard in the perioperative care of hemophilic children in Germany despite the urgent need to cut costs in hemophilia therapy. We present a prospective open-labeled non-randomized study focusing on safety, effectiveness and factor VIII (FVIII) usage compared to bolus injections (BI) in children.

Patients and Methods The study was approved by the local ethics committee. Written informed consent was obtained by all parents and older children. Mann-Whitney-U-tests were performed for statistical analysis. 43 consecutive patients (0.5–17 years of age; median: 9.6 years, for patient characteristics see Table 1) were treated with CI between 1997 and 2003 for different procedures. These included 8 small procedures (i.e. extraction of milk teeth), 24 median procedures (i.e. synovectomy) and 11 major procedures, such as abdominal surgery (see Table 2). The median dose of the bolus to start CI was 69 U/kg (range 40–167 U/kg). The dose was chosen by the treating physician according to the severity of bleeding, the type of hemophilia and the weight of the child, with heavier children tending to need less FVIII concentrate compared to younger children. The rate of infusion was I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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Table 1. Patient characteristics of all consecutive children treated with continuous infusion of FVIII concentrates from 1997 to 2002 (n=43) Age

mean range

9 y 1 mo 8 mo – 17 y

Hemophilia A

mild moderate severe

4 1 38

Genotype

Intron 22 Missense Stop Other Unclear Not tested

9 (20.9 %) 9 (20.9 %) 5 (11.6 %) 2 (4.6 %, 1 Splice, 1 Intron 1) 3 (6.9 %) 15 (34.8 %)

Additional disorders of coagulation

Lupus anticoagulant Past FVIII inhibitor

7 1

Thrombophilia

Prothrombin Mutation het. Factor V Leiden het. Elevated lipoprotein (a) MTHFR homozygous Negative Not tested

7 (16.3 %) 4 (9.3 %) 7 (16.3 %) 2 (4.6 %) 20 (46.5 %) 3 (7.0 %)

FVIII concentrate

Plasma derived Recombinant Switched during CI to Recombinant due to allergic reaction (exanthema)

28 (65.1 %) 14 (32.6 %) 1 (2.3 %)

adapted to the clinical situation and daily factor-VIII-levels. The factor-VIII-levels were kept for surgery above 80% for the first 2 days and then above 50% for the rest of continuous infusion if not dictated higher by the clinical situation. In patients suffering from major bleeding, the levels were kept above 80% until the clinical situation improved. Factor concentrates were diluted in the minimum volume of saline possible. CI was performed until discharge from the hospital and in some cases followed by bolus injections at home. Prior to the study all patients were routinely screened for hematocrit, thrombocyte-count, platelet function (PFA100), liver function, additional Willebranddisease, factor-VIII-inhibitor and lupus-anticoagulant. Routine laboratory tests were used for factor-level (%) and inhibitor (Bethesda-Units) determination every day. Lupus anticoagulant was detected by using lupus-sensitive and lupus-insensitive tests for factor-levels and aPTT. From this study 12 procedure- and age/weight-matched patients having received CI were compared to the next 12 consecutive patients undergoing surgery with bolusinjections of factor concentrates between 2003 and 2004 directly after the CI-study.

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Table 2. Definitions of procedure groups for patients undergoing continuous infusion (n=43) Small procedures (n=8) Factor Use [mean (+/- SEM)] IU/kg/d Total IU/kg Medium procedures (n=24)

Factor Use [mean (+/- SEM)] IU/kg/d Total IU/kg Major procedures (n=11) Factor Use [mean (+/- SEM)] IU/kg/d Total IU/kg

extraction of (milk-)teeth (3), circumcision (3), minor trauma (2) 86.9 (+/- 5.6) 465.7 (+/- 75.9) porth implantation (3), minor abdominal surgery (i.e. appendicitis) (6), synovectomy (11), ENT surgery (4) 110.6 (+/- 13.8) 784.7 (+/- 115.2) major abdominal surgery (i.e. ileus ) (4), abdominal bleeding (4), CNS surgery (3) 109.6 (+/- 13.8) 1057.8 (+/- 209.5)

Results Continuous Infusion Study Safety No direct serious adverse events did occur, but 2/4 patients below 20 exposure days (ED) developed a high-responding inhibitor shortly after CI. Both patients suffered from severe hemophilia. Two patients showed mild thrombophlebitis or rash. 8/43 children needed additional boli, mainly children suffering from serious bleeds or undergoing major surgery (see Table 3). Infusion Rates The rate of infusion was adapted to the clinical situation and the factor-VIIIlevels, starting with a median initial infusion rate of 4.36 U/kg/h (range 2.8 – 9.5 U/kg/h). The highest rates used at any time during continuous infusion were 10.4 U/kg/h, the lowest 1.3 U/kg/h. The severity of hemophilia and the presence of additional disorders of coagulation did not alter the rate of infusion. Product type, i.e. plasma derived or recombinant factor concentrates, did not influence the infusion rate. The infusion rate needed to reach adequate factor-VIII-levels was highly predictable (for reached plasma levels see Fig. 1). Constant reduction was possible during the first three days of treatment, resulting from decreasing clearance especially in older children (see Fig. 2). This was hampered in part by the

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150



140 130

major medium



small

Cell Mean

120 110









100



90



80



70

5

6

FVIII d7

4

FVIII d6

3

FVIII d5

2

FVIII d4

1

FVIII d3

FVIII d2

Bolus

FVIII d1

50

F VIII d0

60 7 [day]

Fig. 1. Reached FVIII-levels [%] for the different procedures

9 8



+ ADC/ Bleeding



- ADC/ Bleeding



[ml/kg/h]

7 6









5







4

 

3 1

2

3

4

5 [day]

Fig. 2. FVIII clearance is influenced by the presence of additional disorders of coagulation (ADC) and/or bleeding

additional presence of a lupus-anticoagulant or bleeding (see Fig. 3). Intra- and postoperative hemostasis was considered very good by all treating surgeons and physicians (see Table 3). Factor Usage Therapy duration and FVIII usage of CI were primarily influenced by the procedure (see Table 2) and tended to be shorter compared to bolus injections (n.s.). The presence of additional disorders of coagulation, especially lupus anticoagulant may have led to false low FVIII-levels in the laboratory. The product used or presence of thrombophilia did not influence the factor usage.

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6 5,5

[ml/kg/h]

5 

< 10 yrs > 10 yrs

 

4,5

*



4 Fig. 3. FVIII clearance is influenced by the patient’s age (patients with ADC excluded)



 



 

3,5

 

3 1

2

3

4

5 [day]

Table 3. Comparison of the characteristics of 12 consecutive patients treated with bolus injections and the 12 matched patients treated before with continuous infusion. Patients were matched for age, severity of hemophilia and procedure but not for genetic background of hemophilia or thrombophilia. Treatment time was adjusted for both groups comparing only the factor usage for the time of continuous infusion before starting bolus injections for the rest of the treatment period with the equivalent number of days in the bolus-injections-group Continuous Infusion

Bolus Injections

Statistical Analysis

* p = 0.02 * p = 0.01 n.d.

FVIII usage [mean (range)] IU/kg/d

all (12) medium (9) small (3)

100.0 100.9 (64 – 160) 97.4 (80 – 113)

141.6 153.9 (119 – 183) 104.7 (78 – 153)

Total IU/kg

all medium small

563.2 556.2 (257 – 800) 584.0 (391 – 796)

812.9 * p = 0.006 861.7 (622 – 1250) * p = 0.009 666.6 (552 – 833) n.d.

Intraoperative hemostasis

excellent

excellent

equal

Concomitant therapy

0/12

0/12

equal

Red blood transfusion

0/12

0/12

equal

Additional boli needed (CI)/change of planned treatment (Bolus)

0/12

0/12

equal

Comparison to Bolus Injections When compared to bolus injections continuous infusion did not differ in respect to safety (apart from inhibitor development in children below 20 ED) and efficacy. CI saved 30% FVIII (p < 0.006, see Fig. 4 and Table 3). This surpasses the calculated reduction of 25% (mean).

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1400 * p = 0.009

1200

Total IU/kg

1000 80 60 40 20 Bolus Injections

Continuous Infusion

Fig. 4. Reduction in factor usage by continuous infusion compared to bolus injections (n = 12 / each)

Conclusion CI has become a widely used method to administer FVIII concentrates in the perioperative period. As shown in a review by Schulman, many concerns regarding safety, stability and efficacy were addressed during the 1990s [8]. However, data on CI in the pediatric population are rare. We therefore aimed to monitor prospectively our experience with CI in our hospital and to compare the results with subsequently treated patients by bolus injections. Our results enhance that CI forms an easy, safe and effective method for perioperative care in children. No problems occurred from the continuous infusion via peripheral access, only 1 patient developed mild thrombophlebitis despite the recommended parallel infusion of saline. 1 patient developed a rush that persisted promptly after switching to another concentrate. Intraoperative hemostasis was excellent in all cases, whereas some of the children undergoing major surgery needed additional boli in the postoperative period. It is noteworthy that one patient bled after the unintended use of an additional filter, resulting in low FVIII levels. The bleeding resolved after the removal of the filter, which stresses the need for strict instructions. Adequate FVIII levels were easily reached and highly predictable. However, during the study period we aimed at rather high FVIII levels which might not be necessary in all patients. Some earlier studies have addressed the problem of inhibitor development after CI. In a recent publication Mulcahy et al. describe two out of 12 patients developing an inhibitor in timely coincidence with CI, both suffering from mild hemophilia [7]. One of these adults developed the inhibitor at the 23rd ED. In our group 2 out of 4 children below 20 ED, both suffering from severe hemophilia developed an inhibitor directly after or during CI. Whereas we can certainly not prove the direct connection between CI and inhibitor development it seems very likely and raises concerns in using CI in children below 20 ED. Our group consisted of only 4 mild

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hemophiliacs, therefore no statement is possible about the discussed even higher risk for these patients [11]. Although the results of our study are limited by the non-randomized design, strictly consecutive inclusion of patients and the well-matched control group let us support the strong potential of CI to save up to 30% FVIII concentrate. These savings might even be emphasized by earlier discharge from hospital, as reported by Campbell [2]. We have also noted a trend towards earlier discharge of our patients after CI (not statistical significant). And, as the minimum FVIII level during CI to maintain excellent hemostasis is unclear, future studies will have to focus on dosage regimen and concomitant therapies to further decrease FVIII usage. Especially false-low laboratory values of FVIII in children with additional lupusanticoagulant might need special attention, although our study was hampered be an unusual high rate of patients positive for lupus-anticoagulant (7/43). In conclusion we found CI to be a save and effective method of FVIII administration in the perioperative period. However, two of our children developed an inhibitor. The costs of the (successful) inhibitor therapy in these two patients outweigh by far the reduction in the overall treatment costs of the entire study. Therefore we cannot recommend continuous infusion for children below 20 exposure days.

References 1. Brinkhous KM (1954) Hemophilia. Bull N Y Acad Med 30: 325 2. Campbell PJ, Rickard KA (1998) Continuous and intermittent infusion of coagulation factor concentrates in patients undergoing surgery: a single centre Australian experience. Aust N Z J Med 28 (4): 440–5 3. Hathaway WE, Christian MJ, Clarke SL, Hasiba U (1984) Comparison of continuous and intermittent Factor VIII concentrate therapy in hemophilia A. Am J Hematol 17 (1): 85–8 4. Hay CR, Doughty HI, Savidge GF (1996) Continuous infusion of factor VIII for surgery and major bleeding. Blood Coagul Fibrinolysis 7 Suppl 1: S15–9 5. Hermens WTH. Dose calculation of human factor VIII and factor IX concentrates for infusion therapy. In: Brinkhous KM, Hemker HC, editors. Handbook of Hemophilia. New York: American Elsevier Publishing; 1975. p. 569–589. 6. Martinowitz U, Schulman S, Gitel S, Horozowski H, Heim M, Varon D (1992) Adjusted dose continuous infusion of factor VIII in patients with haemophilia A. Br J Haematol 82 (4): 729–34 7. Mulcahy R, Walsh M, Scully MF (2005) Retrospective audit of a continuous infusion protocol for haemophilia A at a single haemophilia treatment centre. Haemophilia 11: 208– 215 8. Schulman S (2003) Continuous infusion. Haemophilia 9 (4): 368–75 9. Schulman S, Gitel S, Martinowitz U (1994) Stability of factor VIII concentrates after reconstitution. Am J Hematol 45 (3): 217–23 10. Schulman S, Varon D, Keller N, Gitel S, Martinowitz U (1994) Monoclonal purified FVIII for continuous infusion: stability, microbiological safety and clinical experience. Thromb Haemost 72 (3): 403–7 11. Sharathkumar A, Lillicrap D, Blanchette VS, Kern M, Leggo J, Stain AM, Brooker L, Carcao MD (2003) Intensive exposure to factor VIII is a risk factor for inhibitor development in mild hemophilia A. J Thromb Haemost 1 (6): 1228–36

Decreased Clotting Factor Activity (VIII, IX, XI, and XII) due to Inhibitors with Lupus-like Activity in Childhood V. Aumann, L. Wiens, G. Lutze, and U. Mittler

Introduction Increased activated partial thromboplastin time (aPTT) is a common finding in children and adults. Main reasons are: 1. reduced activity of factors of the intrinsic coagulation system 2. the presence of heparin or hirudin 3. the presence of fibrin-fibrinogen degradation products 4. the presence of pathological inhibitors – Factor VIII (IX)-inhibitors – Lupus anticoagulants The presented study reports prolonged activated partial thromboplastin time (aPTT) in 25 children aged 2 to 14 years. APTT was controlled for various reasons, mostly before planned surgery. In order to confirm the prolonged aPTT these children were referred to our center from different outpatient departments.

Materials and Methods APTT was measured by lupus insensitive STA APTT Kaolin reagent (Roche) and lupus sensitive Platelin LS reagent (Biomérieux). The inhibitor activity was determined with plasma mixing studies using aPTT reagent Platelin LS. Three different tests were performed as confirmatory tests for the lupus anticoagulants: 1. Lupus Anticoagulant Test, Technoclone; 2. PTT-LA, Instrumentation Laboratory; 3. Staclot LA, Stago. The concentration of anticardiolipin antibodies (ACA) was determined with ELISA (Imtec). The activity of clotting factors VIII, IX, XI and XII was measured in plasma without dilution (1:1) and in two plasma dilutions (1:2 and 1:4).

Results A priori, heparin, hirudin, fibrin/fibrinogen split products and factor VIII (IX) inhibitors were excluded as reason for aPTT prolongation by clinical and coagulation tests. I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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80 70

sec

60 50 40 Fig. 1. Determination of aPTT1 using STA APTT Kaolin (lupus insensitive) and aPTT2 using Platelin LS (lupus sensitive) in 25 children aged 2-14 years

30 20

aPTT1

120

120

100

100

80

80

sec

140

sec

140

60

60

40

40

20

20

0

aPTT2

0 0

20 40 60 80 100 Norm al plasm a (%)

0

20 40 60 80 100 Norm al plasm a (%)

determination without incubation

determination without incubation

determination after incubation for one hour

determination after incubation for one hour

Fig. 2. Inhibitor test in plasma mixing studies carried out on two patients with different aPTT prolongation (Platelin LS)

Table 1. Results for LA confirmatory tests in 25 children A Lupus Anticoagulant Test (Technoclone)

B PTTLA (IL)

C Staclot LA (Stago)

Frequency (n)

pos neg neg neg neg

pos pos neg pos neg

pos pos pos neg neg

1 8 3 3 10

Platelin LS (sec)

LA A

LA B

LA C

ACA

VIII (%) 1:1/1:2/1:4

IX (%) 1:1/1:2/1:4

XI (%) 1:1/1:2/1:4

XII (%) 1:1/1:2/1:4

1

54,4

72,5

pos

pos

pos

normal

29/48/56

26/48/60

28/44/52

23/50/76

2

36,3

59,7

neg

pos

pos

normal

101/110/96

58/64/60

89/102/128

88/98/108

3

39,3

54,9

neg

pos

pos

normal

53/58/56

50/52/52

72/78/76

63/60/68

4

37,6

55,2

neg

pos

neg

normal

84/80/84

49/52/52

78/84/72

17/14/16

5

36,6

44,4

neg

neg

neg

normal

66/72/80

47/62/68

73/78/84

58/64/70

6

37,8

51,3

neg

neg

neg

normal

54/58/56

51/52/48

64/68/66

45/46/48

7

39,1

50,6

neg

neg

neg

raised

60/56/60

49/50/52

62/68/60

57/60/60

8

34,1

46,3

neg

neg

neg

normal

76/78/80

50/56/52

68/64/68

27/32/32

bold: inactivating inhibitor effects bold italic: interfering inhibitor effects

V. Aumann et al.

STA APTT (sec)

104

Table 2. Examples for corrected factor activities in three plasma dilutions in 8 children with positive and negative LA confirmatory tests

Decreased Clotting Factor Activity (VIII, IX, XI, and XII) due to Inhibitors

105

Results for aPTT determinations with lupus insensitive STA APTT Kaolin reagent (aPTT1) and lupus sensitive Platelin LS reagent (aPTT2) are shown in Figure 1. There was a significant time difference between the results of the two measurements (p < 0.001). Figure 2 shows the results of inhibitor testing carried out in a plasma mixing study in two patients with different aPTT prolongation. Testing was done immediately and after a one hour incubation period at 37° C. The tests confirmed the presence of inhibitors (immediate type). Frequencies of positive and negative LA confirmatory tests are presented in Table 1. LA confirmatory tests B and C were positive in 15 children. In 9 of these 15 children both tests were positive (in one case additionally with a positive confirmatory test A) and in 3 at a time as a single test. In 10 children all tests (A, B and C) were negative. In 3 children ACA were increased (in two children with positive and in one child with negative confirmatory tests). The four investigated clotting factors showed different activities and demonstrated a wide range between the investigated subjects (Table 2). In plasma of 25 children with positive inhibitory activity in plasma mixing studies normal or decreased activities of the single factors alone or in different combinations were measured in undiluted plasma samples. Usually factor XII activity was involved. In experiments with stepwise diluted plasma samples which consequently have decreased clotting activity, either increased clotting activities with regard to interfering inhibitor effects or nearly equal activities with regard to inactivating inhibitor effects were found.

Conclusions The lupus sensitive Platelin LS reagent showed prolonged aPTT in 25 investigated plasmas compared to the lupus insensitive STA APTT Kaolin reagent. In all investigated plasmas the inhibitor evidence with the plasma mixing study was positive according to immediate inhibition. With selected LA confirmatory tests 15 plasmas with different sensitivity were recognized as LA positive and 10 plasmas were not confirmed as LA positive. It can not be excluded, that other LA confirmatory tests will show positive results. The »lupus sensitive« Platelin LS reagent should be described as »inhibitor sensitive«. The inhibitor effect demonstrated a wide range according to the activities of the single clotting factors and had an interfering as well as an inactivating effect. The patients did not show clinical features of thromboembolic incidents. Despite decreased activities of single factors, increased bleeding was not found. Observations about clinical symptoms and clotting analyses are in progress.

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V. Aumann et al.

References 1. Barthels M, von Depka M. Das Gerinnungskompendium. Stuttgart/New York: Thieme 2003 2. Lutze G, Naumann Ch, Zawta B. Useful facts about coagulation. Mannheim: Roche Diagnostics 2005 3. Poetzsch B, Madlener K. Gerinnungskonsil. Stuttgart/New York: Thieme 2002

VI. Free Lectures

Effect of Activated Recombinant Coagulation Factor VII on the Function of Glycoprotein IIb/IIIa-Inhibited Platelets in Vitro M. Udvardy

Abstract Background Platelet glycoprotein (GP) IIb/IIIa antagonists have been increasingly used in acute coronary artery syndrome to prevent myocardial infarction. The major drawback is that occasionally they may cause severe bleeding complication. The administration of GPIIb/IIIa antagonists induces a condition closely resemble to Glanzmann’s thrombasthenia, an inherited abnormality of GPIIb/IIIa. Activated recombinant coagulation factor VII (rFVIIa) is currently approved for the treatment or prevention of bleeding in patients with Glanzmann’s thrombasthenia. We investigated the effect of rFVIIa on platelet function in the presence of GPIIb/IIIa antagonists. Materials and Methods Abciximab or Eptifibatide was added to normal whole blood samples in the absence or presence of thrombin inhibitor (heparin, hirudin, or PPACK). The effect of rFVIIa at therapeutic concentrations (0.35–7 nmol/l) on GPIIb/IIIa-inhibited platelets was assessed using platelet aggregometry and platelet function analyzer (PFA100). In addition, the effect of rFVIIa on thrombocytopenic samples in the presence of Abciximab was also studied. Results rFVIIa at concentrations used in the study completely corrected the closure times of the samples with GPIIb/IIIa-inhibited platelets, while only a partial correction could have been observed with aggregometry. This effect was also evident in the presence of thrombin inhibitors or thrombocytopenia. Conclusions rFVIIa completely restores the function of GPIIb/IIIa-inhibited platelets in vitro as assessed by PFA-100 experiments. Further studies are needed to correlate this in vitro finding with in vivo conditions. I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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Introduction Platelet glycoprotein (GP) IIb/IIIa is an adhesion receptor which mediates platelet aggregation. GPIIb/IIIa antagonists have been increasingly used in acute coronary artery syndrome to prevent myocardial infarction. The two major groups of GPIIb/IIIa antagonists are a monoclonal antibody – Abciximab; and synthetic compounds that competitively bind to GPIIb/IIIa – like Eptifibatide and Tirofiban [1, 2]. The most common complication caused by GPIIb/IIIa antagonists is bleeding, occasionally in association with thrombocytopenia. The bleeding can be severe and difficult to control. Furthermore, there is no specific antidote to GPIIb/IIIa antagonists, and platelet transfusion is usually required to control severe bleeding [3]. Recombinant coagulation factor VIIa (rFVIIa; NovoSeven, Novo Nordisk A/S, Bagsvaerd, Denmark) is currently approved for the treatment or prevention of bleeding in congenital hemophilia with inhibitor to factor VIII or IX, acquired hemophilia, congenital FVII deficiency, and Glanzmann’s thrombasthenia. Anecdotal data also show that rFVIIa is hemostatically effective in several other clinical conditions associated with bleedings including thrombocytopenia [4–8]. Furthermore, case reports reveal that rFVIIa provides effective hemostasis in patients with heparinand Tirofiban-induced bleedings as well [9]. The effects of rFVIIa on GPIIb/IIIa-inhibited platelets are not clearly elucidated and understood. We studied the effect of rFVIIa on the function of GP IIb/IIIa-inhibited platelets using aggregometry and platelet function analyzer (PFA-100). Two GPIIb/IIIa antagonists were studied: Abciximab and Eptifibatide. Some cases with thrombocytopenia were also included. As heparin or hirudin is usually used concomitantly with GPIIb/IIIa antagonists in patients with coronary interventions, experiments were run in the presence of heparin or hirudin. Moreover, thrombocytopenic samples were also studied.

Materials and Methods Whole Blood Samples Citrated whole blood samples were obtained from six healthy, non-smoker volunteers, known to be free from any medical disorders, or medications for the last four weeks. In addition, samples were also taken from three patients with chronic idiopathic thrombocytopenic purpura (ITP) who had been treated with corticosteroid 1 mg/kg.

GPIIb/IIIa Antagonists Serial dilutions of Eptifibatide (Integrilin, Schering-Plough Corporation, New Jersey, USA) and Abciximab (ReoPro, Eli Lilly, Indianapolis, USA) were carried out in buffered saline to achieve the therapeutic final concentrations of 3 and 15 µg/ml [2, 10].

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rFVIIa To imitate therapeutic concentrations, rFVIIa was dissolved in the solvent provided by the manufacturer and further diluted in buffered saline to obtain final concentrations of 0.35, 1.75, 3.5 and 7.0 nmol/l [11–14].

Analysis of Platelet Function Platelet Aggregation A single channel ChronoLog aggregometer was used for aggregation studies. Platelet rich and platelet poor plasmas (PRP and PPP) were gained by centrifugation from citrated (1:9) venous blood. Measurements with washed platelets [15] were also done. The following activating agents were used: ADP (Sigma, Saint Louis, Missouri, US): 2 mg/ml (5 mM/l final concentration), thrombin (Organon, 0.15–0.3 mg/ml final concentration), epinephrine (Reanal, Hungary, Budapest, 0.05 µg/ml final concentration), collagen (Helena, 2.5–5 µg/ml final concentration) and arachidonic acid (Sigma, Saint Louis, Missouri, USA, dissolved in distilled water, final concentration 165 µg/ml). Platelet function was also measured using a platelet function analyzer (PFA-100, Dade Behring, Marburg, Germany), and following the manufacturer’s instruction. In brief, 800 µl of citrated venous whole blood from normal volunteers were filled into a cartridge coated with collagen/epinephrine, and collagen/ADP. The closure times were recorded. For each whole blood sample, four different combinations were tested: whole blood (+buffer, control), whole blood + rFVIIa, whole blood + Abciximab or Eptifibatide, and whole blood + Abciximab or Eptifibatide + rFVIIa. Moreover, experiments were also carried out with the presence of LMWH (Dalteparin, Fragmin, Pharmacia, Uppsala, Sweden) at a final concentration of 1.25 U/ml, or hirudin (Sigma, Missouri, USA) at a final concentration of 20 µg/ml, or a selective thrombin inhibitor (d-phe-pro-arg-chloromethylketone, PPACK, Calbiochem, Darmstadt, Germany) at a final concentration of 10 µmol/ml.

Thrombin Generation A kinetic chromogenic assay was employed. The principle and details of the assay were previously published [16]. In brief, defibrinated platelet-rich plasma was mixed with Abciximab or Eptifibatide in the presence or absence of rFVIIa. The amount of thrombin generated was measured using a chromogenic substrate S2238 (Chromogenix, Milan, Italy).

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Table 1. The effect of rFVIIa on platelet aggregatory responses of GPIIb/IIIa inhibited platelets

Control rFVIIa Integrilin Integrilin + rFVIIa ReoPro ReoPro + rFVIIa

Collagen response, %

Thrombin response, %

Arachidonic acid response, %

ADP response, %

Epinephrine response, %

86% 54% 4% 1%

88% 42% 0% 0%

83% 83% 0% 0%

83% 75% 0% 0%

75% 81% 0% 0%

5% 6%

13% 3%

6% 0%

0% 5%

5% 3%

Results Platelet Aggregation Platelet aggregatory responses were assessed in a standard way, i.e. the plateau of the individual curves was served as a basis to qualify the responses in percent, and the results are summarized in Table 1. Dilutions of Integrilin (3.15 µg/ml) to 2–50 fold were also checked, however, there was no improvement of aggregation if rFVIIa was also present. The same sort of experiments were carried out with ReoPro dilutions (3 µg/ml up to 50 fold), but there were no aggregatory improvements achieved by NovoSeven in these settings. A more sensitized method of thrombin induced platelet aggregation has also been utilized. In an effort to avoid or reduce the disturbing effect of fibrin clots (occurring during thrombin aggregation) platelet rich plasmas were defibrinated by reptilase (0.1 U/1000 µl PRP). After that defibrinated PRP was mixed with equal aliquots of the identical native PRP (so fibrinogen concentration was on 50% level). The reduced fibrinogen PRP was activated with a low concentration (0.15 mg/ml) of thrombin: in the presence of buffer, ReoPro and/or NovoSeven. In these settings ReoPro also fully blocked thrombin aggregation. However, if 2 nmol/l NovoSeven was also present an aggregatory response of 35% had been documented. If NovoSeven concentration was higher (5 nmol/l) a 45 % response was detected in a quite short time course (2 min).

Results with PFA-100 Results are shown as mean ± SD (Fig. 1). Both Abciximab and Eptifibatide substantially prolonged the closure times. Recombinant FVIIa at all concentrations used in the experiment completely corrected the prolonged closure times to normal levels. Furthermore, the effect of rFVIIa persisted in the presence of hirudin, LMWH, or PPACK (Table 2).

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350 300 collagen/ e pinephr in e

tim e (sec)

250

collagen/ A D P

200 150 100 50 0 abciximab 3 mcg/ml+rFVIIa 7.0 nmol/l

abciximab 3 mcg/ml+rFVIIa 3.5 nmol/l

abciximab 3 mcg/ml+rFVIIa 1,75 nmol/l

abciximab 3 mcg/ml+rFVIIa 0.7 nmol/l

abciximab 3 mcg/ml+rFVIIa 0.35 nmol/l

abciximab 15 mcg/ml

abciximab 3 mcg/ml

rFVIIa 0.35 nmol/l

control

Fig. 1. The effect of rFVIIa on platelet function in the presence of Abciximab measured by the closure times of platelet function analyzer. N=6, results are expressed as mean ± SD Table 2. The effect of rFVIIa on the function of GPIIb/IIIa-inhibited platelets in the presence thrombin inhibitors measured by the closure times of platelet function analyzer. Results are expressed as mean ± SD N=6

Collagen/epinephrine (seconds)

Collagen/ADP (seconds)

Whole blood (control) Whole blood + Hirudin Whole blood + rFVIIa Whole blood + Abciximab Whole blood + Abciximab + rFVIIa Whole blood + Eptifibatide Whole blood + Eptifibatide + rFVIIa

106 ± 11 116 ± 10 109 ± 8 298 ± 3 116 ± 9 289 ± 8 113 ± 9

81 ± 5 86 ± 4 85 ± 7 296 ± 4 84 ± 7 287 ± 6 86 ± 11

Whole blood + LMWH Whole blood + rFVIIa Whole blood + Abciximab Whole blood + Abciximab + rFVIIa Whole blood + Eptifibatide Whole blood + Eptifibatide + rFVIIa

109 ± 7 98 ± 6 298 ± 2 105 ± 7 288 ± 12 113 ± 8

83 ± 9 89 ± 7 296 ± 4 93 ± 9 296 ± 5 90 ± 9

Whole blood + PPACK Whole blood + rFVIIa Whole blood + Abciximab Whole blood + Abciximab + rFVIIa Whole blood + Eptifibatide Whole blood + Eptifibatide + rFVIIa

99 ± 5 98 ± 7 299 ± 3 97 ± 8 291 ± 13 98 ± 8

81 ± 9 84 ± 8 296 ± 5 83 ± 7 296 ± 6 82 ± 6

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300

250

c o lla g e n / e p in e p h r in e c o lla g e n / A D P

time (sec)

200

150

100

50

0 eptifibatide 3 mcg/ml+rFVIIa 7 nmol/l

0 .4 0

eptifibatide 3 mcg/ml+rFVIIa 3.5 nmol/l

eptifibatide 3 mcg/ml+rFVIIa 1.75 nmol/l

eptifibatide 3 mcg/ml+rFVIIa 0.7 nmol/l

eptifibatide 3 mcg/ml+rFVIIa 0.35 nmol/l

eptifibatide 15 mcg/ml

eptifibatide 3 mcg/ml

Fig. 1b

PRP

0 .3 5 PRP + rFVIIa

O D405 4 0 5 nnm m 0D

0 .3 0

PRP + eptifibatide

0 .2 5

PRP + eptifibatide + rFVIIa

0 .2 0 0 .1 5 0 .1 0 0 .0 5 0 .0 0 0

5

10

15

20

time (min) time (min)

25

30

Fig. 2. The effect of rFVIIa on platelet function in the presence of Eptifibatide measured by the closure times of platelet function analyzer. N=6, results are expressed as mean ± SD

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Table 3. The effect of rFVIIa on the function of platelets from patients with idiopathic thrombocytopenic purpura in the presence of GPII/IIIa inhibitor measured by the closure times of platelet function analyzer. Each assay was repeated four times. Results are expressed as mean ± SD Collagen/epinephrine (seconds)

Collagen/ADP (seconds)

Platelet count 60000/µl Whole blood Whole blood + rFVIIa 0.35 nmol/l Whole blood + Abciximab 3 µg/ml Whole blood + Abciximab ± rFVIIa

151 ± 11 111 ± 7 299 ± 12 164 ± 14

86 ± 86 ± 295 ± 88 +

Platelet count 25000/µl Whole blood Whole blood + rFVIIa 0.35 nmol/l Whole blood + Abciximab 3 µg/ml Whole blood + Abciximab + rFVIIa

288 ± 4 221 ± 11 298 ± 4 154 ± 9

282 ± 5 252 ± 8 294 ± 6 123 ± 10

Platelet count 10000/µl Whole blood Whole blood + rFVIIa 0.35 nmol/l Whole blood + Abciximab 3 µg/ml Whole blood + Abciximab + rFVIIa

278 ± 21 198 ± 14 299 ± 2 211 ± 21

289 ± 18 196 ± 13 297 ± 6 262 ± 12

3 4 8 9

Using samples from patients with ITP, we found that at platelet counts of 60,000 and 25,000/µl and in the presence of Abciximab, rFVIIa substantially shortened the closure times compared with the same samples without added rFVIIa (Table 3). However, in the samples with a platelet count of 10,000/µl, the effect of rFVIIa became less obvious.

Thrombin Generation Endogenous thrombin generation capacity was measured by kinetic chromogenic assay. The results of a typical series of measurements are illustrated in Figure 2. Eptifibatide did not modify substantially thrombin generation in PRP, whereas the presence of rFVIIa resulted in increased thrombin generation (p < 0.05). This enhancement was also observed and well preserved, if rFVIIa and Eptifibatide were co incubated in the reaction mixture (p < 0.05).

Discussion Abciximab and Eptifibatide are two commonly used GPIIb/IIIa antagonists for patients with acute coronary artery syndrome. They are frequently administered concomitantly with indirect or direct thrombin inhibitors such as hirudin or LMWH [1, 2]. Furthermore, patients who receive these treatments may experience thrombocytopenia. Bleeding is the most common and frightened complication in patients

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treated with GPIIb/IIIa antagonists. Currently, there are no antidotes for GPIIb/IIIa antagonists; and in the case of severe bleeding platelet transfusions are required. Anecdotal data show that rFVIIa provides effective hemostasis in patients with bleeding caused by LMWH [3, 9, 17] alone or in combination with Tirofiban. Furthermore, rFVIIa may also be effective in bleeding associated with thrombocytopenia [7, 8]. The mechanisms of action of rFVIIa in these bleeding conditions are unknown. Nevertheless, GPIIb/IIIa antagonists induce a condition closely resemble to Glanzmann’s thrombasthenia, an inherited disorder characterized by quantitative or qualitative abnormality of platelet GPIIb/IIIa receptors, resulting in impaired platelet aggregation. In vitro experiments on GPIIb/IIIa-inhibited platelets or platelets from patients with Glanzmann’s thrombasthenia in flow models show that rFVIIa increases thrombin generation leading to enhanced platelet deposition and platelet aggregation independently of GPIIb/IIIa [3, 9, 18]. We used platelet aggregation and platelet function analyzer (PFA-100) to assess the effect of rFVIIa on GPIIb/IIIa-inhibited platelets. Platelet aggregation induced by thrombin, ADP, epinephrine, collagen and arachidonic acid was quickly (within 1 min) and almost completely blocked either by ReoPro or Integrilin. Platelet aggregation was not modified by rFVIIa itself. However, the presence of NovoSeven was not able to correct aggregation of GPIIb/IIIa blocked PRP or washed platelets. The addition of higher concentrations (up to 7 nmol/l) of rFVIIa did not result in better responses. However, in a more sensitized thrombin induced aggregation rFVIIa was able to restore partially aggregatory response in the presence of ReoPro. In PFA-100 experiments pharmacological concentrations of rFVIIa completely corrected the closure times of samples with GPIIb/IIIa-inhibited platelets, and this effect persisted in the presence of several direct or indirect thrombin inhibitors (hirudin, LMWH, or PPACK). Recombinant FVIIa also shortened the PFA-100 closure times of thrombocytopenic samples in the presence of Eptifibatide or Abciximab with platelet counts of 60,000 and 25,000/µl. It has been proposed that the increase in initial thrombin generation leads to faster platelet activation providing catalytic surface for the activation of coagulation factors. Consequently, the few platelets available are being used more effectively and may compensate for the low number [5, 7, 9]. Platelet aggregometry is a very simple, reliable way to analyze if agonist stimulated platelets are able to form a fibrin-platelet network, clumping, aggregation. The system uses PRP or washed platelets gained from anticoagulated blood, but there is a virtual absence of flow conditions and shear powers (only stirring). GPIIb/IIIa blockers inhibit platelet fibrin(ogen) interactions, network formation. Aggregation is almost completely abrogated if ReoPro or Integrilin was present. NovoSeven is not acting directly on GPIIb/IIIa structure, so it is not able to correct abnormal platelet aggregation in these settings. This explains, why we did not find any correction effect of rFVIIa with standard agonist (ADP, epinephrine, thrombin, collagen, and arachidonic acid) induced platelet aggregation. If thrombin aggregation was performed in a sort of sensitized way (partial defibrinization, lower concentrations of thrombin), than some acceleration of aggregation could have been observed with rFVIIa alone or in combination with GPIIb/IIIa blockers. This is in concert with

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recently published data, that rFVIIa was able to improve or correct the aggregatory response of washed platelets gained from Glanzmann’s thrombasthenia patients (after a lag phase), if purified FX, FII and fibrinogen were also added [15]. Platelet function analyzer assesses composite platelet functions in whole blood, i.e. adhesion, aggregation, and a sort of primary hemostatic plug. Although the results from this study indicate that platelet function analyzer may be useful to monitor patients who receive GPIIb/IIIa antagonists or patients with thrombocytopenia, the results should be interpreted with caution as there are some differences between the conditions used in this analyzer and in vivo [3]. PFA-100 tests platelet function in whole blood, in a complexity of platelet dependent closure times with two sets of cartridges: collagen/epinephrine and collagen/ADP. PFA-100 is widely used to test platelet functional disorders, screening for von-Willebrand disease, and also in a variety of other thrombocytopathies, including the effects of antiplatelet medications [19, 20]. However, this device uses a quite high shear-rate flow condition [4500–6000 s–1], which seems to be predominant in arterial vascular disease rather than in thrombosis or diffuse bleedings [3, 21]. Closure times are really reproducible in large series of measurements. If both values gained with the cartridges are normal, than the negative predictive value is 98%, while both test give prolonged closure times the positive predictivity of platelet dysfunction is also about 98% [19, 20]. The mechanism, how rFVIIa is capable to improve platelet functions is far from being completely understood [4, 6, 9, 21, 22]. Certainly, one of the major determinants is the sharp increase in thrombin generation, along with the acceleration of platelet surface bound catalytic coagulatory events [FV/X. etc.], which results in platelet activation at the sites of vascular injuries [23, 24]. Regarding the efficacy of rFVIIa in GPIIb/IIIa deficiency [Glanzmann-thrombasthenia] other factors should also be considered: inhibition of TAFI and fibrinolysis, advantageous changes in the physical structure of clots, improved platelet function caused by the procedure of thrombin generation as a whole and so forth. Some new data call attention to the role of platelet micro particles along with platelet phospholipids and collagen or in concert with platelet associated TF [22, 24]. More recent evidence suggests that rFVIIa is able to increase platelet adhesion, even if GPIIb/IIIa receptors are blocked [15, 25]. A possible way of rFVIIa correction of platelet function in the presence of GPIIb/IIIa blockers is increased thrombin activity due to increased thrombin generation induced by NovoSeven. However, thrombin generation should be more or less limited in our system using PFA-100, as the cartridge contains an in vitro mixture; there is neither vessel wall nor endothelium. However, some earlier studies substantiated the evidence tissue factor independent thrombin activation by rFVIIa and that GPIIb/IIIa deficient platelets are able to form aggregates through a nonspecific physicochemical binding to polymerized fibrin network [17, 26,]. Furthermore it was suggested, that some non GPIIb/IIIa type binding sites on fibrin polymers may also exist and do contribute to platelet attachment [27]. There is also evidence supporting that thrombin activated GPIIb/IIIa deficient platelets may bind to polymerized fibrin via platelet activation by thrombin bound to platelet GPI [28]. We were able to demonstrate that rFVIIa is able to improve substantially endogenous thrombin generation capacity of PRP in the presence of ReoPro. One may assume, whether platelet associated TF, micro particles, platelet collagen interac-

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tions are also contributing to rFVIIa actions on GPIIb/IIIa blocker treated platelets. Platelet-leukocyte interactions may also be accelerated by high shear flow conditions [29]. GPIIb/IIIa inhibitors failed to block platelet-monocyte interactions in vitro. Probably the standard pharmacological concentrations of thrombin inhibitors might also fail to block thrombin action on platelets coupled to leukocytes. As platelet micro particle formation is also enforced by high shear rate, this may contribute to shortening of PFA closure times in the presence of rFVIIa, too. High shear conditions may also accelerate platelet-collagen interactions in the presence of supraphysiological concentrations of rFVIIa in the PFA cartridges. The high shear flow, cell to cell interactions, platelet micro particles may interfere with the standard pharmacological concentrations of direct and indirect thrombin inhibitors used in our settings, allowing enough rFVIIa induced, platelet derived thrombin generation to activate platelets and create a platelet fibrin interaction for sufficient plug formation in PFA. Furthermore 90 µg/kg rFVIIa was able to correct the anticoagulant activities of fondaparinux, a powerful pentasaccaride based anti Xa agent in vivo [30]. We have also demonstrated that rFVIIa enhanced endogenous thrombin generation in platelet rich plasma milieu, even in the presence of a GPIIb/IIIa inhibitor. One may also assume that the thrombin generated in the presence of rFVIIa binds to surfaces such as activated platelets, and this thrombin may be protected from the effect of the standard concentrations of thrombin inhibitors. One should exert extreme caution to extrapolate in vitro findings into clinical bleeding conditions, or to consider PFA 100 experiments as being equal with the clinical bleeding situations, which may occur as complications of GPIIb/IIIa blocker therapy. The feasibility of PFA-100 to assess or predict the correction of platelet functions, achieved by rFVIIa in GPIIb/IIIa blocked patients also needs confirmation on clinical grounds. Our results show that rFVIIa might be a useful hemostatic agent in case of bleeding caused by GPIIb/IIIa antagonists. Further studies are needed to establish the correlation between these findings in this in vitro study and in vivo outcomes. References 1. Crouch MA, Nappi JM, Cheang KI. Glycoprotein IIb/IIIa receptor inhibitors in percutaneous coronary intervention and acute coronary syndrome.Annals Pharmacother 2003; 37:860–871 2. Kondo K, Unemura K. Clinical pharmacokinetics of eptifibatide, a nonpeptide glycoprotein IIb/IIIa receptor antagonist: comparison with the monoclonal antibody abciximab. Clin Pharmacokinet 2002; 41:187–195 3. Li YF, Spencer FA, Becker RC. Comparative efficacy of fibrinogen and platelet supplementation on the in vitro reversibility of competitive glycoprotein IIb/IIIa receptor directed platelet inhibition. Am Heart J 2001; 142:204–210 4. Hedner U. NovoSeven as a universal haemostatic agent. Blood Coagul Fibrinolysis 2000; 11:107–111 5. Hedner U., Ingerslev J. Clinical use of recombinant Factor VIIa. Transfus Sci 1998;19:163– 176 6. Von Depka M. NovoSeven, mode of action and use in acquired haemophilia. Intensive Care Med 2002; 28:S222–S227 7. Kessler C. Haemorrhagic complications of thrombocytopenia and oral anticoagulation. Is there a role for recombinant activated factor VII? Intensive Care Med 2002; 28:S228–S234

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8. Culic S: Recombinant Factor VIIa for refractive haemorrhage in autoimmune idiopathic thrombocytopenic purpura. Br J Haematol 2003; 120:909–910 9. Poon MC, Oiron R. Recombinant activated FVII treatment of platelet-related bleeding disorders. International Registry of rFVIIa and congenital platelet disorder group. Blood Coagul Fibrinolysis 2000; 1:S55–S68 10. Jennings LK, Jacoski MV, White MM. The pharmacodynamics of parenteral glycoprotein IIb/IIIa inhibitors. J Interv Cardiol 2002; 15:45–60 11. Berretini M, Mariani G, Schiavoni M, Rocino A, Paolantonio TD, Longo G, Morfini M. Pharmacokinetic evaluation of recombinant activated factor VII in patients with inherited factor VIII deficiency. Haemtologica 2001; 86:640–645 12. Erhardtsen E: Pharmacokinetics of recombinant activated Factor VII [rFVIIa]. Semin Thromb Hemost 2000; 26:385–391 13. Stachnik JM, Gabay MP. Continuous infusion of coagulation factor products. Annals Pharmacother 2002; 36:882–891 14. Thomsen MK, Diness V, Nilsson P, Rasmussen SN, Taylor T, Hedner U. Pharmacokinetics of recombinant factor VIIa in the rat – a comparison of bio-, immuno- and isotope assays. Thromb Haemost 1993; 70:458–46416. 15. Lisman T, Adelmeier J, Harry F, Heijinen G, de Groot PG: Recombinant Factor VIIa restores aggregation of alphaIIbetaIII deficient platelets via tissue factor-independent fibrin generation. Blood 2004; 103:1720–1727 16. Hemker HC, Beguin S. Thrombin generation in plasma: its assessment via the endogeneous thrombin potential. Thromb Haemost 1995; 74:1388–94 17. McGregor L: Aggregation to thrombin and collagen of platelets from a Glanzmann thrombasthenic patient lacking GpIIb/IIIa. Thromb Haemost 1989; 62:962–967 18. Van Veer C, Mann KG. The regulation of the factor VII-dependent coagulation pathway, rationale for the effectiveness of recombinant factor VIIa in refractory bleeding disorders. Semin Thromb Hemost 2000; 26:367–372 19. Favaloro EJ. Clinical application of the PFA-100. Curr Opin Hematol 2002; 9:407–415 20. Jilma B. PFA-100: a tool to quantify congenital or acquired platelet dysfunction. J Lab Clin Med 2001; 138:152–163 21. Hoffman M. Mechanism of action of NovoSeven, using a cell based model. Bloodline Reviews 2003; 1:5–8 22. Hoffman M, Monroe DM, Roberts HR. Activated factor VII activates factor IX and X on the surface of activated platelets: thoughts on the mechanism of action of acitivated factor VII. Blood Coagul Fibrinolysis 1998; 9(Suppl):S61–S65 23. Galan AM, Tonda R,Altisent C, Maragall S, Ordinas A, Escolar G: Recombinant factor VIIa restores deficient coagulation, experience from an ex vivo model. Semin Hematol 2001; 38:10–14 24. Wilbourn B, Harrison P, Mackie IJ, Liesner R, Machin SJ. Activation of platelets in whole blood by recombinant factor VIIa, a thrombin dependent mechanism. Br J Haematol 2003; 122:651–656 25. Lisman T, Moschatsis S, Adelmeier J, Nieuwenhuis HK, De Groot PG. Recombinant Factor VIIa enhances deposition of platelets with congenital or acquired GpIIb/IIIa deficiency to endothelial cell matrix and collagen under conditions of flow via tissue factor independent thrombin generation. Blood 2003; 101:1864–70 26. Niewiarowski S, Levy-Toledano S, Caen JP: Platelet interaction with polymerizing fibrin in Glanzmann thrombasthenia. Thromb Res 1981; 23:457–463 27. Osdoit S, Rosa JP: Polymeric fibrin interacts with platelets independently from GpIIb/IIIa (abstract). Blood 2001; 98:518abs 28. Soslau G, Class R: Unique pathway of thrombin induced platelet aggregation mediated by GpIb. J Biol Chem 2001; 276:21173–21183 29. Zhao L, Bath P, Losche W, Heptinstall S. Platelet-monocyte interaction and GPIIb/IIIa blockade. Thromb Haemost. 2004; 4:888–90 30. Bijsterveld NR, Moons AH, Boekhold SM, van Aken BE, Fennema H, Peters RJ, Meijers JC, Buller HR, Levi M. Ability of recombinant factor VIIa to reverse the anticoagulant effect of the pentasaccharide fondaparinux in healthy volunteers. Circulation 2002; 106:2250–2254

Mutation Analysis in Hereditary Angioedema Identifies Patients at Risk for Developing Acute and Life Threatening Edema T. Förster, A. Kocot, J. Schröder, W. Kreuz, E. Aygören-Pürsün, I. Martinez-Saguer, K. Bork, I. Scharrer, C. Müller, and J. Oldenburg

Introduction Hereditary angioedema (HAE) (OMIM: 106100) is an autosomal dominant disease due to mutations of the C1 inhibitor gene (C1INH). The C1INH protein, a serin protease inhibitor, is the sole inhibitor of the C1r and C1s components of the classical complement pathway and the major regulator of factors XI and XII and of plasma kallikrein in the contact system (see Fig. 1). Clinically, HAE presents as edema of the extremities, face, trunk, airways or abdominal viscera, often triggered by psychological and/or physical stress. Laryngeal edema may cause suffocation and if not treated properly can be fatal. According to the antigenic plasma level of the C1INH protein two types of HAE are differentiated: In HAE type I, representing 85% of all patients, serum levels of C1 INH are less than 35% of normal. In HAE type II the levels are normal or elevated but the protein is non-functional due to mutations in the reactive center loop. The two types are clinically indistinguishable [1, 2].

C1qrs

Intrinsic coagulation

C1

complement system C1 Inhibitor

Kininogen

FXII

FXIa

FXIIa

Kallikrein

ProKallikrein

FXI Bradykinin contact system of kinin generation

Fig. 1. C1-Inh plays an important role in several pathways of inflammation I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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Patients and Methods Patients suspicious for hereditary angioedema were sent from specialized outpatient clinics in Frankfurt and Mainz. All 8 exons of the C1 Inhibitor gene and the flanking intronic regions were sequenced. The high percentage of large deletions and insertions caused by the frequent intronic alu-sequences was analyzed by Southern Blotting after digestion of genomic DNA with BclI.

Results and Discussion So far 207 HAE families with 347 members have been analyzed for the presence of mutations in the C1 inhibitor gene. In 221 members of 124 HAE families a causative mutation could be identified. 30 (24,2%) of these HAE families showed mutations that were already reported in the literature, while 94 (75,8%) HAE families exhibited a total of 69 different novel mutations in the C1 inhibitor gene. These novel mutations included 27/69 missense mutations, 23/69 small deletions/insertions/ duplications, 11/69 splice site mutations, 8/69 nonsense mutations and 9 large deletions. In 83 of the supposed HAE families no mutation could be detected in the C1inhibitor gene, most likely because the patients in these families suffered from acquired HAE rather than from inherited HAE. The HAEdb (C1 Inhibitor gene mutation database http://hae.biomembrane.hu/) reports on 150 mutations known in the C1 inhibitor gene. These are 38% missense mutations, 8% nonsense mutations, 30% small deletions and 9,3% splice site mutations and 14,7% large deletions [3]. Our data correspond well to those reported in

11,5%

34,6%

29,5%

10,3%

14,1%

Fig. 2. Distribution of mutations found within our cohort

14,7%

1,3% 38,0%

9,3%

Fig. 3. Distribution of mutations reported in literature

28,7%

8,0%

Missens e muta tions n= 27 Non sense m uta tio ns n= 8 Deletions/Insertions n= 23 Splice Site m uta tio ns n= 11 Lar ge de letions n= 9

Missense muta tions n = 57 Nonsense muta tions n= 14 Delet ions/ I nser t ions n= 45 Splice Si t e muta tions n =14 Large d elet ions n= 22 Promoter n =2

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the database which we are now able to enlarge to a total of 219 different mutations in the C1 inhibitor gene, comprising 85/219 (38,8%) missense mutations, 20/219 (9,1%) nonsense mutations, 68/219 (31,1%) small deletions, insertions and duplications, 25/219 (11,4%) splice site mutations and 31/219 (14,2%) large deletions.

Conclusion Routine molecular genetic analysis identified the causative mutations in most of the HAE families. It is very important in diagnosing HAE carriers in affected families prior to clinical manifestation thus allowing the identification of high risk patients for developing acute and life threatening edema.

References 1. Davis AE: Hereditary and acquired deficiencies of C1 inhibitor. Immunodefic Rev. 1989; 1 (3): 207–26 2. Pappalardo et al.: Mechanisms of C1-inhibitor deficiency. Immunobiology. 2002 Sep; 205 (4–5): 542–51 3. Kalmar et al.: HAEdb: a novel interactive, locus-specific mutation database for the C1 inhibitor gene. Hum Mutat. 2005 Jan; 25 (1): 1–5

Recurrent Mutation in ADAMTS13 Gene as a Cause of a Hereditary Thrombotic Thrombocytopenic Purpura in the Czech Republic I. Hrachovinová, P. Salaj, J. Suttnar, Sˇ. Rittich, P. Jinoch, ˇ ulícˇek, and T. Seeman T. Sˇuláková, J. Pták, P. D

Introduction Thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia, microangiopathic hemolysis, neurological abnormality, and renal dysfunction. Similar manifestations also occur in patients with the hemolytic uremic syndrome or other types of disorders. Recent studies demonstrate that severe deficiency of von-Willebrand factor cleaving metalloprotease, ADAMTS13, causes TTP [1–3]. Aim of our prospective study in the years 2002–2004 was to characterize patients sent to our lab with a clinical diagnosis of TTP.

Materials and Methods We investigated 45 consecutive patients with consumptive thrombocytopenia and Coombs negative hemolytic anemia with red cell fragmentation. Samples were taken immediately prior to the first plasma exchange therapy or in a remission of the recurrent form of disease. We measured activity of ADAMTS13 (VWF-CP) with modified method of the quantitative immunoblotting of degraded VWF multimers [4]. Mutation screening was carried out by sequencing all 29 exons and flanking intron regions of the ADAMTS13 gene in patients with a diagnosis of familiar form of the disease.

Results and Discussion Nearly two third of patients (28/45) got a severe VWF-CP deficiency with activity of protease less than 6% of normal (Fig. 1). Antibodies against VWF-CP were found in the 16 of them (57%). Ten patients with severe ADAMTS13 deficiency revealed a hereditary form of TTP. Two patients with severe deficiency without a presence of antibodies are under investigation since they had a normal value of VWF-CP in remission. Eleven of 45 patients had borderline severe or moderate deficiency of VWF-CP activity. After a follow-up was complete, some of these patients had an unexpected diagnosis of an alternative disorder (e.g. sepsis, cancer). Six of 45 patients (13%) displayed VWF-CP values >50%. This group manifested a severe renal failure in I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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45 patients met TTP diagnostic criteria

39 patients - ADAMTS13 level less than 50%

6 patients (13%) - normal level of ADAMTS13

16 patients (36%) - detected inhibitor to ADAMTS13

23 patients without inhibitor against ADAMTS13

12 patients with level ADAMTS13 <10%

10 patients (22%) - recurrent inherited form TTP

11 patients (24%) – level 10–50%

2 patients - normal level in remission

Fig. 1. Chart of study protocol with consideration of ADAMTS13 activity and inhibitor Table 1. Level of VWF-cleaving protease, relapse status and mutation in 10 patients with inherited form TTP #

VWF-cp (%)

Relapse

Mutation

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

2.5 2.0 6.0 3.0 2.5 2.5 2.5 2.0 3.0 <5

+ + + + + + + + + +

4143insA, ? 4143insA homozyg. Arg507Gln, ? 4143insA, ? 4143insA, Arg507Gln 4143insA, Arg507Gln 4143insA, ? 4143insA homozyg. 4143insA homozyg. N.D.

four of six patients. It was significantly higher than in the group with a severe ADAMTS13 deficiency. Mutation analysis of the ADAMTS13 gene in nine VWF-CP deficient patients with familiar form of TTP brought interesting results (Table 1). We found a mutation of one single base frameshift insertion, 4143insA in 7 of 8 unrelated individuals. This is in contrast to the first reports that addressed ADAMTS13 mutations emphasized the considerable heterogeneity of genotypes [3]. The mutation 4143insA was identified also in German, Norwegian, Sweden, Poland and Croatian patients what suspects of a founder mutation [5, 6]. Patients 1, 3, 4 and 7 are not sequenced throughout. We expect to find another causal mutation since TTP phenotype presumes compound heterozygous mutations.

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ADAMTS13 gene: 4284bp, 1427 aa, 29 exons

Exons

5

10

15

20

25

29

4143insA (Cub2): 1520 G-A, Arg507Gln (Cys rich): 1 TH (1994)

2 KH (1996)

Arg507Gln (exon 13) 4143insA (exon 29)

Fig. 2. Mutation analysis in two siblings with TTP

Two female siblings (Fig. 2) with inherited mutations were diagnosed first as thrombocytopenia in the age 1.5 year. The full presentation of disease in the age 5 years was misdiagnosed as hemolytic uremic syndrome. Our investigation contributed to differential diagnosis of disease since the TTP phenotype in childhood can be variable and rapid detection of a mutation is helpful in the prevention of a recurrence of disease. This work was supported by Czech Ministry of Health (IGA MZ NM/7719-3).

References 1. Gerritsen HE, Robles R, Lämmle B, Furlan M. Partial amino acid sequence of purified von Willebrand factor-cleaving protease. Blood. 2001; 98:1654–1661 2. Fujikawa K, Suzuki H, McMullen B, Chung D. Purification of human von Willebrand factor-cleaving protease and its identification as a new member of the metalloproteinase family. Blood. 2001; 98:1662–1666 3. Levy GG, Nichols WC, Lian EC, Foroud T, McClintick JN, McGee BM, Yang AY, Siemieniak DR, Stark KR, Gruppo R, Sarode R, Shurin SB, Chandrasekaran V, Stabler SP, Sabio H, Bouhassira EE, Upshaw JD Jr, Ginsburg D, Tsai HM. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature. 2001; 413:488–494 4. Furlan, M., Robles, R., Solenthaler, M,. Lämmle, B.: Acquired deficiency of von Willebrand factor-cleaving protease in a patient with thrombotic thrombocytopenic purpura. Blood. 91, 1998, s. 2839–2846. 5. Schneppenheim R, Budde U, Oyen F, Angerhaus D, Aumann V, Drewke E, Hassenpflug W, Haberle J, Kentouche K, Kohne E, Kurnik K, Mueller-Wiefel D, Obser T, Santer R, Sykora KW.Von Willebrand factor cleaving protease and ADAMTS13 mutations in childhood TTP. Blood. 2003; 101:1845–1850 6. Assink K, Schiphorst R, Allford S, Karpman D, Etzioni A, Brichard B, van de Kar N, Monnens L, van den Heuvel L. Mutation analysis and clinical implications of von Willebrand factor–cleaving protease deficiency. Kidney International. 2003; 63:1995–1999

Clinical Manifestations of Patients with Dysfibrinogenemia W. Miesbach, V. Catania, M. Boehm, Th. Vigh, and I. Scharrer

Congenital dysfibrinogenemias are characterized by structural abnormalities in the fibrinogen molecule that result in tendencies for hemorrhage and/or thrombosis. Acquired dysfibrinogenemia may carry a worse prognosis than the congenital dysfibrinogenemia due to a severely damaged liver [1]. With rare exceptions, the congenital dysfibrinogenemias are inherited in an autosomal dominant or codominant way. Fibrinogen is a 340000 Da protein which is synthesized in hepatocytes. The molecule is a homodimer; each half consists of three non-identical polypeptide chains that have been termed A alpha, B beta and gamma-chains [2]. The genes for all three chains have been localized to the long arm of chromosome 4 [3]. The amino termini of the three pairs of polypeptide chains are symmetrically arranged into a disulphide knot to form a central E-domain that is flanked by two large Ddomains to form a trinodular structure. Approximately 300 abnormal fibrinogens with unique name designations have been reported to date, and about 83 structural defects have been identified [4]. The most common structural defects involve the fibrinopeptides and their cleaving sites, and the second most common involves the gamma-chain polymerization region. Dysfibrinogenemia can also be acquired secondary to liver cirrhosis or use of certain drugs (e.g. asparaginase).

Patients and Methods We investigated 19 patients from 50 families with dysfibrinogenemia. Of the patients 26/50 (52 %) were female; 24/50 (48 %) were male. There was a median age of 52 years (range 9–89 years). Up to now, in 60 % of the patients an analysis of the fibrinogen gene was performed by Galakanis, New York, and von Depka, Hanover. Each patient was asked about the type of clinical manifestations. In all patients we measured plasma concentrations of fibrinogen using different methods: The functional fibrinogen concentration was determined according to Clauss. The immunological fibrinogen concentration was determined by radial immunodiffusion. Additionally, reptilase time and thrombin clotting times were performed.

Results The median fibrinogen concentration according to Clauss was 51 mg% (range 15–86 mg%, normal value: 150–450 mg%). The median immunological fibrinogen I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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59

60 50

45

40 30 24

21

20

17

10

7

0

ng

isi

Fig. 1. Type of bleeding (%)

sy

u br

ea

m

gu

se

no

o

m

po

45 40 35 30 25 20 15 10 5 0

Fig. 2. Type of thrombosis (%)

st

ive

at

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e

t ns

ru

al

al

in

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t

in

42

25 17

17

. in b. ry inf te ve is em ar p bos ial g l e d r ra de rom lun ca he se yo th rip isea e m p d

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concentration was 244 mg% (normal value: 200–450 mg%); the median concentration of heat fibrinogen according to Schulz was 240 mg% (norm: 200–450 mg%). The median reptilase clotting time was 55 sec. (norm: – 20 sec.). The most common found fibrinogen gene mutation was Alpha 16 Arg->Cys. A tendency towards bleeding was described in 25/50 patients (50 %). The patients mostly suffered from recurrent hematoma (59 %), bleeding after operations or deliveries (45 %), bleedings of teeth (24 %) and nose (21%), menstrual bleeding (17 %) and gastrointestinal bleedings (7%) (Fig. 1). In 12/50 (24 %) of the patients, thrombotic events occurred. 42 % of the patients had peripheral artery disease, 25 % deep vein thrombosis and 17 % lung embolism. 17 % suffered from myocardial infarction and 17 % from stroke (Fig. 2). 6 of 50 patients (12 %) exhibit both, hemorrhage and thrombosis. In 18/50 patients (36 %) from four families, there were no bleeding or thrombotic manifestations of dysfibrinogenemia.

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Table 1. Clinical manifestations of patients with dysfibrinogenemia Manifestation

Patients (%)

without manifestations

18/50 (36 %)

bleeding

25/50 (50 %)

thrombosis

12/50 (24 %)

arterial thrombosis

10/50 (20 %)

simultaneously bleeding

6/50 (12 %)

and thrombosis miscarriages

5/26 (19 %)

Abortions occurred in 5 of 26 female patients from 3 families (19 %). Three of these patients suffered from recurrent abortions. It also could be documented that in four patients with dysfibrinogenemia, abortions could be avoided by continuously administrating fibrinogen concentrates from beginning of the pregnancy in order to achieve fibrinogen plasma concentrations over 100 mg% (Clauss).

Discussion Beside some case reports, there is only a rare number of studies about the rate of clinical manifestations in a large number of patients with dysfibrinogenemia. A meta-analysis of over 260 reported cases of dysfibrinogenemia revealed that approximately 55 % of the patients had no clinical manifestation; approximately 25 % of patients exhibit a bleeding tendency and 20 % had a tendency for thrombosis [5]. In patients with dysfibrinogenemia, thrombosis is usually mild and arterial thrombosis is very rare. In our cohort, however, three patients were suffering from recurrent severe arterial thrombosis. The dysfibrinogenemias most often associated with bleeding tendencies are mutations on the alpha chain, especially in the amino-terminal region (amino acids 17–19) [6]. This region is believed to be a critical size for polymerization between adjacent fibrin monomers. In general, patients with a known history of previous bleeding should be treated by fibrinogen replacement prior to surgery or after trauma. Fibrinogen replacement should be sufficient to raise the plasma level of fibrinogen to hemostatic levels. Antifibrinolytic amino acids are also recommended in patients with dysfibrinogenemia who have bleeding symptoms, but should not be used in patients with dysfibrinogenemia associated with thrombosis. Two mechanisms may be responsible for the occurrence of thrombosis in patients with dysfibrinogenemia [7, 8]: The first is that the abnormal fibrinogen is defective in binding thrombin which results in elevated levels of thrombin. The second is that the abnormal fibrinogen forms a fibrin clot that is resistant to plasmin degradation. In 1995, 51 cases of dysfibrinogenemia associated with thrombosis were reported [9]. Many of the patients also had postpartum bleeding or a history of mild bleeding.

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The variability of clinical manifestations of dysfibrinogenemia may represent the genetic heterogeneity of this syndrome. It could be observed in many cases that patients had family members who experienced the same bleeding or thrombotic manifestation of dysfibrinogenemia. In our study, the rate of clinical manifestations was higher than expected: 50 % of the patients suffered from bleeding symptoms, mostly recurrent hematomas that was not caused by other factors. In patients with thrombotic events, arterial thrombosis was over presented. Surprisingly, 19 % of female patients with dysfibrinogenemia experienced miscarriages. An increased number of spontaneous abortions and stillbirths has been reported previously in patients with dysfibrinogenemia [7]. In our cohort, three of the patients had three consecutive abortions. It could also be shown that by administrating fibrinogen concentrates from beginning of pregnancy, abortions could be avoided. Therapeutic recommendations should be evaluated on the individual basis of the patient. Very careful treatment recommendations have to be provided for patients with simultaneously presentation of both, bleeding and thrombosis. Further studies of the dysfibrinogens may lead to further understanding of hemostasis and thrombosis dependent on the underlying fibrinogen gene mutation.

References 1. Martinez J, Keane PM, Gilman PB, Plascak JE. The abnormal carbohydrate composition of the dysfibrinogenaemia associated with liver disease. Ann N Y Acad Sci 1983;408:388–96 2. Coté HCE, Lord ST, Pratt KP. Gamma-chain dysfibrinogenemias: molecular structurefunction relationships of naturally occurring mutations in the gamma chain of human fibrinogen. Blood 1998;92:2195–2212 3. Henry I, Uzan G, Weil D, Nicolas H, Kaplan JC, Margueric C, Kahn A, Janien C. The genes coding for A alpha-, B beta-, and gamma-chains of fibrinogen map to 4q2. Am J Human Genetics. 1984;36:760–8 4. Martinez J. Congenital dysfibrinogenemia. Curr Opin Hematol 1997;4:357–65 5. Ebert R. Index of variant human fibrinogens. CRC Press, Boca Raton FL, 1994 6. Roberts HR. The dysfibrinogenaemias. Br J Haematol 2001;114:249–57 7. Haverkate F, Samama M. Familial dysfibrinogenaemia and thrombophilia. Report on a study of the SSC Subcommittee on Fibrinogen. Thromb Haemost 1995;73:151–61 8. Galanakis DK. Inherited dysfibrinogenemia: emerging abnormal structure associations with pathologic and nonpathologic dysfunctions. Semin Thromb Haemost 1993;19: 386–95 9. Mosesson MW. Dysfibrinogenemia and thrombosis. Semin Thromb Haemost 1999;25: 311–9

Case Report of a FXIII Inhibitor in a 77-year-old Patient W. Miesbach, M. Boehm, Ch. von Auer, and I. Scharrer

Introduction We report the case of a 77-year-old woman with an acquired inhibitor to FXIII who was admitted with life-threatening bleeding. The development of an FXIII inhibitor is very rare. Only 21 cases have been previously described in the literature and therapeutic procedures have not yet been established [1].

Methods FXIII was measured using Berichrom FXIII reagent from Dade Behring (Marburg, Germany) according to the manufacturer’s instructions. In order to measure the FXIII inhibitor, patient’s plasma samples were mixed in equal parts with a local normal plasma pool. The plasma samples were either neat or had been diluted with imidazole buffer. A reference was produced by diluting normal plasma 1:1 with imidazole buffer. FXIII was measured after incubation for 2 hours at 37°C according to the Bethesda test [2]. The FXIII activity of the test samples was divided by the activity of the reference sample and multiplied by 100 to obtain the percentage residual FXIII activity. In order to quantify the results, the amount of inhibitor was calculated for samples with a residual activity from 25 to 75 and expressed in U/ml, as has been described for coagulation FVIII inhibitors. A sample which inhibited normal FXIII activity by 50 % was assumed to contain inhibitor at a concentration of 1 U/ml.

Case Report A 77-year-old woman was admitted to hospital with suspected thrombosis of the right thigh. Until this event, the patient had not been seriously ill and this was the first time she had been in hospital. There was no previous history of bleeding diathesis. Six months before admission, she had suffered from middle otitis and was treated with Ciprofloxacin. She initially received Warfarin as oral anticoagulant. This had to be stopped due to a spontaneous hematoma of the right thigh. She subsequently underwent surgery I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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due to a bleeding gastric ulcera (Forrest Ib). After the operation, bleeding could not be stopped and the use of multiple units of red blood cells became necessary. After four weeks in a foreign hospital, the patient was referred to our clinic. At admission, laboratory tests showed a low level of hemoglobin at 7.2 mmol/L (normal range: 12–16 mmol/L) and a mild thrombocytemia of 135000/µl (normal range: 150000–440000/µl). The coagulation profile, liver enzymes and renal values were normal. Autoantibodies, e.g. anti-nuclear factor or antiphospholipid antibodies were not found. Other possible causes of bleeding, such as von-Willebrand disease or dysfunction of the thrombocytes, were excluded. There were multiple painful hematomas distributed over the entire body of the patient, particularly on the right thigh. Examinations of heart, lungs, and abdomen were unremarkable, except for a little ascites. We found a FXIII deficiency of 9 % and administered FXIII concentrates as the patient was in deep shock due to the continuous loss of blood. However, FXIII activity could not be normalized, despite administration of 2500 E Fibrogammin. We subsequently tested the plasma for an inhibitor to FXIII and detected an inhibitory activity of 19 BU/ml. The recurrent bleedings of the soft tissues and muscles were subsequently treated with activated recombinant FVII (rFVIIa) (dose: 120 µg/kg). We started daily plasma exchange, immunosuppression with steroids (100 mg/day), and two doses of Cyclophosphamide (300 mg/m2). Intravenous immunoglobulins were administered (1g/kg for four days), as well as antibiotics Imipenem and Cilastatin. Daily plasma exchange continued for 26 days. In spite of this multimodal treatment, continuous substitution of red blood cells and fresh frozen plasma (FFP) was necessary due to recurrent soft tissue bleeding. Surgical removal of the hematoma of the right thigh was carried out four times under rFVIIa. During the course of this treatment, the patient’s condition gradually improved. A thrombosis in the left internal jugular vein was detected coincidentally. The time point of occurrence could not be determined, and the patient did not complain about pain or swelling. Another thrombosis of the right popliteal vein from an even earlier date was also detected. This was probably due to compression by the huge hematoma of the right thigh. The patient was put on mild anticoagulants with low molecular heparin. During this anticoagulant treatment, FXIII activity remained unchanged, although it is known that high doses of Heparin may reduce FXIII activity. Screening for hereditary or acquired thrombotic risk factors was negative. CT scans of the thorax, brain and abdomen as well as biopsy of the bone marrow showed no abnormalities and no signs of malignancy. The level of FXIII inhibitor decreased during immunosuppressive treatment, but FXIII activity remained unchanged. The patient received four weekly doses of Rituximab (375 mg/m2). This was tolerated without any evidence of side-effects. Peripheral B-cell depletion was found consequently. Remarkably, FXIII activity began to increase markedly one week after the last course of Rituximab. No bleeding episodes occurred after this point in time. After being treated for seven weeks at our clinic and after an interval of two weeks without any bleeding, the patient was transferred to a hospital for rehabilitation. During the second week of rehabilitation, an infection of the urinary tract was

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treated with Ciprofloxacin. During this treatment, the FXIII activity decreased from 32 % to 9 % and FXIII inhibitor raised from 17 U/ml to 50 U/ml. Twelve days after treatment with Ciprofloxacin the patient reported severe headache. She felt very tired and it was noticed that the patient’s co-ordination was impaired. The next day she vomited blood and could not be woken. A cranial CT showed massive cerebral bleeding from which she died one day later.

Discussion Recommendations for the treatment of inhibitor mediated FXIII deficiency are very rare, and are based on published case reports. High and frequent doses of FXIII concentrates may partly increase FXIII levels in patients with FXIII inhibitor and reduce mortality [3]. In our patient, we observed no increase of FXIII activity after treatment with Fibrogammin (9% before, 6% after Fibrogammin). Therefore, we chose to administer rFVIIa repeatedly. The efficacy of Rituximab for the treatment of patients with acquired hemophilia A has recently been emphasized [4]. Rituximab is a specific anti B-cell chimeric monoclonal antibody which rapidly eliminates most circulating B cells. It has been initially indicated for treatment of CD 20-positive non-Hodgkin’s lymphoma. Our patient with FXIII inhibitor responded positively to treatment one week after the administration of Rituximab. Not only did the bleeding stop, but FXIII activity also increased. The sudden death of the patient due to cerebral bleeding was, however, unexpected. The patient did not exhibit any symptoms of bleeding immediately prior to her death. Her FXIII level had, however, decreased within one week from 32% to 9% and FXIII inhibitor raised from 17 U/ml to 50 U/ml. This leads us to conclude that there may be variable FXIII levels, even after an initial response to Rituximab and to the multimodal therapy which was performed with the aim of eliminating the FXIII inhibitor. The second treatment cycle with Rituximab may have positively influenced the FXIII activity. The influence of Ciprofloxacin on the FXIII activity may also be a possible cause for the decrease in the FXIII activity.

References 1. Nijenhuis AVM, van Bergeijk L, Huijgens PC, Zweegman S. Acquired factor XIII deficiency due to an inhibitor: a case report and review of the literature. Haematologica 2004;89: 14–19 2. Kaspar CK. Laboratory tests for factor VIII inhibitors, their variation, significance and interpretation. Blood Coagul Fibrinolysis 1991;2:7–10 3. Daly HM, Carson PJ, Smith JK. Intracerebral hemorrhage due to acquired factor XIII inhibitor – successful response to factor XIII concentrate. Blood Coag Fib 1991;2:507–14 4. Stasi R, Brunetti M, Stipa E, Amadori S. Selective B-cell depletion with Rituximab for the treatment of patients with acquired hemophilia. Blood 2004;103:4424–8

VII.

Poster

VIIa: Hemophilia and Hemorrhagic Disorders

The ABC’s of Hemophilia German Working Group of Hemophilia Nurses (»Arbeitskreis der Hämophilieassistentinnen der Hämophiliezentren in Deutschland«)

This guide was developed by the Association of Hemophilia Nurses of the World Federation of Hemophilia (WFH) in 1981 and has been translated into German by the German Working Group of Hemophilia Nurses (»Arbeitskreis der HämophilieAssistentinnen«). »The ABC’s of Hemophilia« is directed to anyone interested in the treatment of hemophilia, and in particular to hemophilia patients and their families. It is designed to provide basic information on the most important aspects of the disease and its treatment. Providing patients and their families with information on the disease is a necessary and important part of therapy. The original version of this guide has already been used in numerous countries all over the world. Any input received so far has been implemented into this German version. This guide is characterized in particular by its clear visualization, flexibility and diverse applicability. The German Working Group of Hemophilia Nurses hopes that it will prove to be a useful tool for many people, helping them to provide optimal care to hemophilia patients. This guide is available from the German Hemophilia Society (»Deutsche Hämophiliegesellschaft«) in Hamburg.

I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

The Long Journey to Being Diagnosed as a Carrier of Hemophilia A – A Woman with Abnormally Prolonged Bleeding after Myocardial Infarction W. Miesbach, B. Putz, Ch. von Auer, and I. Scharrer

Hemophilia A is caused by a deficiency of factor VIII (FVIII). The disease results from mutations in the FVIII gene which are heterogenous both in type and position within the gene. Carriers of hemophilia A are often admitted with normal clotting factor VIII levels, but occasionally also report a considerable tendency to bleeding. It has recently been found that decreased concentrations of factor VIII and IX in carriers of hemophilia A and B are associated with favorable effects on blood coagulation and hemostasis which contributes to cardiovascular protection and a decreased mortality from ischemic heart disease [1]. In this report, we report the case of a woman who was first diagnosed at the age of 60 years as a carrier of hemophilia A. She was suffering from abnormally prolonged bleeding after an angiographic intervention that was performed due to a myocardial infarction. The patient had a noticeable history of bleeding events since childhood. A one stage method was used to test for factor VIII activity (Instrument ACL 300, normal range: 64 %–167 %). The fact that the patient was a carrier of hemophilia A was confirmed by analysis of the factor VIII gene mutation (performed by PD. Dr. J. Oldenburg, Würzburg/Frankfurt). The patient was born in 1943. The patient was suffering since childhood from bruising and nose bleeds. Due to abnormally long and heavy menstrual bleedings, she had suffered from several abrasions of the uterus. Life-threatening bleedings occurred twice after operations: after teeth extraction and after surgical procedures on the cervical spinal column. In an emergency case, she had to be treated by tracheostoma due to acute dyspnea. Bleeding stopped just a few days after receiving a large number of blood transfusions. Furthermore, heavy bleeding after delivery was reported following two pregnancies. In 2001, she suffered from acute myocardial infarction and heavy bleedings occurred after angiographic intervention. The tendency towards bleeding did not change during the course of years. There is no known history of hemophilia or other bleeding diseases in her family. Laboratory investigations showed normal values of PT (101 %), aPTT (36 sec), fibrinogen (272 mg %) and thrombocytes (244000 µl). The FVIII activity was lower than normal at 44 % (normal level: 64–167 %). A test for a FVIII gene mutation was carried out, and a missense mutation Thr2291IIe (6929C>T) was found. Additionally, the level of plaminogen activator inhibitor-1 (PAI-1) activity was also lower than normal, which could have intensified the bleeding tendency (PAI I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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activity: 0 U/ml, normal value: 0–22 U/ml; PAI-antigen: 3.4 ng/ml, normal value: 3–25.8 ng/ml). The parameters for the von-Willebrand syndrome and of platelet function were normal. No thrombophilic risk factors were found. This report shows that carriers of hemophilia A may show serious bleeding complications similar to those of a patient with mild or moderate hemophilia A. And however, the diagnosis of the carriers may often be made too late if there is no known family history of hemophilia.

Reference 1. Srámek A, Kriek M, Rosendaal FR. Decreased mortality of ischemic heart disease among carriers of haemophilia. Lancet 2003;362:351–4

Clinical Investigation of Orthopedic Outcome in Patients with Severe Hemophilia – Advantage of an Early Prophylactic Treatment? S. Meister, K. Christensen, A. Weidenhammer, T. Spranger, and G. Auerswald

Introduction A substantial goal in treatment of hemophilia is the avoidance of hemophilic arthropathy secondary to recurrent hemarthroses and chronic synovitis. In order to prevent joint damage effective prophylaxis should be started in an early age. Efficacy of early prophylactic treatment has been shown within recent years. Physical examination and joint status was performed to determine whether early long-term prophylaxis is favorable.

Patients Examination of all regularly treated patients in our hemophilia center (residual activity < 2%, born between 1980 and 1999) was conducted with the exception of one patient who had previously suffered from a cranial hemorrhage and spastic hemiplegia. At the time of investigation, all patients, except one with a high titer inhibitor and failed immune tolerance, completed prophylactic substitution treatment with different factor concentrates.

Physical Examination Altogether 59 patients were examined, 52 patients with hemophilia A and 7 patients with hemophilia B, the average age of the patients being 12.81 years, the prophylactic substitution treatment was begun at an average patient age of 4.58 years. A detailed study showed that 25 patients had restricted movement in one or more joints. Patients who were administered the prophylactic substitution treatment later in life were particularly affected. Therefore three groups were formed: patients in the group 1 were treated with prophylaxis at the age of 7 years or older. Group 2 started the prophylactic treatment in the age of 3–6 years, group 3 was less than three years old when prophylaxis was started.

I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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Treatment group with start of prophylactic treatment

Group 1 > 7 years

Group 2 3-6 years

Group 3 0-3 years

Number of patients

19

20

19

Age (median) / range ()

15.6 years (9-23)

11.2 years (6-22)

9.2 years (5-18)

Start of prophylactic treatment (median)

8.7 years

4.6 years

1.4 years

If one compares patients who started the prophylactic substitution treatment early in life (0–3 years, group 3) with patients who started the prophylactic substitution treatment at the age of 7 years or older (group 1) then a clear difference between the groups emerges. From a group of 20 patients who were administered the prophylactic substitution treatment earlier in life, only 20% had slight movement restrictions, whereas more noticeable movement restrictions were observed in 73.7% of patients who were administered the prophylactic substitution treatment later in life (Fig. 1). 65% of patients in group 2 had no movement restrictions. Regarding the severity of loss of full range of motion (FROM) we found great differences between the groups. All affected joints in group 3 showed just a discreet loss of FROM, whereas loss of FROM was usually greater in group 1 (Fig. 2). 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

>3 joints 3 joints 2 joints 1 joint None

Group 1

Group 2

Group 3

Fig. 1. Joints with limited ranges of motion in each patient

Fig. 2. Loss of full range of motion (FROM)

Number of affected joints

40 35

Group 1

30

Group 2

25

Group 3

20 15 10 5 0 Total

Discreet

up to 10°

11-20°

21-30°

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Number of affected joints

40 35 30 25

Group 1

20

Group 2

15

Group 3

10 5 0 Total

ankle

knee

elbow

hip

Fig. 3. Location of affected joints

The ankle joints were affected most often with movement restrictions. Especially in group 1 loss of FROM was often found in the elbow. Hip and knee were affected less often (Fig. 3). Location of bleeds stated in the case histories were similarly distributed within the groups, but patients of group 1 ware affected more often than patients of group 2 and 3. Most bleeds were reported in the ankle joints and knees. Less bleeds occurred in hip, hand and feet (Fig. 4).

ankle knee elbow hip hand foot

Fig. 4. Location of bleeds (all groups)

Summary This clinical investigation supports former data regarding the efficacy of early prophylactic treatment (Astermark et al., Fischer et al., Kreuz et al.). Comparing our three treatment groups, a clear difference between the groups emerges. Patients with early effective prophylaxis reported less bleeds and joints were clearly less affected by loss of range of motion. In our center, prophylactic treatment was started earlier within recent years. This accounts for the age difference between the three groups. But group 3 also includes some older patients which you can see by the range of age. In conclusion it appears that early long-term prophylaxis is strongly recommended in children with severe hemophilia.

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References 1. Astermark et al.: Primary prophylaxis in severe hemophilia should be started at an early age but can be individualized. Br J Haematol 1999; 105: 1109–13 2. Fischer et al.: The effects of postponing prophylactic treatment on long-term outcome in patients with severe hemophilia. Blood 2002; 99: 2337–41 3. Kreuz et al.: When should prophylactic treatment in patients with haemophilia A and B start? – The German experience. Haemophilia 1998 Jul;4(4):413–7

The Relevance of Thrombophilic Risk Factors on Bleeding Tendency in Hemophilia A Patients M. Krause, A. Hahn, F. Peyvandi, and I. Scharrer

Introduction By assuming the hypothesis, that a high coinheritance of genetic hemostatic abnormalities influences the bleeding tendency in patients with coagulation disorders, we analyzed adult patients with hemophilia A.

Patients and Methods A total of 42 patients with hemophilia A (median age: 44 years, range: 22–78 years) and 80 sex-matched healthy subjects (median age: 26 years, range: 16–58 years) were studied in our hemophilia treatment center (Table 1). In addition to factor VIII activity, FV G1691A mutation and the FII G20210A variant were investigated by PCR amplification and digestion of the fragments by MnII and HindIII. Table 1. Characteristics Patients [n = 42]

Controls [n = 80]

Median age [years]

44 [22–78]

26 [16–58]

Hemophilia A: severe moderate mild

17 10 15

Results In our study the prevalence of FV G1691A was higher among patients (5/42 patients) than among control subjects (2/80 controls) [12% vs. 2.5%; p = 0.04 OR 4.7]. We found no differences in the FII G20210A variant in 1/42 patient as compared to 2/80 among controls [2.4% vs. 2.5%; p = 0.96 OR 0.95]. In none of our patients both defects together were identified (Table 2). Within the last year the onset of bleeding was not different in thrombophilic hemophilia patients (n = 6) 48 bleeding episodes/year compared with non-carriers (n = 36) 251 bleeding episodes/year [p = 0.76, OR 0.87] (Table 3). I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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Table 2. Results I Patients [n=42]

Controls [n=80]

p

OR [95% CI]

FV G1691A

5 [12%]

2 [2.5%]

0.04

FII G20210A

1 [2.4%]

2 [2.5%]

0.96

4.7 [1.00 – 22.48] 0.95 [0.08 – 10.91]

Table 3. Results II

n number of bleeding episodes/year [range] bleeding episodes/ year/patient

non-carriers

FV G1691A/ FII G20210A

36

6

251

48

[0–60]

[0–21]

7

8

p

OR [95% CI]

0.76

0.87 [0.34–2.18]

Conclusion – The FV G1691A was found significantly more frequent in the hemophiliacs than in the general population. – In our small study group we could not demonstrate a relevant influence of the FV G1691A on bleeding tendency. – Further studies are needed to confirm whether FV G1691A plays a role on the response of FVIII replacement therapy in hemophilia A patients. References 1. Arbini AA, Manucci PM, Bauer KA. Low prevalence of the factor V Leiden mutation among ›severe‹ hemophiliacs with a ›milder‹ bleeding diathesis. Thromb Haomost 1995; 74:1255–1258 2. Dargaud Y, Meunier S, Negrier C. Haemophilia and thrombophilia: an unexpected association! Haemophilia 2004; 10:319–326 3. Escuriola Ettingshausen C, Halimeh S, Kurnik K, Schobess R, Wermes C, Junker R, Kreuz W, Pollmann H, Nowak-Göttl U. Symptomatic onset of severe hemophilia A in childhood is dependent on the presence of prothrombotic risk factors. Thromb Haemost 2001; 85: 218–220 4. Franchini M. Thrombotic complications in patients with hereditary bleeding disorders. Thromb Haemost 2004; 92: 298–304 5. Lee DH, Walker IR, Teitel J, et al. Effect of the factor V Leiden mutation on the clinical expression of severe hemophilia A. Thromb Haemost 2000; 83: 387–391 6. Nowak-Göttl U, Escuriola C, Kurnik K, Schobess R, Horneff S, Kosch A, Kreuz W, Pollmann H. Haemophilia and thrombophilia. What do we learn about combined inheritance of both genetic variations? Hämostaseologie 2003;23:36–40 7. Van Dijk K, van der Bom JG, Fischer K, Grobbee DE, van den Berg HM. Do prothrombotic factors influence clinical phenotype of severe haemophilia? A review of the literature. Thromb Haemost 2004; 92:305–310

Magnetic Field Therapy in Patients with Severe Hemophilia – Motion Analysis and Quality Control A. Seuser, G. Schumpe, T. Wallny, and H.-H. Brackmann

Material and Methods Ten subjects with severe hemophilia and severe hemarthropathy of both knee joints were treated for three months with a magnetic field applicator (manufacturer: Schober). The patients performed no other physiotherapy during this period. Their prior pain management regimen and factor VIII supply were continued unchanged. Motion analysis on a treadmill and during knee bends was performed for both knees before and after starting magnetic field therapy. Patients were provided with the Schober Medicare GmbH Enzymed 5004 magnetic field system consisting of a controlling device, plug and an applicator worn as a cuff on the extremities. The system produces a pulsating magnetic field, primarily in an oscillation range of 2 to 22 Hz. The impulse is a 5-fold sawtooth ramp signal. The treatment period per application is 20 minutes. The device is programmed to automatically produce a gradually beginning and gradually ceasing magnetic field effect at a range of 40 to 90 microtesla. Volunteers use the system 12 times daily on the affected knee joints for a period of three months.

How the Magnetic Field Works 1. In the vicinity of a magnetic field, more oxygen is delivered from the blood to the surrounding tissue. The increased oxygen supply widens the circulatory capillaries, resulting in better perfusion and hence a better supply of nutrients and hormones. This is a particular advantage in inflamed tissue. 2. Deflection of positive and negative ions in the magnetic field in proportion to the magnetic flow density and size of charge shifts the charge environment on cell membranes with a positive impact on intracellular metabolic processes. 3. Every organism has electrical conductivity. A magnetic field that changes over time creates a voltage in a conductor. The size of the voltage depends not on the magnetic flow density but on how it changes as a function of time. Rectangular and sawtooth impulses are therefore used to influence the tissue. The sawtooth impulses are more powerful. 4. Hydrogen ions are moved the most in the displacement current of the charge bearers in the magnetic field. This results in a pH shift with release of calcium ions and activation of NO synthesis. This in turn dilates the veins and activates macrophages. Regeneration processes are significantly speeded up. I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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The interaction of these physiological changes helps to reduce inflammation, relieve pain, and regenerate affected joints in the musculoskeletal system. A number of papers have demonstrated the positive effect, including double-blind studies in subjects with knee problems. These effects were to be studied in a group of hemophilic patients on the basis of a motion analysis.

Results 80% of patients reported a subjective improvement in their hemarthropathy symptoms. Motion analysis study disclosed an improvement of the functional endpoints investigated – angle, velocity and acceleration – in all the patients. A score was developed for more precise evaluation of the functional improvement, both for gait analysis and knee bends. Figure 1 (left) shows the results of gait analysis in all patients before and after treatment in lateral views from the right and left and in a view from behind. The functional score increased almost two-fold in both motion planes during the threemonth treatment period. Figure 2 shows distribution between the right and left knee and conclusively shows that both the right and left knee joints improved substantially in terms of their lateral stability and stance phase function. Figure 3 shows the right knee of patient FA during treadmill walking. It is interesting to note that stance phase activity is almost absent (top two figures) both before (figure on the left) and after treatment (figure right). Major improvements can be seen in the angle acceleration phases (bottom two figures). Angular acceleration decreased by almost half and describes a regular rhythmic, sinus curve. This is consistent with a significantly reduced joint strain. Patient MB presents a similar situation (Fig. 4). In this patient’s case, the stance phase of the knee joint (left – before treatment, right – after treatment) almost 2,5

improvment

2

after treatment

1,5

after treatment

1

before treatment

0,5

lateral view (right + left) 0

Fig. 1

before treatment view from behind (right + left)

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2,5

improvment

2

after treatment after treatment

1,5

1

after treatment after treatment

before treatment

before treatment

0,5

lateral view (right)

view from behind (right)

before treatment lateral view (left)

before treatment view from behind (left)

0

Fig. 2

doubled in terms of the angular action. The entire gait is more regular overall. The larger stance phase angle means better distribution of force over a larger joint area, thus helping to spare the joint. The positive impact of magnetic field therapy was particularly evident in gait analysis. Knee joint function during the strain imposed by knee bends benefited little.

Conclusion and Discussion Magnetic field therapy is a treatment modality that has no side effects, causes virtually no discomfort, and involves little restriction on freedom. Its role in orthopedic treatment is increasing. Our study shows that both subjective and objective outcome measures were improved by magnetic field therapy. The improvement is due to the physiological effects of magnetic field therapy as described above, which have been determined in many studies (see references). An improvement in blood flow and supply of nutrients to the joints helped to reduce inflammation in almost all patients. This in turn resulted in better muscular interaction and better joint control during gait. Given that no other physiotherapy was performed during the magnetic field treatment period, this effect appears to be attributable solely to magnetic field therapy. The fact that the positive effect has no impact on the knee bend outcome is easily explained. The latter imposes a strain on the knee joint that has more to do with strength than with coordination, and as such can only be safely and reliably affected by suitable weight training in addition to the above-mentioned physiological changes in the interior of the knee. We intend to include magnetic field therapy as a convenient and successful adjuvant in the treatment of chronically impaired joints. Magnetic field therapy in combination with coordination training and physiotherapy will make it possible to obtain even greater functional improvement for the patients concerned.

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Fig. 3

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Fig. 4

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References 1. Barovic, J., Fischer, G., Steigerung der Beweglichkeit und Schmerzlinderung der Erkrankungen des Bewegungsapparates durch Magnetfelder, Landeskrankenhaus Maribor und Universität Graz 2. Basset, C.A., Pawluk, R.I., Pilla, A.A., (1974), Acceleration of fracture repair by electromagnetic fields. A surgically noninvasive method. Annals NY, Acad.-Science 238,pp. 242–-261 3. Basset, C.A.L., Beneficial effects of electromagnetic fields, J. Cell. Biochem. 51, 1993 pp. 387–393 4. Capenter, David O., Ayrapetyan Sinerik, Biological effects of electric and magnetic fields – sources and mechanism, Volume 1, Academic Press, Inc., San Diego (1994) 5. Cohen, D., Detection and Analysis of Magnetic fields produced by Bioelectric Currents in Humans, Science 156 (1967), pp. 652–654 5. Goodman R., Chizmadzhev Y., Henderson A., Electromagnetic fields and cells, J. cell. Biochem. 51 (1993) pp. 436–441 6. Grigorieva, V.D., Tsarfis. P.G., Gerasimenko, V.N., Fiveiskaya, A.A., Shubina, A.V., Proskurova C.J., Volkova, L.P., Use of a Low-Frequency Permanent Magnetic Field in Patients with Deforming Osteoarthrosis and Rheumatoid Arthritis, Vopr.kurotol.fizioterapi, 4, 29–33 7. Ludwig, W., Therapie mit elektromagnetischen Feldern, Naturheilpraxis 09/200 8. McCleod, K.J., Donahue, H.J., Levin, P.E. Fontaine, M.A., Rubin, C.T., Electric fields modulate bone cell function in a density-dependent manner, J. Bone Miner. Res. 8 (1993) pp 977– 984 9. Rosen, A.D., Membrane response to static magnetic fields: Effect of exposure duration, Biochem. Biophys. Acta 1148 (1993) pp. 317–320 10. Stemme, O., Physiologie der Magnetfeldbehandlung – Grundlagen, Wirkungsweise, Anwendung. Dr. Otto Stemme Verlag, München 1992, ISBN 3–980–3094 11. Trock, D.H., et al., A Double-Blind Trial of the Clinical Effects of Pulsed Electromagnetic Fields in Osteoarthritis, Journal of Rheumatology, 1993:20, pp. 456–460

The Clinical Course of two Patients Receiving High Dose Factor VIII – Replacement Therapy K.H. Beck, and M. Mohrmann

Background The risk of thrombosis in conjunction with elevated factor VIII (FVIII) plasma levels is well known. Individuals exceeding plasma levels of 150% exhibit a risk for thromboembolic complications six times higher than those ranging below 100%. Perioperative FVIII plasma levels (bolus injection) in replaced adults should normally be close to 100% preoperatively, 50 % in the initial postoperative period and 30% thereafter. The overall replacement should be continued for 12 to 14 days (Aledort, Haemostasis-Forum [1]). The guidelines issued by the Federal Chamber of Physicians [2] are formulated less stringently. However, both attitudes represent an expert consensus on the basis of evidence. The physician in attendance takes his/her therapeutic decisions on the basis of the patient’s current clinical symptoms and his laboratory results. On the basis of §§ 12 and 275 SGB V [3] the Medical Service of the Health Care Insurances has to examine by random testing the necessity and economic efficiency of this replacement therapy. In the course of our examinations two comparable cases attracted our attention.

Patients A seventy-one year old man with an acquired von-Willebrand Syndrome suffering from a coxarthrosis received a total endoprothesis of his hip. Body weight was 76 kg, and FVIII:C amounted to 15 % plasma activity,VWF:RCof. 2 % and VWF:Ag 12,5 %. During the initial 6 days 64,000 U Haemate HS were replaced on a daily basis. Thereafter, the daily dose was lowered (see Fig. 1). Additionally, low molecular weight heparin was applied subcutaneously. The second case concerns a seventy-six year old woman with an unsecured vonWillebrand disease and without any family history. She underwent an osteosynthesis with a proximal femur nail because of a pertrochanteric femur fracture. During the initial postoperative period Haemate HS was replaced with a daily dose of 5,000 to 6,000 U, thereafter in a reduced amount. Perioperative heparinization was abandoned because of the potential bleeding risk. No clotting factor determinations in parallel with the replacement therapy were performed.

I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

The Clinical Course of two Patients Receiving High Dose Factor VIII

151

1200 IUx10e2 Haemate HS/day NovoSeven IU

1000 800 600

VW F:Ag (%)*

400 200

FVIII:C (%)*

* vWF:Ag=400 i.e. > 400,

9. 7.

7. 7.

5. 7.

3. 7.

1. 7.

29 .6 .

27 .6 .

25 .6 .

23 .6 .

21 .6 .

0

aPTT/10 (sec)

F VIII:C=500 i.e. > 500

Fig. 1. Perioperative replacement therapy of an aquired VWS with coxarthrosis and a total endoprothesis of the hip

IU x10Haemate

700

HS/day

600 VWF:Ag (%)

500 400

VW F:Rcof (%)

300 200

FVIII:C (%)

100 aPTT/10 (sec) 02 .1 0. 20 03 04 .1 0. 20 03 06 .1 0. 20 03 08 .1 0. 20 03 10 .1 0. 20 03 12 .1 0. 20 03 14 .1 0. 20 03 16 .1 0. 20 03

0

Fig. 2. Perioperative replacement therapy of an unsecured VWD with a femur fracture and consecutive osteosythesis

Clinical Course In the first case FVIII:C-levels higher than 500% were obtained, the VWF:Ag ranged beyond 400% (Fig. 1). The clinical course was without incidence. In the second case the preoperative FVIII:C-level was 242%, the VWF:Ag 214% (Fig. 2). No further factor VIII plasma levels were documented. The recorded aPTT values ranged from 25 to 27 seconds (normal range: 23-36 seconds). After the termination of the replacement therapy the VWF:Ag level was just beyond 400%, VWF:RCof. was 224%. Nine days postoperatively a fulminant lung embolism with a right ventricular heart failure and congestive hepatitis occurred. Lysis of the embolus was performed together with intensive care actions.

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Conclusion In the light of the data provided by the literature and with respect to the described cases it should be discussed if it could be reasonable to issue directives which are more emphatic and more detailed than the present guidelines.

References 1. Aledort L, Haemostasis-Forum/Question-Panel: 27-02-2003. Protocol existing for dose and time of duration for the factor needed for surgery in haemophiliacs 2. Leitlinien zur Therapie mit Blutkomponenten und Plasmaderivaten, Bundesärztekammer, Deutscher Ärzteverlag, Köln 2003 3. Sozialgesetzbuch V, § 12 – Wirtschaftlichkeitsgebot, § 275 – Begutachtung und Beratung

Determination of the Factor VIII Plasma Activity of Hemophilia A Patients Treated with a New Recombinant Factor VIII Concentrate K.-W. von Pape, R. Klamroth, U. Kyank, and J. Bohner

Introduction There are two assays suitable for the determination of the factor VIII activity in the patient’s plasma, both of which are commercially available and standardized: the onestage assay and the chromogenic assay. In clinical practice the one-stage assay has gained widespread acceptance due to its simplicity and level of automation. It is based on aPTT and assesses fibrin formation. The two-stage assay, though principally identical to the chromogenic assay, has been almost completely replaced by the latter in clinical practice. The rather more complex chromogenic assay utilizes the photometric detection of a dye which is released from a chromogenic substrate and in its formation speed correlates with factor VIII activity. This assay yields reproducible measuring results and is less sensitive to interference factors than the one-stage assay. It is recommended as a reference test by the ISTH and the European Pharmacopoeia [1]. The one-stage assay determines lower activities for recombinant factor VIII concentrates than the chromogenic assay. This difference is all the greater the more the concentration and composition of the phospholipids used in the aPTT reagent deviate from the physiological state [2]. Interlaboratory experiments showed that if the standardized test protocol of ISTH/SSC is complied with, the discrepancies between one-stage and chromogenic assay are limited to a maximum of 10 % with recombinant concentrates [1]. This difference should not be of any relevance in daily clinical practice. The study presented here is intended to investigate whether those results also apply to ADVATE, a new recombinant factor VIII concentrate. ADVATE only differs from its precursor Recombinate in the elimination of human or animal derived additives during manufacturing, and the introduction of the S/D procedure. For that reason, one-stage assay should be equally suitable for treatment control as with Recombinate. Therefore, we have compared four different aPTT reagents using the one-stage assay. As a reference we used the measuring results obtained by means of a chromogenic assay.

Material and Methods Five patients with severe hemophilia A were treated with one dose of ADVATE each. The dosages were chosen so as to reach an activity of at least 50 %. Blood samples I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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were taken prior to as well as 30, 60, 120 and 240 minutes after infusion of ADVATE. After centrifugation of the citrated blood the factor VIII activity in the plasma was determined using both the chromogenic and the one-stage assay. The chromogenic assay was performed with Coamatic Factor VIII by Chromogenix. Calibration was done using a Hitachi 917 (Roche). The one-stage assay was performed on each plasma sample using Dade Bering’s Blood Coagulation System (BCS) and four different aPTT reagents: Actin FSL (Dade Behring), Dapttin (TC/Technoclone), SynthASil and SP (Instrumentation Laboratory). Dade Behring Standard-Human-Plasma was used as a calibrator.

Results and Discussion Tables 1–3. One-stage assay (4 different aPTT reagents) vs. chromogenic assay (Coamatic, Chromogenix) at three time points after ADVATE infusion (30, 60, 240 min). Potencies have been calculated as a percentage of those obtained with the chromogenic method 30 min post infusion Potencies (%) with 4 different aPTT Reagents Patient

Actin FSL

Dapttin

SynthASil

SP

1 2 3 4 5

76 78 104 86 92

83 78 105 92 98

79 85 86 93 94

67 88 90 83 86

60 min post infusion Potencies (%) with 4 different aPTT Reagents Patient

Actin FSL

Dapttin

SynthASil

SP

1 2 3 4 5

89 85 100 84 86

86 85 98 88 98

98 87 90 82 94

73 91 86 71 90

240 min post infusion Potencies (%) with 4 different aPTT Reagents Patient

Actin FSL

Dapttin

SynthASil

SP

1 2 3 4 5

87 106 103 91 88

77 103 93 91 93

80 100 90 87 90

80 103 90 85 86

Determination of the Factor VIII Plasma Activity of Hemophilia A Patients

Patient 2, 2000 IU Advate

80

80

70

70

60

60

Factor VIII [%]

Factor VIII [%]

Patient 1, 2000 IU Advate

50 40 30 20 10

50 40 30 20 10

0

0 0

50

100 150 Time [min]

200

250

0

Patient 3, 2000 IU Advate

50

100 150 Time [min]

200

250

Patient 4, 2000 IU Advate

80

80

70

70

60

60

Factor VIII [%]

Factor VIII [%]

155

50 40 30 20 10

50 40 30 20 10

0

0 0

50

100 150 Time [min]

200

250

0

50

100 150 Time [min]

200

250

Patient 5, 1250 IU Advate 80

Factor VIII [%]

70

Actin FSL Dapttin SynthAsil SP Coamatic F VIII

60 50 40 30 20 10 0 0

50

100 150 Time [min]

200

250

Figs. 1a–e. Temporal progression of the factor VIII activities of five patients following one ADVATE infusion, measured using one-stage and chromogenic assay

Our studies, too, confirm that the average results of factor VIII determination using a one-stage assay are lower than those obtained by means of chromogenic assay. In Tables 1–3 the factor VIII measurements obtained using the one-stage assay with 4 different aPTT reagents are represented as a percentage of the activity values measured by means of the chromogenic assay, with the exemplary time points of 30, 60

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Table 4. Average and range of the measuring values of each of the three blood samples taken after 30, 60 and 240 min, expressed as a percentage of the respective value from the chromogenic assay

30 min post infusion 60 min post infusion 240 min post infusion

Mean of percentage

Range of percentage

87 88 91

67 – 105 71 – 100 77 – 106

and 240 min after ADVATE infusion. One can see that many measuring values are close to the chromogenic value, sometimes even exceeding it. The average and the range of the values represented as a percentage of the chromogenic measuring values (defined as 100 %) are shown in Table 4. The four different phospholipid reagents we used in the one-stage assay in order to determine factor VIII activity after ADVATE infusion resulted in equivalent values for the five patients at all time points investigated. This is clearly illustrated in Figure 1. Thus, the one-stage coagulation tests we have assessed, are all equally suitable for monitoring and controlling treatment with the new recombinant factor VIII concentrate, ADVATE.

References 1. Barrowcliffe T.W. Standardization of FVIII & FIX assays. Haemophilia July 2003; 9: 397–402 2. Mikaelsson M, Oswaldsson U, Sandberg H. Influence of phospholipids on the assessment of factor VIII activity. Haemophilia March 1998; 4: 646–650

Socio-Economic Aspects of Hemophilia Treatment in Romania D. Mihailov, M. S¸erban, B. Lippert, W. Schramm, and S. Arghirescu

Introduction Hemophilia is a model of chronic disease having a treatment which is limited by several factors, the economic ones being the most important. In our country, »on demand« substitution is the mean treatment modality, using especially fresh-frozen plasma (FFP) and cryoprecipitate. These products (especially cryoprecipitate) are not available in all medical centers which offer treatment for hemophiliacs. In the absence of hemophilia comprehensive care centers and of home therapy programs, early treatment of bleeding episodes is not possible, the patients traversing long distances to a hospital where they can receive substitutive therapy. Due to their high costs, clotting factor concentrates are administrated only for severe hemorrhagic episodes and surgical interventions, often in suboptimal doses. Inadequate »on demand« treatment, as well as the absence of prophylactic therapy lead to unfavorable long and short-term consequences, affecting the social integration of the patients.

Aim of the Study The aim of our study was to assess the costs of hemophilia treatment and its socioeconomic consequences.

Material and Methods Our study group consisted of 224 hemophilia patients (84.38% hemophilia A and 15.62% hemophilia B), registered and treated in the Hemophilia Center Timis˛oara and in the Clinical Center for Evaluation and Rehabilitation »Cristian S¸erban« Buzias¸. Most patients had severe hemophilia (61.61%), 20.53% had moderate and 17.86% presented mild form. 11.61% of hemophiliacs were from Timis˛oara, 10.27% were from Timis county and 78.12%, from other counties. The patients were followed-up during January 1997-December 2003. We evaluated: – direct medical costs: therapy and hospitalization costs of all hemorrhagic episodes, secondary prophylactic therapy and surgical interventions; I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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– direct non-medical costs: home-hospital travel costs; – indirect costs: morbidity costs; loss of income of family members who forfeit paid employment in order to offer home care for hemophilia patient; average number of days off at school or work; – socio-economic consequences of the treatment: social integration of the patients, treatment compliance. Data regarding hemorrhagic episodes, secondary prophylactic therapy, surgical interventions and their replacement therapy and hospitalization duration were obtained from retrospective patients’ charts review. Knowing that in a developing country an economic analysis is difficult to realize, because of high inflation rate, unitary cost of clotting factor and hospitalization were expressed in €, at average exchange rate communicated by National Romanian Bank for 2003. Direct medical costs were calculated starting from the following unitary values (according to the National Catalogue of Medication Price, and the data offered by the Accounting Department of the hospitals, respectively): – 1 hospitalization day in the IIIrd Pediatric Clinic ≈ 99 € – 1 hospitalization day in a Surgical Clinic ≈ 101 € – 1 cryoprecipitate/FFP Unit ≈ 10 € – 1 IU FVIII/FIX ≈ 0.5 € – 1 IU APPC ≈ 1 € – 1 kIU rFVIIa ≈ 12.8 € For direct non-medical costs, indirect costs and socio-economic consequences of the treatment evaluation, a questionnaire containing questions regarding these aspects was administered to the patients who expressed their agreement to participate in our study. After data collection, we considered 177 questionnaires which offered complete answers to all questions.

Results Direct Medical Costs Therapy costs represented 54.56% of direct medical costs in hemophilia A patients without inhibitors 67.13% of them in hemophilia B patients, and 87.63% in hemophilia A patients with high-titer inhibitors. Pseudotumour was the most costly complication in hemophilia A patients with or without high-titer inhibitors (Table 1 and 2), and complicated hematomas consumed the highest financial resources in hemophilia B patients (Table 3). Secondary prophylaxis was administrated only in hemophilia A patients without inhibitors: 10 cases with chronic synovitis and 8 cases with psoas hematoma. Secondary prophylaxis could be administrated only for short periods (an average of 4.3 weeks for chronic synovitis and 4.13 weeks for psoas hematoma respectively), which leads to unfavorable consequences, especially in the case of psoas hematoma.

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Table 1. Direct medical cost in hemophilia A patients without high-titer inhibitors Hemorrhagic episode*

Joint bleeding Uncomplicated hematoma Complicated hematoma Pseudotumour Dental extraction Head trauma Wound bleeding Gastrointestinal bleeding Epistaxis Hematuria Bone fracture/fissure/strain

No. of episodes 501 134 60 1 96 31 23 9 8 39 14

DIRECT COST/EPISODE (€) Substitution

Hospitalization

TOTAL

391.69 844.74 1 366.53 78 420.00 404.53 656.92 403.48 3 030.00 60.00 379.49 1 729.32

437.89 618.38 1 036.20 21 087.00 464.06 648.29 396.00 1 188.00 358.88 467.08 926.36

829.58 1 463.12 2 402.73 99 507.00 868.59 1 305.21 799.48 4 218.00 418.88 846.56 2 655.68

*35 combined hemorrhagic episodes Table 2. Direct medical cost of hemorrhagic episodes in hemophilia A patients with high-titer inhibitors Hemorrhagic episode

Joint bleeding Uncomplicated hematoma Complicated hematoma Pseudotumour Dental extraction Wound bleeding Epistaxis Hematuria Bone fracture

No. of episodes 39 14 1 5 1 2 3 1 1

DIRECT COST/EPISODE(€) Substitution

Hospitalization

TOTAL

4 054.56 8 064.86 30 772.00 47 664.80 11 000.00 9 296.00 3 649.33 1 000.00 2 400.00

408.69 1 173.86 1 980.00 8 236.80 198.00 1 584.00 924.00 495.00 297.00

4 463.25 9 238.72 32 752.00 55 901.60 11 198.00 10 880.00 4 573.33 1 495.00 2 697.00

Table 3. Direct medical cost of hemorrhagic episodes in hemophilia B patients Hemorrhagic episode

Joint bleeding Uncomplicated hematoma Complicated hematoma Dental extraction Wound bleeding Head trauma Epistaxis Hematuria Bone fracture

No. of episodes 70 8 8 1 1 3 3 2 1

DIRECT COST/EPISODE (€) Substitution

Hospitalization

TOTAL

699.14 782.50 1 360.00 1 200.00 60.00 270.00 20.00 1 395.00 360.00

308.31 420.75 618.75 198.00 198.00 429.00 660.00 396.00 594.00

1 007.45 1 203.25 1 978.75 1 398.00 258.00 699.00 680.00 1 791.00 954.00

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Fig. 1. Orthopedic and surgical interventions in hemophilia patients

Mean direct medical costs of secondary prophylaxis were 1 922.11 € (866.11 €-substitution cost and 1 056 €-hospitalization cost). During the study period, there were 54 orthopedic and surgical interventions in 37 hemophilia patients (Fig. 1). Costs of surgical interventions represented 37.89% of total direct medical costs (1 146 118 €): 45.43% (1 044 613 €) of total substitution costs and 14% (101 505 €) of total hospitalization costs.

Direct non-Medical Costs Means of transportation used by our patients were: car (72.31% of cases), train (26.55%), bus (23.16%), taxi (22.03%) and ambulance (31.07%). Home-hospital travel costs represented important percents of mean family or patient income (Table 4) and lead to a poor treatment compliance. Table 4. Direct non-medical costs Mean of transportation

CAR

TRAIN

BUS

TAXI

AMBULANCE

Travel cost (€)

6.58

10.12

2.35

1.18

Supported by society

% of MFI* % of MFI/MB** % of MPI***

3.84 11.73 18.38

6.77 20.92 35.05

1.50 4.97 7.12

0.63 2.00 2.96

* mean family income/month ** mean family income/no. of family members/month *** mean personal income/month

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Indirect costs a) Morbidity costs. Mean personal income per month in our patients was: preschool children-10.17 € (SD = 1.31), students-12.18 € (SD = 6.87), university students-32.3 € (SD=9.7), employees-142.63 € (SD = 48.05), handicapped with social support34.35 € (SD = 4.76). Monthly morbidity cost of hemophilia patients was 108.28 €. b) Loss of income of family members who forfeit paid employment in order to offer home care for hemophilia patient is another important consequence of hemophilia and its treatment. In our study group, in 62.71% of the cases, one family member forfeit paid employment in order to offer home care for hemophilia patients. Distribution of the hemophiliacs who need home care, according to hemophilia severity was: 68.47%-severe, 22.52%-moderate and 9.01%-mild form. Mean income was lower in these families, the income difference being 81.88 €/month (Fig. 2). c) Average number of days off at school or work was 46.64/year (Table 5), hemophilia severity having an important influence on these absences, probably due to higher bleeding tendency of hemophiliacs with severe form. Table 5. Average number of days off at school or work CATEGORY

Average no. of days/year Mean

SD

p

Socio-professional status

Student University student Employee

45.23 53.00 47.84

24.02 22.27 24.30

0.5417

Hemophilia severity

Mild Moderate Severe

19.56 31.48 59.30

8.60 16.08 19.20

<0.0001

Age group

< 18 years >18 years

50.24 44.65

20.78 23.73

0.2471

46.64

23.79

TOTAL 240 200 160 120 80 40 0

with forfeiture

(62,71%)

–

without forfeiture (37,29%)

MFI*-Mean (€)

137,74

219,62

MFI*-SD

32,51

92,61

MFI/MB**-Mean (€)

38,13

81,19

MFI/MB**-SD

14,86

45,84

p<0,0001 p<0,0001

Fig. 2. Comparison of mean family income and mean family income/no. of family members, according to paid employment forfeiture of a family member

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Socio-Economic Consequences of the Treatment Socio-professional status of adult hemophiliacs was: 62.36% – handicapped with social support, 22.35% – employed and 15.29% – university students. Marital status of the adults was: 85.88%-single, 9.41%-married, 3.53%-divorced and 1.18%-widower. 77.4% of the patients missed hospitalization for financial reasons, on an average of 9.89 times/year (SD = 8.26), and 78.53% of the patients came too late at the hospital, for financial reasons, on an average of 9.12 times/year (SD = 7.57). Mean family income was lower in these families, the differences being statistically significant (Table 6, 7). Table 6. Mean income of patients who missed hospitalization for financial reasons, compared to patients who didn’t miss hospitalization Mean income/month

MFI (€) MFI/MB (€) MPI (€)

Patients who missed hospitalization

Patients who didn’t miss hospitalization

mean

SD

mean

SD

155.36 50.08 31.46

57.43 33.50 33.71

212.50 68.26 44.63

101.47 43.77 63.10

p

< 0.0001 0.0056 0.0835

Table 7. Mean income of patients who came too late at hospital, for financial reasons compared to patients who didn’t came too late Mean income/month

MFI (€) MFI/MB (€) MPI (€)

Patients who came too late

Patients who didn’t come too late

mean

SD

mean

SD

155.82 49.65 31.77

57.85 33.35 34.04

213.81 70.78 44.21

102.53 43.75 63.81

p

< 0.0001 0.0015 0.1086

Conclusions and Discussions Low proportion of therapy costs reported to total direct medical costs were due to extensive use of FFP and cryoprecipitate, which are cheap products, but offer a suboptimal therapy. In hemophilia A patients with high-titer inhibitors, limited access to therapeutic products (due to their high costs) lead to their delayed administration, in suboptimal doses, leading to supplementary hospitalization. Pseudotumour was the most costly complication in hemophilia A patients (both with or without inhibitors), while in hemophilia B, the other complicated hematomas consumed the highest financial resources. Because both complications resulted from suboptimal treatment of muscular hematoma, we conclude that »savings« achieved by administration of cheap products (FFP and cryoprecipitate), in low doses, lead to costly complications.

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Secondary prophylactic substitution was administered only in 10 hemophilia A patients with chronic synovitis and 8 patients with psoas hematoma, the major disadvantage being that it could be achieved only in hospital, for short periods (mean period = 4.22 weeks), so that the results were unfavorable for psoas hematoma. We can notice that the need for patients’ hospitalization in order to therapy administration had a negative influence not only on direct non-medical costs and indirect costs, but it also increases direct medical costs (they represented 19 008/34 598 = 54.94% of total direct medical costs of secondary prophylaxis, also because FFP and cryoprecipitate assured most of the substitution). From the sum corresponding to direct hospitalization costs, 38 016 IU FVIII proceeding from factor concentrates could have been purchased. Starting from the observation that all complications which required surgical interventions also resulted from suboptimal substitutive therapy, and their direct medical costs represented important percents of total costs, we can conclude that adequate resources allocation, which allows an optimal treatment of hemophilia patients, lead to other financial resources savings. Direct non-medical costs (travel costs to and from the hospital) represented important percents from mean family income/month, mean family income/number of family members/month and mean personal income/month; social protection measures from our country didn’t cover these costs for hemophilia patients, leading to a poor treatment compliance. The majority of hemophilia adults are handicapped with social support, single and unable to have a paid job, because of the disease and its complications. Poor social integration of the patients leads to unfavorable consequences, and high morbidity costs are responsible for treatment compliance problems, which can result in supplementary complications and increase treatment costs for the provider. Inadequate financial resources allocation, absence of Hemophilia Comprehensive Care Centers and incomplete evidence of hemophiliacs are responsible for inadequate hemophilia treatment in our country, which leads to costly complications, affecting life expectancy and social integration of these patients, resulting in important loss of income for both hemophiliac and family members and also in a poor treatment compliance. All these factors present a strong interaction, need additional financial resources and affect life quality of the patients. References 1. Aledort LM. Orthopedic Outcome Studies and Cost Issues. Semin Thromb Hemost 2003; 29(1):55–60 2. Colgrove RW, Huntzinger RM. Academic, behavioral, and social adaptation of boys with hemophilia/HIV disease. J Pediatr Psychol 1994; 19:457–473 3. Ekert H, Brewin T, Boey W, et al. Cost-utility analysis of recombinant factor VIIa (NovoSeven) in six children with long-standing inhibitors to factor VIII or IX. Haemophilia 2001; 7(3):279–285 4. Kvist B, Kvist M, Rajantie J. School absences, school achievements and personality traits of the haemophilic child. Scand J Soc Med 1990; 18(2):125–132 5. Lundberg O. Childhood living conditions, health status, and social mobility: a contribution to the health selection debate. Eur Soc Rev 1991; 7:149–162

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6. Markova I, McDonald K. Integration of hemophilic boys into normal schools. Child Care Health Dev 1980; 6:101–109 7. Olch D. Effects of hemophilia upon intellectual growth and academic achievement. J Genet Psychol 1971; 119:63–74 8. Rosendaal FR, Smit C, Varekamp I et al. Modern haemophilia treatment: medical improvements and quality of life. J Intern Med 1990; 228(6):633–640 9. Schramm W, Berger K. Linking medicine and economics: health economics and quality of life in haemophilia care. Haemophilia 2002; 8:217–220 10. Schramm W, Royal S, Kroner B, et al. Clinical outcomes and resource utilization associated with haemophilia care in Europe. Haemophilia 2002; 8:33–43 11. van de Mheen H, Stronks K, Looman CWN, Mackenbach JP. Role of childhood health in the explanation of socioeconomic inequalities in early adult health. J Epidemiol Community Health 1998; 52:15–19 12. Weitzman M. School absence rates as outcome measures in studies of children with chronic illness. J Chronic Dis 1986; 39:799–808 13. Woolf A, Rappaport L, Reardon P, et al. School functioning and disease severity in boys with hemophilia. J Dev Behav Pediatr 1989; 10:81–85

Immunosuppressive Treatment in Acquired von-Willebrand’s Syndrome H.-H. Wolf, A. Harba, A. Frühauf, and H.-J. Schmoll

Introduction In contrast to congenital von-Willebrand (VW) disease acquired VW syndrome is a rare bleeding disorder representing 0.4 % of VW patients. Mostly complicating either hematologic neoplasias like myeloproliferative syndromes and Non Hodgkin’s lymphomas, autoimmunologic diseases, or drug administration, degree of clinical manifestations may be various [1, 2]. Clinical and laboratory data are different to hereditary VW disease [3]. Presenting in patients without any history of bleeding diathesis acquired VW syndrome is characterized by loss of high molecular multimeric structure of VW proteins. In most patients accelerated clearing of plasmatic VW factor (VWF) is found, either due to circulating anti VW protein autoantibodies, shear-induced selective adsorption of VWF multimers onto malignant cells or platelets, or proteolysis. ADAMTS13 may be a protease that is able to cleave VWF multimers [4].

Patient’s Characteristics We report a 72-year-old female patient without history of hemorrhages admitted to the hospital for anemia, severe spontaneous nasal bleeding, and sugillations after minor injuries. The patient’s condition was reduced because of obesity and arterial hypertension. Physical examination was without any pathological findings. There were no lymphomas and no hepatosplenomegaly.

Lymphoma Staging Thoracic and abdominal computed tomography showed normal findings. Invasive diagnostic procedures like bone marrow biopsy or gastroscopy could not be performed because of severe hemorrhagic diathesis. Cytological and immunological bone marrow examination revealed a 5 % bone marrow infiltration of B cell lymphoma; flow cytometry proved the cells positive for I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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CD 20+, CD 19+ and FMC7+, but CD 23– and CD 5– negative. Therefore, histopathological diagnosis was marginal zone staining B cell lymphoma. MRI revealed 3 focal lesions of the thoracic spine.

Hematological Parameters At time of admission we found microcytic anemia (Hb 3,8 mmol/l), abnormal aPTT and PFA tests as well as low plasma concentrations of VWF antigen,VWF Ristocetin cofactor, and VWF collagen binding activity, respectively. VWF CBA/VWF antigen ratio was 0.41. Multimeric analysis revealed VW syndrome type 2B and loss of major multimers (Prof. Budde, Hamburg). Coombs test was negative, no paraproteins have been found neither in serum nor urine. HLA antibodies as well as platelet glycoprotein anti- Ia/IIa, IIb/IIIa, Ib/IX autoantibodies could be detected. Plasma dilution test did not reveal increasing plasma concentrations of VW protein. Substitution Therapy After admission substitution therapy with plasma-derived FVIII-VWF concentrate (dosage 60 IU/kg body weight twice daily) had been started without any success. Acute hemorrhages could not be stopped. 16 packed red cells had to be administered. We started immunoglobulin therapy 0.1 g/kg body weight intravenously once a week over a three months period and prednisolon 1 mg/kg body weight orally in order to enable bone marrow puncture. Spontaneous hemorrhages and frequency of transfusion slowly improved, but parameters of coagulation did not normalize. Immunosuppressive Therapy Aggressive cytotoxic chemotherapy could not be administered because of the patient’s poor condition. We decided to start therapy with monoclonal anti CD20 antibody Rituximab 375 mg/m2 body surface area intravenously six times within a three months period (day 1, 8, 15, 22, respectively, followed by one single infusion every 4 weeks). Immunoglobulin therapy was administered every 4 weeks. After three weeks of therapy coagulation parameters normalized. For plasma concentrations of VWF proteins see Table 1. Radiation therapy of vertebral infiltrations was started (total dosage 36 Gy). Maintenance therapy with azathioprine had been administered over a 6 months period. Remission duration of Non Hodgkin’s Lymphoma was 14 months. Due to progressive bone marrow involvement aggressive chemotherapy had to be started. Paraneoplastic von-Willebrand`s disease disappeared completely.

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Table 1. Changes in coagulation parameters meanwhile therapy VWF antigen

admission 25 immunoglobulins 37 before Rituximab 51 after Rituximab 176

VWF Ristocetin cofactor %

VWF FVIII collagen % binding activity %

PFA COL/ADP sec

aPTT platelets sec Gpt/l

8 2 2 >170

15 14 15 98

> 263 > 196 > 214 127

34 36 31 27

21.1 28.5 24.1 146.8

109 88 126 107

Conclusions Immunoglobulin therapy combined with prednisolon proved to be an appropriate therapy of acquired VW syndrome in order to enable invasive diagnostic procedures but not sufficient in order to control bleeding disorders. In our patient there was no chance improving hemorrhagic diathesis without immunosuppressive therapy [5]. Combined Rituximab and steroid therapy was able to rise VWF plasma concentrations within a short period of time probably due to effective B cell suppression rather than reduction of plasmatic clearance [6, 7]. We conclude that bone marrow examination is essential in patients with acquired VW syndrome. Diagnostic procedures should be performed prior to immunosuppressive therapy to reveal underlying hematologic malignancies.

References 1. Association of Hemophilia Clinic Directors of Canada (AHCDC): Hemophilia and von Willebrand`s disease: 2. Management. CMAJ 153, 147–57, 1995 2. Shinagawa A., Kjima H., Berndt M.C., et al.: Characterization of a myeloma patient with a life-threatening hemorrhagic diathesis: presence of a lambda dimer protein inhibiting shear-induced platelet aggregation by binding to the A1 domain of Willebrand factor. Thromb Haemostas 93 / 3 3. Lollar P.: Pathogenic antibodies to coagulation factors. Part II. Fibrinogen, prothrombin, thrombin, factor V, factor VXI, factor XII, factor XIII, the protein C system and von Willebrand factor. 4. Rieger M., Mannucci P.M., Kremer Hovinga J.A., et al.: ADAMTS13 autoantibodies in patients with thrombotic microangiopathies and other immunomediated diseases. Blood online, May 17, 2004-11-4490 5. Lipkind H.S., Kurtis J.D., Powrie R., et al. : Acquired von Willebrand disease: management of labour and delivery with intravenous dexamethasone, continuous factor concentrate, and immunoglobulin infusion. Am J Obstet Gyencol 2005, 192, 2067–70 6. Fakhouri F., Vernant J.-P., Veyradier A., et al.: Efficiency of curative and prophylactic treatment with rituximab in ADAMTS13 deficient-thrombotic thrombocytopenic purpura: a study of 11 cases. Blood online, June2, 2005-03-0848 7. Miura S., Li C.Q., Cao Z., et al.: Interaction of a von Willebrand Factor Domain A1 with platelet glycoprotein Ib alpha-(1-289): slow intrinsic binding kinetics mediate rapid platelet adhesion. J Biol Chem 275, 7539–46, 2000

HCV-Infection in HIV-Infected and Non-Infected People with Hemophilia – A Retrospective Study: Medical Aspects H. Krebs, M.M. Schneider, and W. Schramm

Objectives HIV- and HCV-infections are still dominating issues in treatment of patients with hemophilia. Cumulated German data since 1982 of deceased patients with hemophilia showed a significant increase of patients dying of liver disease and cancer in recent years. While improved therapy has changed the long-term outcome of HIVinfection significantly, a rising number of liver cirrhosis and cancer is responsible for a combined 44% of all reported deaths in the 02/03 survey [3]. To investigate which medical and psychosocial factors can influence long-term survival we initiated a retrospective study in 100 patients with severe hemophilia A or B with and without HIV/HCV-infection.

Methods Based on data over the last 18 years we retrospectively compared medical and psychosocial parameters of 100 patients with hemophilia A (n = 89) or B (n = 11).A portion of 37 patients has died mainly of AIDS during observation time while 63 patients are still alive (see Fig. 1 and Table 1 for distribution of causes of death). The distribution of infections (HIV/HCV) is given in Table 2. HCV-treatment with IFN-a2b or PEG-IFN-a-2a and Ribavirin was given to 13 out of 91 HCV+ patients. In five cases treatment was successful (41,7% »sustained response«: > 6 months after end of treatment without evidence of HCV-RNA), one patient still is under treatment. Almost all of the patients received psychological support in the past two decades. We clustered data into four time frames: (1986–1989, 1990–1995, 1996–2000, 2001–2004) for statistical analysis. We compared time dependent CD4+-status, HIVand HCV-RNA levels, HCV-genotype, ALT-levels, parallel performing of a HIV- or HCV-ART and certain psychosocial parameters derived from in-depth psychological interview-technique and group rating (see article about psychosocial aspects).

Results As shown in Figure 2 the difference of HIV+/HCV+ patients regarding ALT-levels amongst periods 1986–1989 and 2001–2004 is highly significant, while HIV-/HCV+ I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

HCV-Infection in HIV-Infected and Non-Infected People with Hemophilia

Fig. 1. Causes of deaths Table 1. Distribution of causes of death 1986–1989

1990–1995

1996–2000

2001–2004

HIV bleeding liver disease other unknown

1 0 1 0 0

13 3 1 2 0

5 4 0 0 1

2 1 2 1 0

total*

2

19

10

6

* for one patient we do not know the exact date of death Table 2. Distribution of infections HCV+

HCV–

unknown

S

HIV+

62

4

3

69

HIV–

29

2

0

31

total*

91

6

3

100

169

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b)

a)

Fig.2a,b. ALT-levels [IU/l] of HCV infected patients with and without HIV between 1986 and 2004

patients do not show a relevant discrepancy in the same time frame (HIV+/GOT: p =0.001; HIV-/GOT: p =0.685; HIV+/GPT: p <0.001; HIV-/GPT: p =0.224; calculated with paired t-test). Those HIV+ patients receiving HIV-ART show higher mean ALT-levels especially in period 2001–2004 than untreated patients (GPT: p =0.044; GOT: p =0.066; calculated with t-test; see Fig. 3). HIV+ patients who died within the

a)

b)

Fig. 3. ALT-levels [IU/l] of HIV+/HCV+ patients with and without HIV-ART between 1986 and 2004

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Fig. 4. CD4+ cells/ml of HIV+ patients alive vs. deceased

Table 3. Success of HCV-treatment HCV-treatment

N

HIV+

%

N

HIV–

%

N

success no success

2 6

25 75

3 1

75 25

5 7

total*

8

100

4

100

12

S

% 41.7 58.3 100

* one patient is still under HCV-treatment

observation period showed significant lower CD4+ cells/ml already in the first time frame 1986 – 1989 than those patients still alive (p =0.002; calculated with t-test; see Fig. 4). HCV-treatment was successful in 75% of HIV- patients but only in 25% of HIV+ patients (see Table 3). It is striking that these medical results correspond highly to psychosocial parameters showing the same significant differences between HIV+ alive and deceased patients within the same time frames (see poster about psychosocial aspects). These clues are obvious indications for a higher survival rate of HIV+ patients with good psychosocial parameters.

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Conclusion Despite mortality from HIV in patients with hemophilia is keeping on decreasing, HIV still remains an important factor as an HIV/HCV co-infection seems to increase risk of progression of liver disease to cirrhosis and hepatocellular carcinoma [1]. As HIV+/HCV+ patients show higher mean ALT-levels than HIV-/HCV- and an accompanying HIV-ART even seems to booster this effect, there are evident arguments for an early onset of an effective HCV-therapy in spite of still limited treatment possibilities and low success rates especially in HIV/HCV co-infected patients [2, 4]. Furthermore our findings show that there are evident medical and psychosocial parameters triggering the progression of HIV and influencing survival rates. More quality of life research should be initiated to be able identifying those patients with a bad prognosis more early in future. The basis for quality of life research are validated instruments.

References 1. Darby SC, Ewart DW, Giangrande PL, Spooner RJ, Rizza CR, Dusheiko GM, Lee CA, Ludlam CA, Preston FE: Mortality from liver cancer and liver disease in haemophilic men and boys in UK given blood products contaminated with hepatitis C. UK Haemophilia Centre Directors’ Organisation. Lancet. 1997 Nov 15;350(9089):1425–31 2. Lee C, Dusheiko G: The natural history and antiviral treatment of hepatitis C in haemophilia. Haemophilia. 2002 May;8(3):322–9 3. Schramm W, Krebs H. HIV Infection and Causes of Death in Patients with Hemophilia in Germany (Year 2001/2002 Survey). In: Scharrer I, Schramm W (Hrsg) 33rd Hemophilia Symposium Hamburg 2003, pp 3–12, Springer-Verlag, Berlin 4. Yee TT, Griffioen A, Sabin CA, Dusheiko G, Lee CA: The natural history of HCV in a cohort of haemophilic patients infected between 1961 and 1985. Gut. 2000 Dec;47(6):845–51

HCV-Infection in HIV-Infected and Non-Infected People with Hemophilia – A Retrospective Study: Psychosocial Aspects M.M. Schneider, H. Krebs, and W. Schramm

Objectives Since the enormous improvements in HIV-treatment there is a distinct decrease in mortality connected with HIV and a reported increase of dying of liver diseases connected with HCV among hemophilia patients. The fact, that almost everyone (about 98%) of our patients with hemophilia is HCV-infected, points out the importance of this dangerous infection. So we tried to find out if there are certain medical and/or psychosocial factors influencing the course of diseases in people with hemophilia, in order to support the patients in question in their fight with lifethreatening illness. Thus we initiated a study in 100 of our HCV-infected patients. As we didn’t get every single parameter from each (either medical or psychosocial data) we recur on this article on a total amount of 94 patients. alive

deceased

S

HIV+

37

20 (AIDS) 10 (liver/others)

67

HIV–

22

5 (liver/others)

27

total

59

35

94

Methods Our investigation of these 94 valid patients (about 2/3 of them are HIV-co-infected) is based on retrospectively raised medical data (HIV, HCV, ALT, ART etc.) over the last 19 years and on psychosocial parameters derived (also retrospectively) from indepth psychological interviews with the following personality-factors »Stable Self Esteem« (SSE), »Future Orientation« (FO), »Inner Resources« (IR), »Psychic Disturbances« (PD), »Social Disturbances« (SD) and »Adaptive Coping« (AC). These parameters were gained by group-rating. Since 1986, more than 30% of our patients had died, mainly in the 90ies, mainly of AIDS. We divided our patients into two maingroups (alive ones vs. deceased ones), both grouped in HIV-infected and non-infected (comp. above). We analyzed our data material (clustered in 4 time-breaks, each I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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about four until five years) by statistical tools, searching for significant correlations and differences between the different groups (compare also our article about medical aspects).

Results 1. In 1986 we found high differences (p < 0.001) in all psychosocial parameters between HIV+ and HIV– patients; all of them are HCV-infected. We found low SSE, low FO, low IR, high PD, high SD and low AC within the group of the HIV+ (n = 67) in comparison to the group of HIV– (n = 27). At this point of view all of our investigated patients were still alive (comp. Fig. and Table 1). 2. In the next time-period it came to an approximation over all parameters among both groups (HIV+ vs. HIV–). We found higher scores in SSE, FO, IR, lower scores in PD and SD, and in addition a higher score in AC in 1990 over all investigated persons (67 HIV+ and 27 HIV–) with none significant differences (comp. Fig and Table 2). 3. After getting conscious of HCV (1996), a general break-in for all (HIV+ and HIV–) patients occurred. The HIV-non-infected persons were suddenly confronted with the meaning of a most dangerous infection whereas the HIV-infected ones were additional hit. Our ratings show, that obviously the HIV+ could deal a little ›better‹ with the ›new‹ disease, although there are no significant differences between both groups. In the meantime 20 of the HIV+ (most of them of AIDS) and one of the HIV– had died (comp. Fig and Table 3). 4. Comparing the psychosocial parameters of meanwhile 37 HIV+ and 22 HIV– in the last time cluster (2001–2004), you may see that since the all involving threat of HCV (1996–2000) there is a steadily equalization like above (as shown in result 3) among all alive hemophilia patients (either HIV-co-infected or not), but now with clear improvements over all psychosocial factors (comp. Fig. and Table 4). 5. Comparing the alive HIV+ vs. the deceased HIV+ over the time-periods, there are high-significant differences regarding the factors IR, PD and AC already in the beginning in 1986-89 (comp. Fig. and Table 5). During the next three time periods these differences between the two groups vanish – may be due to the psychosocial support to nearly all of our patients. The distinct higher (›better‹) scores of the alive HIV+ in these psych. factors at the first time cluster can also be shown in some medical parameters, especially CD4-counts in comparison to the deceased HIV+ in course of observation (compare again our article about medical aspects). 6. Comparing all the alive ones (HIV+ and HIV–) vs. all the deceased ones we found again only in the first period (1986–89) the same high-significant differences regarding the same factors (comp. Fig. and Table 6), like we found when we compared the alive HIV+ vs. the deceased HIV+. These significant differences already in the beginning between alive people with hemophilia and later deceased ones (th. i. who will die several years later) show the importance of psychosocial factors and the necessity of paying attention to these often underestimated factors in order to identify in time the patients’ need for a more healthy and satisfying quality of life.

HIV

mean

p

T

df

SSE

HIV+ HIV-

32,84 47,78

< 0,001

7,967

39,206

FO

HIV+ HIV-

32,09 47,78

< 0,001

8,268

39,731

Psychosocial factors

IR

HIV+ HIV-

32,69 44,81

< 0,001

7,083

41,374

1986 – 1989

PD

H IV+ HIV-

50,30 38,15

< 0,001

6,956

62,641

SD

HIV+ HIV-

32,99 23,33

< 0,001

5,742

68,645

AC

HIV+ HIV-

29,85 41,11

< 0,001

5,597

42,568

psych. factors

HIV

SSE

HIV+ (n = 67)

50

HIV - (n = 27)

40

67 HIV+ and 27 HIV-

30 20 10 0

Tab./Fig. 1

mean

p

T

df

HIV+ HIV-

48,21 53,33

,028

-2,226

46,486

FO

HIV+ HIV-

44,63 51,11

,016

-2,457

52,193

IR

HIV+ HIV-

41,79 46,67

,027

-2,240

41,599

1990 – 1995

PD

HIV+ HIV-

37,16 31,85

,005

2,846

50,652

67 HIV+ and 27 HIV-

SD

HIV+ HIV-

24,63 21,11

,027

2,248

70,146

AC

HIV+ HIV-

41,79 44,81

,242

-1,178

50,272

SSE1

FO1

IR1

PD1

SD1

60

HIV+ (n = 67)

50

HIV - (n = 27)

AC1

Psychosocial factors 40 30 20 10 0

Tab./Fig. 2

SSE 2

FO2

IR2

PD2

SD2

AC2

175

Fig. and Tables 1–2

60

HCV-Infection in HIV-Infected and Non-Infected People with Hemophilia

psych. factors

SSE

mean

p

T

df

HIV+ HIV-

34,47 32,69

,381

,884

43,742

FO

HIV+ HIV-

34,04 33,08

,666

,434

49,224

IR

HIV+ HIV-

32,34 31,15

,502

,676

52,776

1996 – 2000

PD

HIV+ HIV-

49,36 50,00

,730

-,346

52,597

47 HIV+ and 26 HIV-

SD

HIV+ HIV-

26,38 29,23

,081

-1,776

56,982

AC

HIV+ HIV-

33,19 31,92

,560

,587

50,081

mean

p

T

df

60

Psychosocial factors

HIV - (n = 26)

40 30 20 10 0

Tab./Fig. 3

psych. factors

HIV

SSE

HIV+ HIV-

50,00 49,13

,760

,308

34,069

FO

HIV+ HIV-

47,11 44,35

,296

1,060

36,720

IR

HIV+ HIV-

44,21 43,48

,752

,318

39,304

2001 – 2004

PD

HIV+ HIV-

33,16 34,35

,491

-,693

54,449

37 HIV+ and 22 HIV-

SD

HIV+ HIV-

18,16 17,83

,803

,251

57,809

AC

HIV+ HIV-

47,89 46,52

,657

,447

40,789

Fig. and Tables 3–4

HIV+ (n = 47)

50

Psychosocial factors

SSE 3

FO3

IR3

PD3

SD3

60

HIV+ (n = 37)

50

HIV - (n = 22)

AC3

40 30 20 10

Tab./Fig. 4

0 SSE 4

FO4

IR4

PD4

SD4

AC4

M.M. Schneider et al.

HIV

176

psych. factors

HIV+

mean

p

T

df

SSE

de ceased alive

31,50 34,59

,169

-1,410

29,071

FO

deceased alive

30,50 33,78

,127

-1,559

39,031

IR

deceased alive

29,50 35,41

,003

-3,229

35,063

PD

deceased alive -

54,00 47,30

,005

3,030

28,983

SD

deceased alive

35,50 30,81

,051

2,043

26,052

AC

deceased alive

27,50 32,97

,009

-2,772

38,376

mean

p

T

df

60 deceased (n = 20) 50

1986 – 1989

40

37 HIV+ alive and 20 HIV+ deceased in course of observation Tab./Fig. 5

psych. factors

N

SSE

deceased alive

33,71 39,15

,020

-2,385

64,850

FO

deceased alive

33,14 38,64

,019

-2,408

70,443

IR

deceased alive

31,71 38,81

,000

-3,737

65,733

PD

deceased alive -

51,71 43,90

,000

3,887

54,666

SD

deceased alive

34,00 27,97

,003

3,164

51,812

AC

deceased alive

28,00 36,10

,000

-3,993

69,413

alive (n = 37)

30 20 10 0 SSE 1

FO1

IR1

PD1

SD1

AC1

PD1

SD1

AC1

60 deceased (n = 35)

Psychosocial factors 1986 – 1989

50

alive (n = 59)

40 30

59 HIV+/- alive and 35 HIV+/- deceased in course of observation

20 10 0

Tab./Fig. 6

SSE1

FO1

IR1

177

Fig. and Tables 5–6

Psychosocial factors

HCV-Infection in HIV-Infected and Non-Infected People with Hemophilia

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M.M. Schneider et al.

Conclusion Our results show evidently that the psychosocial factors are of high importance besides the medical suppositions. We have to concede that part of our psychosocial data was gained retrospectively, so these results should be evaluated by standardized measurement tools, like they are used for example in the ESCHQoL-study. But even so, the necessity of identifying those psychosocial constrictions at the earliest, the patients in question are afflicted with, is shown by the fact that the alive people with hemophilia (either HIV+ or HIV–, but all HCV-infected) dispose of »better« psychosocial conditions (and of »better« medical conditions) in comparison to the (later) deceased patients already in 1986–1989, at an early date, when all our investigated patients were alive.

Testing Factor VIII Activity by Using the Chromogenic Assay in Carriers of Hemophilia A W. Miesbach, Th. Vigh, and I. Scharrer

Bleeding tendencies in carriers of hemophilia A are related to factor VIII activity and the underlying factor VIII gene mutation. In earlier investigations, it could be shown that carriers were less likely to report a tendency for bleeding when timely diagnosis and treatment for carriers of hemophilia A is able to prevent the tendency to bleeding [1]. It could be shown that for younger carriers, fewer carriers had a history of bleeding events than the older carriers. This correlation was in inverse proportion to the FVIII concentration. There are two different test systems available for assessing factor VIII activity. The one-stage clotting assay was described more than 50 years ago by Langdell, et al. [2]. After preincubation of activated partial thromboplastin, FVIII-deficient plasma and the test plasma were mixed resulting in contact activation of plasma leading to a fibrin clot formation. This assay became the most widely used and least expensive method for measuring FVIII activity in patients and in therapeutic FVIII concentrates. The chromogenic assay can be viewed as a descendent of the two-stage FVIII clotting assay which was developed in the UK and which is rarely used. For this test, no hemophilic or FVIII deficient plasma is required. The test substance is first added to a mixture of bovine FIXa, FX and thrombin in the presence of phospholipids, leading to the activation of FX. In the second stage, a chromogenic substrate specific for FXa is added, and the absorbance is measured [3]. In order to focus on the value of chromogenic factor VIII assay in carriers of hemophilia A, we compared the results of chromogenic factor VIII testing with the factor VIII levels found using the one-stage method. We tested whether one of the two tests correlates more sensitively with bleeding symptoms in 25 carriers of hemophilia A.

Patients and Methods We investigated twenty-five carriers of hemophilia A with a median age of 40 years (range: 18–77 years). For all carriers, FVIII activity was measured using both, the one stage clotting assay and the chromogenic assay. In 17 carriers, an analysis of the FVIII gene was performed by PD Dr. Oldenburg, Frankfurt. There was a single point mutation in 13 carriers, in three carriers an intron 22-inversion and in one carrier a small deletion. Each carrier was asked about the frequency and type of bleeding I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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26% 35%

after operation easy bruising menstrual teeth no bleeding 18% 9%

Fig. 1. Rate of bleeding events in carriers of hemophilia A

12%

events. The following symptoms were noted: easy bruising, nose bleeding, gum bleeding, bleeding after small or larger traumata, bleeding after surgical procedures, long or heavy menstrual bleedings. The one stage assay was carried out using an ACL 300 (IL). The chromogenic assay was carried out with reagents from Immunochrom.

Results A tendency towards bleeding was described in 12/25 carriers (48%). The carriers mostly suffered from bleeding after operations or deliveries, and also from easy bruising (Fig. 1). The median result of the FVIII activity using the one stage assay was 53 % (range: 4–102 %, normal value: 64–155 %). The median result of the FVIII activity using the chromogenic assay was 60 % (22–104 %, normal value 60–150 %). 80 70

68 64

60 50

47 44

40

decreased FVIII in all carriers decreased FVIII in carriers with bleeding

30 20 10 0 one stage assay

chromogenic assay

Fig. 2. Rate of reduced FVIII activity in all carriers and in carriers with tendency for bleeding

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181

Table 1. Frequency of bleeding events in carriers with reduced FVIII activity

Factor VIII Ø FVIII act. median (normal value) Bleeding events

One-stage assay

Chromogenic assay

17/25 (68 %) 42 % (4–62 %) (64–167 %) 8/17 (47 %)

11/25 (44 %) 46 % (22–59 %) (60–150 %) 7/11 (64 %)

The one stage assay measured a lower level of FVIII activity in more of the carriers than the chromogenic assay (17/25 carriers vs. 11/25 carriers; Table 1). Of the carriers for whom the chromogenic assay showed a low level of FVIII activity, bleeding events occurred more often than in carriers with a low level of FVIII activity measured by the one stage assay (7/11 carriers vs. 8/17 carriers). Although decreased levels of FVIII were more commonly found using the one-stage clotting assay, FVIII levels found using the chromogenic assay were lower than those found by the one stage clotting method in carriers with a tendency for bleeding (FVIII activity 47 %, 22–104%, versus FVIII activity 51.5 %, 4–104 %; Fig. 2).

Discussion The comparison of both assays showed that in carriers with a tendency to bleeding the results from the chromogenic assay seem to be closely associated to the bleeding events. The rate of low FVIII levels in carriers of hemophilia A, however, was higher using the one-stage clotting assay than the chromogenic assay. It is known that the simplicity of the one-stage clotting test belies a high degree of variability and is dependent on reagents and other circumstances performing the test. Variations in FVIII levels within laboratories were around 10–20% or even higher [4]. In international studies, the chromogenic assay has consistently demonstrated less variability between laboratories than the one-stage method. Marked differences were observed between the potency of therapeutic products as assessed by the one-stage and the chromogenic assays. Very low residual FVIII activities cannot be measured accurately using the chromogenic assay due to a limited sensitivity. FVIII activity is mostly lower when measured by the one-stage assay. Therefore, FVIII concentrates must be tested using both the one stage clotting assay and the chromogenic assay. In our study, the median FVIII activity was lower measured by the one stage than the chromogenic assay in all of the carriers. In carriers with tendency to bleeding, however, the chromogenic assay measured lower FVIII activity levels than the one-stage assay and seems to be more closely related to the patient’s clinical condition. This is confirmed by the results of a recent study that has shown that the chromogenic assay is more physiological than the one-stage assay, because the result of the one-stage assay may be influenced by lipid artifacts [5]. In conclusion, the chromogenic assay should be considered for use for the diagnosis of carriers of hemophilia A. Further studies are needed to establish the rela-

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tionship between test results and the tendency to bleeding in patients with hemophilia A.

References 1. Miesbach W, Oldenburg J, Stier-Brück I, Vigh T, Scharrer I. Bleeding tendency of carriers of hemophilia A – dependent on the age of the carriers? In: I. Scharrer/W. Schramm (Ed). 34th Hemophilia Symposium Hamburg 2003, Springer Medizin Heidelberg Verlag 2005, 191–195 2. Langdell RD, Wagner RH, Brinkhous KM. Effect of antihemophilic factor on one-stage clotting tests. J Lab Clin Med 1953;41:637–47 3. Barrowcliffe TW. Methodology of the two-stage assay of Factor VIII. Scand J Haematol 1984;33:25–38 4. Over J. Methodology of the one-stage assay of Factor VIII. Scand J Haematol 1984; 33: 13–24 5. Mikaelsson M, Oswaldsson U, Sandberg H. Influence of phospholipids on the assessment of Factor VIII activity. Haemophilia 1998;4:646–8

Hip Replacement in Hemophilic Patients – A 30 Years Single Center Experience A. Kurth, Ch. Eberhardt, L. Hovy, M. Krause, and I. Scharrer

Introduction Hemophilic arthropathy of the hip is less common in patients with bleeding disorders. If the destruction of the hip joint is severe enough replacement of the joint is indicated.

Materials and Methods We performed a retrospective analysis of the cases done at our center to determine the outcome of hip replacements in this specific group of patients. In July 1972 the first elective total hip replacement (THR) in a hemophilic patient was performed at the University Hospital in Frankfurt. Between then and 2002 in 13 patients 15 hip joints were found to be necessary to replace. In 10 hemophilic patients and 3 VWD patients a hip replacement was performed. The mean age of the patients was at the time of surgery 42 years. During that time we changed the implants used for the replacement. At the beginning we used only cemented cups and stems. Later hybrid THR was used with a cemented stem and a cementless cup. Recently we use only cementless implants even in those high risk patients. The charts of the patients were evaluated for complications after surgery, early complications during surgery, survival of the implants, cases of revision surgery, and infections. To determine the functional outcome the widely accepted Harris Hip Score was calculated for the time before surgery and at the time of follow up. The earliest follow up was 12 months after surgery.

Results Good long term results after a follow up time of 132 months (12–363) were found. No perioperative complications like bleeding or infection were found. Only one aseptic loosening of a cemented cup occurred after 14 years as well as one septic loosening 14 months postoperatively in a HIV positive patient. Another HIV positive patient developed a hematogenic abscess on both operated hips without loosening. I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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The calculated survival time after a mean follow up of 11 years was 86.7 %. The Harris Hip Score increased remarkably from 48 points (range 32-66) preoperatively to 89 points (range 76–100) at the time of follow up. We found excellent and good results in 72 %. Fair results in 19 %, and bad results in 9 % of the cases.

Conclusion Despite FVIII prophylaxis and better joints in hemophilic patients this group of patients will get older and arthritis of the hip is an increasing problem in the older generation. Regardless hemophilic arthropathy patients will develop hip and knee arthritis and the need for surgery will increase. We conclude after more than 30 years of experience with THR in hemophilic patients that for advanced severe hemophilic hip arthropathy, THR results in painrelief and in improvement of quality of life. Thus, total hip replacement should be considered regardless the age of the patient and the surgical intervention should be performed in specialized centers with experience in the treatment of bleeding disorders.

VIIb. Inhibitors in Hemophilia

Successful Therapy with anti CD20 Monoclonal Antibody Rituximab in Patients with Acquired Hemophilia against Factor VIII M. Krause, Ch. von Auer, I. Stier-Brück, and I. Scharrer

Introduction Immunomodulatory therapy regimens with normalization of FVIII levels and elimination of the inhibitor are reasons for the treatment in patients with acquired hemophilia. The aim of the present study was to evaluate the response to Rituximab in the first line or second line therapy.

Patients and Methods In 4 patients (2 females, 2 males; age: 70–81 years) FVIII activity levels were < 1% with the inhibitor titer from 9 to 156 BU (Table 1). Rituximab was administered at the dose 375 mg/m2 once weekly for 4 weeks with combination of Prednisone therapy.

Characteristics Table 1 patient

sex

age [years]

FVIII [%]

FVIII inhibitor [BU]

associated diseases

1# 2# 3# 4#

female female male male

75 81 70 76

1 0 0 6

17 [max. 22] 156 [max. 188] 35 [max. 50] 9 [max. 9]

breast cancer idiopathic colon cancer dysplasia of colon

Results In all patients we documented complete remission (CR) 4, 5, 23 and 32 weeks after begin of Rituximab. The inhibitor titer was 0 BU and the factor VIII activities were 73%, 90%, 77% and 98% in this patients (Fig. 1). The period of response in patients, which shows higher inhibitor titer (max. 50 BU; 188 BU), was much later than in I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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patients (23 or 32 vs. 4 or 5 weeks) with lower inhibitor titers (max. 9 BU; 22 BU) (Fig. 2).

Results I patients

FVIII activity [%] 200

1# Complete remission

†

160

2#

120 80

3# 40 0 0

20

40

60

80

100

4#

time [week ]

Begin of Rituximab

Fig. 1

Results II p atients FVIII inhibitor [BU] 200

1#

160

Complete remission 2#

120 80

3# 40

† 0

4# 0

20

40

60

80

100 time [ week ]

Begin of Rituximab

Fig. 2

Successful Therapy with anti CD20 Monoclonal Antibody Rituximab in Patients

189

Conclusion – The therapy with Rituximab shows high response rates and appears to be effective in the elimination of inhibitors. – Whether the success of the inhibitor elimination by Rituximab correlated to the inhibitor titer is unknown. – Further studies are needed to confirm whether Rituximab has a role in the first line therapy compared with other therapy regimens, or only in cases unresponsive to other immunomodulatory treatments.

References 1. Cohen AJ, Kessler CM. Acquired inhibitors. In: Lee CA, ed. Haemophilia. Bailliere’s Clinical Haematology 1996; 9: 331–354 2. Green D, Lechner K. A survey of 215 non-hemophilic patients with inhibitors to factor VIII. Thromb Haemost 1981; 45: 200–203 3. Kain S, Copeland T-S, Leahy M F. Treatment of refractory autoimmune (acquired) haemophilia with anti-CD20 (Rituximab) Br J Haematol 2002; 119: 572–580 4. Karwal MW, Schlueter AJ, Zenk DW, Davis RT. Treatment of acquired factor VIII deficiency with rituximab. [abstract] Blood 2001; 98: 533a 5. Kessler CM, Nemes L. Acquired inhibitors to factor VIII. In: Rodriguez-Merchan EC and Lee CA Inhibitors in patients with haemophilia. Blackwell Science 2002; 98–112 6. Shapiro SS, Hultin M. Acquired inhibitors to the blood coagulation factors. Sem Thromb Haemostas 1975; 1:336–385 7. Stasi R, Brunetti M, Stpa E, Amadori S. Selective B-cell depletion with rituximab for the treatment of patients with acquired hemophilia. Blood 2004 8. Wiestner A, Cho HJ, Asch AS, Michelis AM, Zeller JA, Peerschke EI, Weksler BB, and Schechter GP. Rituximab in the treatment of acquired factor VIII inhibitors. Blood 2002; 100: 3426–3428

Unusual Prolonged Course of an Immune Tolerance Therapy (ITT) in a Patient with Severe Hemophilia A and a High-Titer Inhibitor Development S. Meister, T. Spranger, K. Christensen, H.-H. Brackmann, and G. Auerswald

Objective A frequently encountered complication of therapy given to patients with severe hemophilia A is the development of antibodies (inhibitor) to infused factor VIII. Incidence of FVIII-inhibitor development is reported as 20–30%. Common treatment is the induction of immune tolerance by administration of high doses of FVIII twice daily according to the »Bonn Protocol«. This protocol sets itself two targets: inhibitor elimination and avoidance/therapy of bleeds. Inhibitor elimination is successful in up to 90% (Kreuz et al.). Without complications or interruptions in the course of ITT, therapy is successful within four months in median (Kreuz et al.). Intermittent therapy, and possibly also infections of central venous lines, can result in a prolonged course or failure of therapy (Oldenburg et al.). We report a now 17-year-old patient with severe hemophilia A and high-titer inhibitor development. The inhibitor elimination was complicated with several infections, consecutive surgery and severe bleeds. The ITT took an unusual prolonged course; inhibitor elimination was possible even after years.

Case Report Severe hemophilia A (FVIII rest-activity < 1%) was diagnosed post partum due to positive family history. At the age of eight months, first FVIII therapy was given to stop a mucosal bleed. Other FVIII substitutions followed on demand. In September 1989, at the age of two years, he developed a high-titer inhibitor. An immune tolerance therapy (ITT) was started with 180 IU/kg FVIII (plasma derived (pd), containing VWF) twice daily according to the »Bonn Protocol«. The inhibitor first increased to 16 Bethesda units/ml (BU/ml) and decreased afterwards to 1 BU/ml. FVIIIactivity was detectable whereas the FVIII-recovery did not normalize. FVIII administration got more difficult, a central venous line was implanted. The course of therapy was complicated by several infections of the central venous line, thrombosis, sepsis and severe joint bleeds. The central venous line had to be taken out twice; in addition, compliance was difficult. Therapy was not given continuously. The inhibitor titer ranged between 1 and 88 BU/ml over several years. Since severe bleeds could not be avoided, therapy was continued with aPCC twice daily in addition to FVIII. After six years of ITT and still a high-titer inhibitor, the FVIII and I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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aPCC substitutions were reduced to once daily. This was done because there was no FVIII activity detectable 10 minutes after FVIII administration, and FVIII administration was still difficult and stressful for the family. Acute bleeds were treated with aPCC, later with rFVIIa, but they did not occur more often. First the inhibitor increased immensely up to more than 256 BU/ml and decreased afterwards. In September 1995, the pd FVIII concentrate was changed to a recombinant FVIII concentrate. Therapy was continued with 80 IU/kg, later on with 40 IU/kg FVIII and aPCC once daily. Acute bleeds were treated with rFVIIa (first 90 µg/kg, later 300 µg/kg). Factor administration became less difficult and compliance increased. At the same time, the patient had significantly less bleeds and the inhibitor-titer fell slowly. Almost 12 years after starting ITT, the inhibitor was not detectable anymore; again, 1.5 years later, FVIII-activity could be measured for the first time (23 hours after FVIII administration with 5%). Therapy with aPCC was stopped. The inhibitor-titer is still negative, but FVIII recovery did not normalize so far. The patient had no joint bleeds in the past year, and he self-administers the FVIII regularly once daily. This has resulted in an enormous benefit for quality of life.

Anamnestic Data Date of birth Genetics First FVIII therapy Therapy »on demand« FVIII-inhibitor und begin of ITT

08. July 1987 Intron 22 Inversion March 1989 (mucosal bleed) until July 1989 July 1989

Used FVIII Concentrates Haemate Hemofil M Octa SD plus Kogenate

since March 1989 since July 1989 since Sept. 1990 since Sept. 1995

Bypass-Concentrates aPCC (Feiba) rFVIIa (NovoSeven)

since May 1993 since Oct. 1997

Maximum inhibitor titer: Inhibitor not detectable

> 250 BU/ml since June 2001

250

Good Compliance

Irregular F VIII-substitution Implantation of port, severe hematoma ematoma postoperatively

150

Infection of port and explanation, Renal bleeding

Severe bleeds

100 50

Implantation central venous line

Sepsis Catheter explanation Thrombosiss Thrombosi

Measurable rable FVIII-recovery I ery

0 Jul De Dec Jun De Dec Jun De Dec Jun De Dec Jun De Dec Jun De Dec Jun De Dec Jun De Dec Jun De Dec Jun De Dec Jun De Dec Jun De Dec Jun De Dec Jun De Dec Jun De Dec Jun De Dec Jun De Dec Jun 87 87 88 88 89 89 90 90 91 91 92 92 93 93 94 94 95 95 96 96 97 97 98 98 99 99 00 00 01 01 02 02 03 03 04

F VIIIconcentrates FVIII concentrates

r-Factor VIII r-Factor VIII 1 1x x 40 40 IU/kg IU/kg

pd-Factor VIII 2 x 200 IU/kg

aPCC

2x 2 x 60 60 IU/kg IU/kg

1x 1 x 60 60 IU/kg IU/kg

rFVIIa (on demand)

90 µg/kg 90

Fig. 1. Factor VIII-inhibitor titer development and factor VIII-rest-activity. Treatment is mentioned below

300 µg/kg 300

S. Meister et al.

[BU/ml]; [%]

Factor VIII-activity 12 hours after infusion in % %

Infection of central venous line, Catheter explanation, Catheter implantation

200

192

Factor VIII-inhibitor titer titre development in BU/ml

Unusual Prolonged Course of an Immune Tolerance Therapy (ITT)

193

Discussion This long course of ITT shows that inhibitor elimination could be achieved by regular FVIII exposure even after years. Different circumstances have been described to cause a prolonged course of ITT: especially discontinuation of ITT and irregular FVIII exposure. Also, infections and surgery can cause a prolonged course of ITT. Intron 22 inversion might be another possible cause for prolongation. Elimination of the inhibitor provides tremendous relief for the patient and his family. It also improves the quality of life. Last but not least, successful ITT is cheaper than ongoing administration of bypass-concentrates. After failure of ITT, FVIII administration at regular intervals might achieve elimination of the inhibitor.

References 1. Kreuz et al.: Epidemiology of inhibitors and current treatment strategies. Haematologica. 2003 Jun;88(6):EREP04 2. Kreuz et al.: Immune tolerance therapy in paediatric haemophiliacs with factor VIII inhibitors: 14 years follow up. Haemophilia. 1995 Jan.; 1: 24-32 3. Oldenburg et al.: Induction of immune tolerance in haemophilia A inhibitor patients by the ‘Bonn Protocol’: predictive parameter for therapy duration and outcome. Vox Sang. 1999;77 Suppl 1:49-54

Successful Major Surgery with Minimal Dosage of rFVIIa in a Hemophilia A Patient with High Level of Alloantibodies to Factor VIII M. S¸erban, P. Tepeneu, S. Arghirescu, R. Badet˛i, C. Jinca, D. Mihailov, D. Lighezan, and W. Schramm

Summary The experience of a favorable outcome of an elective major orthopedic surgical procedure performed in a severe case of hemophilia A with high titer inhibitors is presented. Specific for the presented case is the minimal dosing schedule of rFVIIa (7 days’ consumption of 68,960 mg vs. standard recommended dosage of 270,000 mg), encouraging for better expectation of success in life-threatening events, despite a low availability to an indispensable, costly therapy. Major surgery in hemophiliacs remains a difficult medical problem in developing countries. It is considered of particular concern, due to the need of a sufficient hemostatic control for a long postoperative period, in order to permit wound healing. Major surgical procedure encounters catastrophic potential risk in the case of complicated hemophilia with high level of alloantibodies to FVIII or IX. Lack of appropriate hemostatic agents in an adequate dosage, due to financial restraints is life threatening for this group of patients. This report aims at presenting a favorable experience with major surgery in a high responder inhibitor hemophilic patient, undertaken with a minimal dosage of therapy with rFVIIa [1, 2].

Case Report We describe a 13 years old child with severe form of hemophilia A and high level of FVIII inhibitors (180–320BU), who experienced a history, started during infancy, with numerous, severe, often life-threatening bleedings (dental, oral wound, joint, ileo-psoas muscle). A major functional impact has been determined by its bone hemorrhages, causing multiple pseudotumours (PT) developed distally in the legs and presenting a proximal progression. At 8 years of age the child had a left shank amputation followed by a right shank amputation at the age of 11, due to septic, complicated, spontaneously discharged, uncontrolled bleeding pseudotumour. The postoperative evolution was complicated with frequent life-threatening bleedings, with severe posthemorrhagic anemia, imposing a long (more than 6 months) hospital admission and intensive care. Afterwards he slowly recovered, but unfortunately, progressively growing pseudotumours of the amputated stump developed (Fig. 1a, b). The lack of therapeutic I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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a

195

b

Fig. 1a, b. The pseudo-tumor developed distal in the right shank (front) (a) and (profile) (b) – preoperative state

Fig. 2. Bilateral femoral amputation at the inferior 1/3 level of the femur – postoperative state

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Preoperative rFVIIa bolus 120 µg/kg

110 µgkg 12h

18.3µg kg/h

Day

0

1

90 µgkg 12h

2

90 µgkg 12h

90 µgkg 12h

3

4

90µg kg

5

90µg kg

6

7

Legend Bolus administration of rFVIIa Continuous infusion of rFVIIa Tranexamic acid, 25 mg/kg Fig. 3. Bilateral femoral amputation – treatment regimen

control due to shortage of bypassing agents and a complicating osteomyelitis imposed a bilateral amputation, this time, at the inferior 1/3 of the femur (Fig. 2). The hemostatic control was performed with rFVIIa (Fig. 3). Preoperatively, a bolus administration of 120 µg/kg was initiated, followed over the first 24 h by a continuous infusion with an hourly rate of 18.29 µg/kg/h. In the next days, boluses of 110 µg/kg/12h on day 2 and 90 µg/kg/12h rFVIIa on day 3, 4 and 5 were administered. In the following 2 days only one daily bolus administration of 90 µg/kg/24h was performed. Tranexamic acid (25 mg/kg) was administered q8h in the first day and q12h in the next days. Changing of dressing or drainage tube positioning were preceded by a single bolus administration of rFVIIa (90 µg/kg) and tranexamic acid (25 mg/kg) application on the wound. Total amount of rFVIIa consumed in 7 days was 68,960 µg, in comparison with 270,000 µg as recommended in standard regimens (Fig. 4). The perioperative blood loss was comparable to that during similar surgery in the normal population. The hemoglobin level ranged between 8–10 g/dl 300000 250000 200000 150000 100000 50000 0 our exper ience

standard regimen

Fig. 4. rFVIIa consumption

Successful Major Surgery with Minimal Dosage of rFVIIa in a Hemophilia A Patient

197

with the need of two units of blood transfusion. No evidence of systemic activation of coagulation was assessed.

Discussion The development of inhibitors against factor VIII or IX is the most common and most serious complication of replacement therapy in patients with hemophilia A or B. Surgery in hemophilic patients with inhibitors presents a particular challenge to hemostasis management. rFVIIa is an optimal hemostatic agent, that can be used in these patients. It represents an important advance in the treatment of hemophiliacs with inhibitors. The first report of surgical intervention in patients with severe hemophilia A and inhibitor successfully performed under cover of rFVIIa was published in 1988. Since then, the use of rFVIIa for elective surgical procedures has been increasingly reported [3, 4, 5]. However, while rFVIIa is considered a safe and effective treatment for bleeding episodes in hemophilia patients with inhibitors, the optimal and minimal dose regimen have yet to be established [6]. Our experience regarding hemostatic treatment of patients with high-titer inhibitors allowed us to perform safely a major devastating orthopedic surgery with a special dosing schedule. In the reported case it was evident, that even in reduced number of administration, but with standard dosage (90–120 µg/kg) rFVIIa gives new perspectives, allowing emergency or elective surgery. The favorable outcome of our patient under the conditions of minimal dosage of rFVIIa is encouraging for developing countries, confronted with important restraints and shortages regarding mainly costly treatments.

References 1. Hvid I, Rodriguez-Merchan EC: Orthopaedic surgery in haemophilic patients with inhibitors: An overview. Haemophilia 8:288–291, 2002 2. Ingerslev J: Efficacy and safety of recombinant factor VIIa in the prophylaxis of bleeding in various surgical procedures in hemophilic patients with factor VIII and factor IX inhibitors. Semin Thromb Hemost 26: 425–432, 2000 3. Ludlam CA, Smith MP, Morfini M, et al: A prospective study of recombinant activated factor VII administered by continuous infusion to inhibitor patient undergoing elective major orthopaedic surgery: A pharmacokinetic and efficacy evaluation. Br J Haematol 120: 808–813, 2003 4. Rodriguez-Merchan EC, de la Corte H: Orthopaedic surgery in haemophilic patients with inhibitors: A review of the literature, in Rodriguez-Merchan EC, Goddard NJ, Lee CA (eds): Musculoskeletal Aspects of Haemophilia. Oxford, UK, Blackwell, 2000, pp 136–142 5. Schulmann S: Joint surgery in patients with inhibitors, in Rodriguez-Merchan EC (ed): The Haemophilic Joints: New Perspectives. Oxford, UK, Blackwell, 2003, pp 56–62 6. Kenet G, Lubetsky A, Luboshitz J, Martinowitz U: A new approach to treatment of bleeding episodes in young hemophilia patients: a single bolus megadose of recombinant activated factor VII (NovoSeven); J of Thromb and Hemost, 1:450–455, 2003

Inhibitor Development after Continuous Infusion of Factor VIII: A Retrospective Study in Germany Ch. VON Auer, J. Oldenburg, M. Krause, W. Miesbach, I. Scharrer, and CI-Study Group

Introduction The continuous infusion (CI) of coagulation factor VIII (FVIII) concentrates has been used since the early 1990s. Compared to the traditional way of factor application by episodic bolus infusions (BI) the CI had several advantages such as keeping a steady level of missing coagulation factor, avoiding of unnecessary high peaks of FVIII, 30% saving of FVIII [1] and therefore reduction in treatment cost. Reports of the occurrence of inhibitors after treatment with CI have raised concerns about this method of factor-application.

Method We conducted a retrospective study in Germany to investigate inhibitor development after CI of FVIII. Two questionnaires were developed and sent to 100 hemophilia treating colleagues in Germany. One to collect general information about the treatment modalities in the specific center, the other one contained queries about the patients with inhibitor development.

Results Out of 100 colleagues 41 answered the questionnaires. In 25 hemophilia treatment centers CI was used as treatment modality, in 4 of them it is still used today. The remaining 16 centers never used CI for their patients. Altogether 190 CI were conducted in 110 patients. In 12 of these patients inhibitor development was reported. – Age of the patients: Median 27 years – Severity of hemophilia: five pts. with severe hemophilia, one pt. with moderate hemophilia and six pts. with mild hemophilia – Exposure days: Median 14.5 days – Thrombophlebitis: found in 2 patients – Family history: one patient’s cousin also developed an inhibitor. I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

Inhibitor Development after Continuous Infusion of Factor VIII

199

– Genotype: missense-mutations (4), intron-22-inversions (2), small deletion (1), five were unknown (unk). – Indication for CI: treatment of serious bleeding and surgical procedures – Infused FVIII before inhibitor development: between 4300 and > 100000 IE – Infusion sets: varied from minipumps (2) to common infusion sets (7), three unk – Inhibitors: alloantibodies, low responding (4) and high responding (8) – Immune tolerance therapy: done in 7 patients, 4 with successful outcome – Factor concentrates: plasma derived (8) and recombinant (4) concentrates. Age H em.A Anamn. HR/LR p./t. 7 Mo severe PUP high perm. 3.5 Y severe PTP hi gh trans. 5.5 Y severe PTP low trans. 8Y mild PTP high perm. 11 Y mild PUP low perm. 19 Y mild PTP high trans. 35 Y mild PTP high perm. 45 Y severe PTP hi gh perm. 45 Y severe PTP low perm. 57 Y moderate PTP high perm. 72 Y mild PTP low ? 73 Y mild PTP high ?

ED 13 1 10 5 28 16 30 >100 >100 >100 >7 4

FVIII plasm plasm rec pla sm plasm pla sm rec rec plasm rec plasm pla sm

Genotype Sma ll D eletion Intron-22-Inversion Intron-22-Inversion Missense-Mutation Missense-Mutation Missense-Mutation unknown no I ntron-22-Inversion Missense-Mutation unknown unknown unknown

Comparing reported data of patients with or without inhibitor development after CI we found the following results: Question

Inhibitor

No Inhibitor

Hemophilia A (Pat) severe moderate mild

12

91

(5) ( 1) ( 6)

( 76) ( 8) ( 7)

Hemophilia B severe moderate mild Age ( Years) PUP ( Pat.) PTP ( Pat.) ED ( Days) Change of Product Blood Transfusion Infections Vaccination

0

7

(0) ( 0) ( 0) 6 -73 2 10 6 - 65 1 1 2 0

( 5) ( 2) ( 0) 7-55 2 91 50 - 200 7 3 10 0

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Conclusion and Discussion In 41 German hemophilia treating centers 190 CI’s were conducted in 110 patients. Strikingly 12 of these patients developed an inhibitor against factor VIII after CI. In only two of the reported patients we found the so called null-mutations which prevent synthesis of FVIII protein (here Intron-22-inversion). These patients exhibit an inhibitor prevalence of 21–88% [2]. Therefore only the first three patients in our table belong to that group of patients where most commonly an inhibitor development is seen (genotype/PUP and severe hemophilia). In our study strikingly the inhibitors developed very often in patients with mild or moderate hemophilia and genotypes that exhibit an inhibitor prevalence of < 10 % [2]. The portion of this patient group is especially high in comparison with the distribution of severity types in patients with no inhibitor development after CI. Our findings agree with published results (19 case reports) [1, 3, 4, 5]. Most reported cases were patients with mild hemophilia and/or PTP’s. Our investigation may suggest that there might be an uncommon inhibitor pathomechanism, resulting in a peculiar group of patients developing the inhibitor after CI. On the other hand one could suggest that patients with mild hemophilia might exhibit a much higher prevalence of inhibitor development when treated with an »intensive FVIII-treatment« such as CI. Due to different documentation systems in the hemophilia centers retrospective data collection appears to be difficult and time consuming. Therefore a prospective multicenter study to investigate the inhibitor development after CI should be conducted.

References 1. Batorova A, Martinowitz U. Continuous Infusion of coagulation factors. Haemophilia 2002;8:170–177 2. Oldenburg, J. & E. Tuddenham. 2002. Genetic basis of inhibitor development in severe haemophilia A and B. In Inhibitors in Patients with Haemophilia. E.C. Rodriguez-Merchan & C.A. Lee, Eds.: 21–26. Blackwell Publishing. Oxford, UK 3. Hermans C, Thyn Yee T, Perry D, Lee C. Development of inhibitor in haemophilia patients treated with continuous infusion. Haemophilia, 2002;8:541 4. Koestenberger M, Raith W, Muntean W. High titre inhibitor after continuous factor VIII administration for surgery in a young infant. Haemophilia 2000;6:120 5. White B, Cotter M, Byrne M, O`Shea E, Smith OP. High responding factor VIII inhibitors in mild haemophilia – is there a link with recent changes in clinical practice? Haemophilia 2000;6:113–115

Inhibitors in PTP’S: A Retrospective Study in Germany Ch. von Auer, J. Oldenburg, M. Krause, W. Miesbach, I. Scharrer, and PTP-Study Group

Introduction Today the development of an inhibitor against FVIII represents the most serious complication in hemophilia treatment. Previously treated patients (PTP), who have not developed an inhibitor so far, are considered to be tolerant to factor VIII or IX and at low risk for inhibitor development. The risk for inhibitor development is highest in children under 5 years of age with few exposure days (ED). After 20 ED and/or in children between 6–10 years of age the risk is already leveling off [1]. Older patients with more than 50–100 ED have a much lower risk to develop an inhibitor. Inhibitor detection in such a patient should raise concerns about the concomitant variables such as product neoantigenicity or way of application [2].

Method To learn about the current situation of inhibitor development in PTP’s , not product related data of topical interest were hard to find. We therefore conducted a retrospective study onto inhibitor development in PTP’s in Germany in the last 5 years. We designed a questionnaire with 14 retrospective questions. It was sent to 88 hemophilia treating hospitals/offices in Germany. The following questions were asked about the patients: – age – severity of hemophilia – exposure days – genotype – factor concentrate – indication – infused amount – change of product – way of application – concomitant medication – concomitant diseases – concomitant blood transfusion – vaccination I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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– inhibitor: – high responding (HR) or – low responding (LR) – permanent/transient – type I/type II – ITT – ITT-Success

Results So far 47 centers have answered the questionnaire. 32 of them reported no inhibitor in PTP’s in the past 5 years. 15 colleagues counted altogether 37 PTP’s with inhibitor development. Evaluating data, it became obvious that there is a variety of existing PTP definitions in Germany, referring to age of patient, number of ED and former change of product. In literature we could not find a standard definition for PTP. For our own research we determined PTP as patient who has more than 20 ED or at least one change of product or severe hemophilia and is at least ten years old. With this definition we registered 28 PTP-inhibitors, 9 reported patients had to be excluded. The following survey shows reported data concerning age of the patient, severity of hemophilia, gene mutation, estimated risk of inhibitor development concerning gene mutation, exposure days (ED), factor concentrate, given amount, date of inhibitor detection, former change of product, date of change of product, way of application (bolus/continuous infusion) and BU of inhibitor (high responder/low responder).

Conclusion Most of the 28 patients were over 18 years old (17/28) and had severe hemophilia (18/28). The distribution of gene mutations showed an unexpected high number of missense mutations (25%) with a low risk for inhibitor development. 23 patients had more than 20 ED when the inhibitor developed, 9 of them even more than 100 ED. 15 patients received recombinant factor concentrates before or during inhibitor development, 12 patients were treated with plasma derived concentrates. Most factor concentrates were given as bolus infusion. Indication was spontaneous bleeding or trauma and/or surgery (18/28). Seven inhibitors were detected during routine testing. Most inhibitors were high responding (17/28). 16 patients had at least one change of product before inhibitor detection, whereas vaccination or therapy with erythrocyte concentrates seemed to have no important influence. Concerning concomitant diseases, Hepatitis C was found most frequently, nevertheless most patients were non-infected patients (19/28). Recorded concomitant medication showed no significant coherence. 19 patients received immune tolerance induction, 11 of them were successful.

Severity

Gene Mutation

s m s s m s s m s s s m

Intron 22 Inversion ? Small Deletion Intron 22 Inversion ? Intron 22 Inversion ? ? Intron 22 Inversion Big Deletion Big Deletion Missense Mutation

Inh.Risk% (3) 21 21 21 21

21 25 25 25

35

m

?

14 15 16 17

35 9m 15m 3

m s s s

? Intron 22 Inversion ? Small Deletion

21

18

19

m

Missense Mutation

<10

19 20 21 22 23 24 25

45 45 57 60 63 75 55

s s ms s s ms s

No Intron 22 Inversion Missense Mutation ? Missense Mutation ? ? Stop Mutation

26

39

s

?

27

15

s

Insertion

28

70

m

?

21

<10 <10

17

3

6 10 11 13 18 21 21 21 23 25 25 26

FVIII Date of Amount/ IU rec./plasm. Inh.Det. r 3000 5/03 r 38000 6/00 p 5250 12/01 r 6000 7/00 p 430000 8/99 p ? 4/00 ? ? 11/01 p >42000 2/01 r 35000 6/99 p 10500 1/01 p 94.500 7/03 r 25000 3/03

Change of Product 1 1 1 2 1 0 ? ? 10 0 1 0

Date of Change 12/02 12/99 9/01 12/99 8/99 0 ? ? 6/99 0 1/01 0

BI/CI

BU

BI both BI BI CI BI BI CI both BI BI BI

LR HR LR HR HR HR LR LR HR HR HR HR LR trans. HR HR HR HR HR trans HR LR HR LR LR LR HR LR trans. LR trans. HR

29

r

48348

1/04

0

0

BI

30 35 37 60

r p r r

100001 20000 24000 43000

11/98 12/99 1/98 5/98

? 0 0 2

? 0 0 ?

CI both BI BI

79

p

30500

7/99

?

?

CI

101 101 101 101 112 112 468

r p r r p r r

>70000 >37500 150000 80000 74000 40000 100001

2/99 11/99 3/98 5/02 4/98 7/96 11/01

2 0 2 2 0 1 1

9/98

7/96 ?/97

both both both BI BI BI BI

1001

p

1000001

1/04

1

?/91

BI

1501

r

90000

?

3

?

BI

?

p

10000

11/99

2

?

BI

?/90 ?

203

13

ED

Inhibitors in PTP’S: A Retrospective Study in Germany

Age/Y . 1 15m 2 ? 3 17m 4 3 5 40 6 49 7 27 8 73 9 15 10 2 11 58 12 10

No

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To what extent this patient cohort represents typical PTP’s is a question of definition. Altogether the number of patients is still too small to draw any conclusions. In our retrospective study it became obvious that inhibitors in PTP’s are still a serious and underestimated problem in hemophilia treatment today. A definition from the SSC of the ISTH for the term »PTP« would be helpful, especially to evaluate international data. Secondly the use of the German register for drug side effects or the development of a German Hemophilia Register would make it easier to evaluate data in the future. A prospective, not product related study should be conducted.

References 1. Wight J, Paisley S. The epidemiology of inhibitors in haemophilia A: a systematic review. Haemophilia 2003;9:418–435 2. White GC et al. Utilisation of Previously Treated Patients (PTPs), Noninfected Patients (NIPs), and Previously Untreated Patients (PUPs) in the Evaluation of New Factor VIII and Factor IX Concentrates. Thromb Haemost 1999;81:462 3. Oldenburg J, pers. communication

Elective Orthopedic Surgery in Inhibitor Patients – the Frankfurt Concept A. Kurth, U. Stumpf, B. Habermann, M. Krause, and I. Scharrer

Inhibitors against FVIII or FIX in patients with hemophilia are a common and serious complication. Until recently elective surgery was despite of a substitution therapy mostly associated with major bleeding. Only emergency medical care was done if necessary. We report about 5 patients with hemophilia A and inhibitors who underwent 12 orthopedic interventions. Because of severe joint destruction 1 arthrodesis of the knee joint, 1 arthrodesis of the upper ankle joint and 4 total knee replacements were performed. In 1 patient a close reduction of a distal femur fracture, an osteosynthesis and a mega-prosthesis was necessary. Furthermore, 2 hematomas and 1 pseudotumor were removed. Before the elective surgery the patients underwent through diagnostic procedures. In the early patients a preoperative immunadsorbant therapy with Therasorb was done to eliminate the inhibitors. Currently we perform elective surgery only with a therapy of recombinant factor VIIa and if necessary we change to Feiba. In inhibitor patients we did not see increased bleeding during or after surgery compared to our experiences with non-inhibitor hemophilic patients. In addition, the reduction of the fracture was done without any bleeding complications. In summary, we changed our approach for the treatment from preoperative decrease of inhibitors and perioperative substitution with factor VIII to an only treatment with recombinant factor VIIa in inhibitor patients undergoing an elective orthopedic procedure. Dosage NovoSeven 2 h preoperative: postoperatively: 1.–3. post-op day: 4. post-op day:

140–180 µg/kg (»thrombin-burst«) 140 µg/kg every 2 h/24 h 120 µg/kg continuous infusion 40–50 µg/kg/h

Tranexamic acid 3x 1 Amp. à 500 mg daily control : Hb, D-Dimere, FVII (1,000–2,000 %) Feiba 200 U/kg/d I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

VIIc. Clinic and Casuistics

The Frequency of Venous Thromboembolism in Women with FV Leiden in Association with Pregnancy and Puerperium P. Dulícˇek, J. Maly´, M. Pecka, and M. Beránek

Introduction Venous thromboembolism (VTE) is a multifactorial disease. Pregnancy is a well recognized thrombophilic condition which is associated with 6–10-fold higher risk of VTE and the risk is even higher after delivery [1–3]. Resistance to activated protein C (APC-R) with FV Leiden is the most common inherited thrombophilia currently known and individuals with heterozygous form have 3–7-fold higher risk of VTE [4–7], in homozygous form is the risk even 10-fold higher.

The aim and Type of the Study The aim of our study was the assessment of the frequency of VTE in females with FV Leiden ( in heterozygous and homozygous form ) in association with pregnancy and puerperium and according these results and available data to formulate the principles of thromboprophylaxis in these settings. It is the retrospective case control study.

Investigated Groups We have assessed the frequency of VTE in 3 cohorts. 1. Cohort contains 224 women with FV Leiden in heterozygous form (460 pregnancies). None of these pregnancies was covered by pharmacological thromboprophylaxis with either LMWH or UFH. These females were recruited from the patients who were recommended to our clinic for evaluation of suspicious inherited thrombophilia after the fulfillment of at least one of the following criteria spontaneous VTE or VTE in association with risk situations before the age of 45 years recurrent VTE (deep venous thrombosis or pulmonary embolism) recurrent superficial thrombophlebitis (without varices) – at least 5 events positive family history of venous thromboembolism with the first event before the age of 45 years venous thrombosis at unusual sites (v. portae, lienalis etc.) Thrombosis was objectively confirmed in every case (ultrasound, spiral CT, lung scanning). In every woman APC-R with FV Leiden was diagnosed and other thrombophilic conditions were excluded (antithrombin deficiency, protein C and protein S defiI. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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ciency, hyperhomocysteinemia, FII20210A and antiphospholipid syndrome). These criteria fulfilled 134 females. Another 90 females were included into this group. There are family relatives of those 134 females, when FV Leiden was diagnosed during the evaluation of this mutation in the first- degree relatives of affected women. 2. Cohort contains 40 women with FV Leiden in homozygous form (70 pregnancies) and without thromboprophylaxis as well. These females were recruited according the same inclusion criteria as in the first cohort. 3. Cohort contains 201 women without FV Leiden in association with 422 pregnancies and also without thromboprophylaxis in pregnancy and puerperium. 111 females out of 201 women in this group were recruited after the exclusion of FV Leiden during the evaluation of this mutation in the first-degree relatives of affected females. Another 90 women were included into this group. In these females FV Leiden was not proven during the assessment of prevalence of F Leiden in the East Bohemian region in 1996. The assessment of frequency of VTE in pregnancy and puerperium was done within the period 1996–2003. The results were statistically assessed by Fisher’s exact test in program NCSS 2004. Results 1. Cohort VTE occurred 44–fold this group. In 17 cases VTE developed in pregnancy (once in I. trimester, twice in II. trimester and l4-fold in III. trimester), in 27 women VTE occurred after delivery and always within the first 10 days. In l6 cases VTE developed after delivery with caesarian section. Proximal DVT was detected in 34 females and 5 times was complicated by pulmonary embolism. Left extremity was affected in 82% in pregnancy and after delivery both extremity was affected similarly (15-fold left leg, 12-fold right leg). The majority of VTE (30 cases) occurred in the subgroup of 134 females. The frequency of VTE is 9,6% in this group 2. Cohort VTE occurred in 14 cases (once in II. trimester, 4-fold in III. trimester and 9-fold after delivery and always within the first 2 weeks. In all cases proximal deep venous thrombosis developed and in 2 cases was complicated by pulmonary embolism. The frequency of VTE is 20% in this group 3. Cohort Pregnancy was complicated by VTE only in case – in III. trimester. The frequency of VTE is 0.24% in this cohort. Frequency of VTE in the both cohorts of females with FV Leiden reached the statistical significance in comparison with women without FV Leiden mutation. The results are shown in the Table 1.

The Frequency of Venous Thromboembolism in Women with FV Leiden

211

Table 1

No. of women No. of pregnancies No. of VTE / pregnancy

FV Leiden in heterozygous form

FV Leiden in homozygous form

Females without FV Leiden

224 460 44 (9.6%)

40 70 14 (20%)

201 422 1 (0.24%)

Discussion The data about the frequency of VTE in women with FV Leiden in association with pregnancy are variable and depend on the design of study. Among females with VTE in pregnancy the frequency of FV Leiden is higher (20%–30%) [8, 9] than in the groups, when prevalence of VTE is assessed prospectively (2%) [10]. The frequency is almost 10% in our cohort of women with FV Leiden in heterozygous form. That is caused by inclusion criteria because nowadays is known, that individuals with thrombophilia and positive family or personal history of VTE have higher risk of VTE than individuals with thrombophilia without positive history of VTE [11]. This fact also explains the higher frequency of VTE (20%) in our females with homozygous form (10%–20% according literature) [12].

Conclusion We do not recommend LMWH in asymptomatic women with FV Leiden in heterozygous form during pregnancy, but coumarin is recommended within 2 months after delivery. In females with personal history of VTE we recommend LMWH in pregnancy. The dosage and time of administration depend on the assessment of personal history of VTE and the assessment of all other risk factors of VTE. Coumarin is also taken 2 months after delivery. For all women we recommend the physical activity with both legs and elastic compressive stockings. In females with homozygous form LMWH is recommended in all cases and also in this case the dosage and time of introduction depend on personal history of VTE and after careful assessment of all other well known risk factors of VTE in pregnancy (age, obesity, varices etc.). Coumarin is also taken at least within 2 months after delivery.

References 1. Kierkegaard, A.: Incidence and diagnosis of deep vein thrombosis associated with pregnancy. Acta Obstet. Gynecol. Scand., 1983, 62, p. 239–243 2. Treffers, P.E., Huidekoper, B.L., Weenink, G.H., et al.: Epidemiological observation of thrombo-embolic disease during pregnancy and in the puerperium, in 56,022 women. Int. J. Gynecol. Obstet., 1983, 21, p. 327–331

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3. Greer, I.A.: Haemostasis and thrombosis in pregnancy. In Bloom, A.L., Forbes, C.D., Thomas, D.P., Tuddenham, E.G.D., editors. Hemostasis and Thrombosis, 1994, Vol. 2., p. 987–1015 4. Rosendaal,F.R., Koster, T., Vandenbroucke,J.P., et al.: High risk of thrombosis in patients homozygous for factor V Leiden (activated protein C resistance). Blood, 1995, 55, p. 1504–1508 5. Koster, T., Rosendaal, F.R., de Ronde, H., et al.: Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden Thrombophilia Study. Lancet, 1993, 342, p. 1503–1506 6. Kontula, K., Ylikorkala, A., Miettinen, H., et al.: Arg 506Gln factor V mutation (factor V Leiden) in patients with ischaemic cerebrovascular disease and survivors of myocardial infarction. Thromb. Haemost., 1995, 73, p. 558–560 7. Price, D.T., Ridker, P.M.: Factor V Leiden mutation and the risks for thromboembolic disease: a clinical perspective. Ann. Intern. Med., 1997, 127, p. 895–903 8. Bokarewa, M.I., Breme, K., Blombäck, M.: Arg 506 – Gln mutation in factor V andrisk of thrombosis during pregnancy 9. Hallak, K., Senderowicz, J., Cassel, A., et al.: Activated protein C resistance (factor V Leiden) associated with thrombosis in pregnancy. Am. J. Obstet. Gynecol., 1997, 176, p. 889–893 10. Martinelli, I., Bucciarelli, P., Margagline, M.: The risk of venous thromboembolism in family members with mutations in the genes of factor V or prothrombin or both. Br. J. Haematol., 2000, 111, p. 1223–1229 11. Lensen, R.P., Rosendaal, F.R., Koster, T. et al.: Apparent different thrombotic tendency in patients with factor V Leiden and protein C deficiency due to selection of patients. Blood, 1999, 8, p. 4205–4208 12. Pabinger, I., Nemes, L., Rintelen, C., et al.: Pregnancy associated risk for venous thromboembolism and pregnancy outcome in women homozygous for factor V Leiden. The Hematology Journal., 2000, 1, p. 37–41

VIId. Diagnostics

Spectrum of Molecular Defects and Mutation Detection Date in Patients with Severe Hemophilia A N. Bogdanova, A. Markoff, U. Nowak-Göttl, R. Eisert, C. Wermes, H. Pollmann, A. Todorova, A. Eigel, B. Dworniczak, and J. Horst

Introduction Hemophilia A is a common X-Linked bleeding disorder caused by various types of mutations in the factor VIII gene. The most common intron 22- and intron 1-inversions are responsible for about 40–50% of the severe hemophilia A cases while large deletions, point mutations and small rearrangements are responsible for the disease in the rest of the patients. Here we report on the mutation detection rate, the spectrum of mutations in a cohort of 87 patients with severe hemophilia A, as well as their distribution through the F8 protein.

Patients and Methods Eighty seven severely affected unrelated patients of German origin, previously tested negative for the common intron 22-inversion, were included in the study. Information about the presence of F8 inhibitors was systematically collected. Genomic DNA was extracted from peripheral blood lymphocytes using salting procedure. The intron 1 inversion was analyzed with breakpoint PCR using the oligonucleotides and conditions as published [1]. For sequencing analysis of the F8 gene PCR and sequencing reactions were performed as previously described [2]. Southern blot analysis of Taq I digested DNA, hybridized with F8 cDNA probe was performed to screen for the presence of gross deletions [3]. The newly detected amino acid substitutions were scored for potential gross or local conformational changes and influence on molecular stability for every single F8 domain with available structures, using homology modeling [4–6].

Results and Discussion Spectrum and Distribution of the Mutations Using the techniques outlined above we were able to identify the disease causing mutation in all screened patients. A full spectrum of mutations including missense, nonsense, small and gross rearrangements and splice mutations was in our patient group. The molecular defects are systematically presented in Figure 1. I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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Fig. 1. Spectrum and distribution of the mutations in the F8 gene, identified in 87 male patients with severe hemophilia A, negative for the common intron 22-inversion.

The missense mutations account for 34 (39%) of the cases. They are distributed throughout the whole sequence of the factor VIII gene, but predominantly in the A1 and A2 domains. 19 of them are novel, namely in A1 domain: V10G, T49A, T49P, L58P, D116E, C179G, S207C and H281L; in A2 domain: Y395C, K425N, R427P, L547F and C528W; in B domain: P1641L; in A3 domain: E1875K and S1959R; in C1 domain: P2948L and P2052S and R2052L in the C2 domain. These novel amino acid substitutions were scored for potential conformational changes and influence on the molecular stability, using homology modeling. Figure 2 illustrates the effects of the mutations V10G (shifting in the hydrogen bonding patterns of the A1 domain and K425N (rendering the charged residue not being to participate in the ionic bond with E581 (A2 domain). Protein truncating point mutations were present in 45 (51.7%) of the patients and more than the half of them are located in exon 14 (B domain) of the factor VIII gene. The majority of these type molecular defects are small deletions or insertions, causing frameshift. Five such mutations, c.205–206delCT, c.1578delA, c.3385delC, c.3055-3058delACAA and c.3090-3093delAAAG as well as the nonsense mutations L168X, Q1317X, L952X, Q1304X, K1263X, W1029X, Y1748X and Y1786X are described for the first time. Gross rearrangements were detected in 6 (7%) of the cases. Three of these patients (3.5%) carry the intron 1 inversion. Splicing errors were detected in only two patients.

Development of Factor VIII Inhibitors None of the patients with missense/nonsense/splicing mutations or gross rearrangements developed inhibitors during their treatment with rFVIII. All four inhibitors positive patients (4.6%) are carrying small rearrangements, namely c.2610delT,

Spectrum of Molecular Defects and Mutation Detection Date in Patients

217

Fig. 2. Dreiding model images of the F8 A1 domain (upper left panel) with the mutation V10G (upper right panel) and of the F8 A2 domain (lower left panel) with the mutation K425N (lower right panel). Protein chains are colored in light blue, selected amino acids are dark blue. Disulfide bonds are presented in yellow, hydrogen bonds in red and ionic bonds in magenta. Residues participating in ionic bonds are highlighted in green (where not selected in dark blue).

c.4665–4678del/c.4664/4678insAAGGAA and c.3385delC in exon 14 and c.5961delA in exon 18 of the factor VIII gene.

Discussion It is well known, that the common intron 22 inversion of the factor VIII gene accounts for 40-50% of the severe hemophilia A cases. Therefore the purpose of this study was to determine the mutation spectrum and detection rate in a large severe hemophilia A patient sample, previously tested negative for the intron 22 inversion.

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The disease causing mutation was identified in all tested patients. Based on our results, the expected mutation detection rate in severely affected male patients with hemophilia A is approaching 100% when testing for the common inversions and big deletions, followed by sequencing analysis of the negative probands. However, the detection rate in female carriers of severe hemophilia A is about 96%, since gross deletions would not be detected in women. Nearly half of the mutations, observed in this study, are located in the A1 and A2 domains. The vast majority of protein truncating mutations affects exon 14 of the factor VIII gene (B domain). Therefore, one should probably start the genetic analysis in severely affected patients with screening these parts of the gene. Although the number of inhibitors-positive patients is too small to draw definite conclusions, the results of our study give evidence for a possible correlation between this important complication and the presence of small deletions in the factor VIII gene. References 1. Bagnall RD, Waseem N, Green PM, Giannelli F. Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia Blood. 2002; 99(1):168–74 A 2. Bogdanova N, Markoff A, Pollmann H, Nowak-Göttl U, Eisert R, Dworniczak B, Eigel A, Horst J. Prevalence of small rearrangements in the factor VIII gene F8C among patients with severe hemophilia A.. Hum Mutat. 2002; 20(3):236–7 3. Millar DS, Zoll B, Martinowitz U, Kakkar VV, Cooper DN. The molecular genetics of haemophilia A: screening for point mutations in the factor VIII gene using the restriction enzyme TaqI. Hum Genet. 1991; 87:607–612 4. Lund O, Frimand K, Gorodkin J, Bohr H, Bohr J, Hansen J, Brunak S. Protein distance constraints predicted by neural networks and probability density functions. Protein Eng. 1997; 10: 1241–1248 5. Pemberton S, Lindley P, Zaitsev V, Card G, Tuddenham EG, Kemball-Cook G. A molecular model for the triplicated A domains of human factor VIII based on the crystal structure of human ceruloplasmin. Blood. 1997; 89(7):2413–21 6. Stoilova-McPhie S, Villoutreix BO, Mertens K, Kemball-Cook G, Holzenburg A. 3-Dimensional structure of membrane-bound coagulation factor VIII: modeling of the factor VIII heterodimer within a 3-dimensional density map derived by electron crystallography. Blood. 2002; 99(4):1215–23

Hemophilia Patients and Prothrombotic Gene Mutation A. Hlusˇí, V. Krcˇová, P. Novák, and L. Slavík

Introduction Various prothrombotic gene mutations have been described as risk factors for thromboembolism. These molecular hemostatic defects increase risk of thrombosis in carriers of mutations. In hemophilia patients, thrombotic events are rare, due to congenital factor deficiency. There is considerable variability in bleeding patterns of severe hemophiliacs. Knowledge of these determinants could help to optimize treatment strategy and estimate clinical phenotype of hemophilia.

Aim To evaluate the clinical phenotype in hemophiliac A and hemophiliac B patients and its relation to presence of prothrombotic gene mutations.

Subjects and Methods In a group of 26 severe hemophilia patients (19 with hemophilia A, 7 with hemophilia B, none with inhibitor) we evaluated the presence of inherited prothrombotic genetic factors – Factor V Leiden (G1691A), prothrombin mutation (G20210A), methylentetrahydrofolate reductase mutation (C677T) and plasminogen activator inhibitor gene mutation (4G/5G). TC genotyping kit and 4G/5G polymorphism PCR testing with Bsl I restriction endonuclease were used. In clinical phenotype, we observed the number of bleeding episodes per year requiring therapy, factor concentrate utilization and history of thromboembolism in patients and their families. Five of these patients were treated by intermediate dose prophylaxis, others through on-demand treatment. There were no patients with thromboembolism history, but one patient with positive family history of pulmonary embolism.

Results Heterozygous gene mutations were found in 16 patients, including 5 with combined defects. In accordance to prevalence in normal population, MTHFR mutation was I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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the most frequent one. Heterozygosity for this mutation is not associated with an increased risk for thrombosis.

Conclusion In our group of patients with hemophilia we have not found carriers of homozygous prothrombotic gene mutations. Heterozygous defects were found only. There was no influence on clinical phenotype in single defects. We observed a statistically insignificant effect on clinical phenotype in combined defects. Our experience is limited by the small group of patients under examination. The clinical phenotype may also be influenced by other factors than those involving clot formation. Further studies are needed to confirm an exact role of prothrombotic factors.

gAla82Gly Represents a Common Fibrinogen Chain Variant in Caucasians V. Ivaskevicius, E. Jusciute, M. Steffens, Ch. Geisen, P. Hanfland, Th. F. Wienker, E. Seifried, and J. Oldenburg

Abstract Screening of 200 blood donors for the presence of polymorphisms in 3 fibrinogen genes (FGA, FGB, FGG) revealed two individuals with a heterozygous missense mutation (c.323C > G, gAla82Gly) in the FGG gene. This mutation has been reported previously to cause mild hypofibrinogenemia. Analysis of an additional 416 blood donors showed two more heterozygous gAla82Gly mutations, resulting in an overall gAla82Gly allele frequency of 0.0032. Haplotype analysis demonstrated that the gAla82Gly mutation originated from a common founder. From these data we estimated that homozygous individuals for gAla82Gly should occur at a frequency of 1:95,000, suggesting that hypofibrinogenemia represents a more frequent condition in the population than believed so far.

Introduction Fibrinogen is an important component of the coagulation cascade, as well as a major determinant of blood viscosity and blood flow. Moreover it plays a critical role in physiopathological processes, such as inflammation, atherogenesis and thrombogenesis. Fibrinogen is a soluble plasma glycoprotein, comprised of three pairs of nonidentical polypeptide chains (Aa, Bb and g), linked to each other by disulfide bonds at their N-termini. The fibrinogen protein chains are encoded by three genes (OMIM: FGA +134820, FGB *134830, FGG *134850) grouped in a cluster of about 50 kb on chromosome 4q23-32 [1]. Mutations affecting the fibrinogen genes result in absence (afibrinogenemia), reduction (hypofibrinogenemia) or dysfunction (dysfibrinogenemia) of the corresponding protein. To date approximatly 150 different gene mutations, predominantly missense mutations, have been involved in a database for human fibrinogen variants (http://www.geht.org/databaseang/ fibrinogen) [2]. In this study we show for the first time that the rare gAla82Gly variant, causing mild hypofibrinogenemia, represents a founder mutation that is present in the normal population. Moreover, the gAla82Gly variant is in complete linkage disequilibrium with the b(-854) G>A polymorphism in the FGB promoter.

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Patients, Materials and Methods Subjects In the present study we included two cohorts of blood donors from the region of Bonn in Germany. All blood donors were of Caucasian origin. The first cohort consisting of 200 individuals (100 females and 100 males) was investigated for the presence of polymorphisms in the 3 fibrinogen genes FGA, FGB and FGG. HardyWeinberg equilibrium was proven for the identified SNPs (data not shown). The second cohort consisting of 416 individuals (221 females and 195 males) was analyzed only for the presence of the gAla82Gly variant and the b(-854)G>A polymorphism, in order to allow a more exact estimation on their frequencies. From the blood donors no plasma samples for the testing of clotting parameter were available. Additionally, three patients exhibiting the gAla82Gly mutation were included to prove the founder effect by haplotype analysis of the FGA, FGB and FGG genes. These patients were sent for genetic testing of the fibrinogen genes, because they suffered from clinically symptoms as epistaxis or recurrent miscarriages due to a mild hypofibrinogenemia with fibrinogen levels in the low normal range of 1.4 to 2.4 mg/ml (normal 1.5–4.0 mg/ml) (Table 1). All blood donors and patients gave informed consent according to the declaration of Helsinki. Table 1. Comparison of the genotypes of the individuals carrying gAla82Gly mutation Phenotype

C/C C/C C/C C/C

G/G G/G G/G G/G

A/A A/A A/A A/A

G/G G/G G/G G/A

C/C C/C C/C C/T

G/A A/A G/A G/A

G/G G/G G/G G/A

C/C C/C C/T C/C

C/C C/C C/C C/T

C/C C/C C/C C/T

G/G G/G G/G G/A

Sex

BbArg448Lys, c.1433 G>A

BbSer159Ser, c.567 C>T C/C C/C C/C C/T

BbTyr345Tyr, c.1125 C>T

b(-148) C>T

b(-249) C>T

b(-455) G>A

Thrombin clotting time (sec) normal range 18–22 sec.

C/C C/C C/C C/C

b(-854) G>A

n.d. n.d. n.d. n.d.

C/G C/G C/G C/G

b(-993) C>T

n.d. n.d. n.d. n.d.

A/A A/A A/A A/G

AaThr312Ala, c.991A>G

n.d. n.d. n.d. n.d.

A/A A/A A/A A/A

b(-1420) G>A

4 5 6 7

a(-58) G>A

21.5

a(-128) C>G

21

g3'UTR, 8537 C>T

1.4

gAla82Gly, c.323 C>G

31 A/A A/G C/G C/C C/C G/G A/A G/G C/C G/A G/G C/C C/C C/C C/C G/G F

g(-239) A>G

n.d. 22

g( -773) A>T

18 n.d.

Subject No

11 A/A A/G C/G C/C C/C G/G A/A G/G C/C G/A G/G C/C C/C C/C C/C G/G M 2.4 21 A/A A/G C/G C/C C/C G/G A/A G/G C/C G/A G/G C/C C/C C/C C/C G/G F 1.4

M M F F

Clinical symptoms

FGB SNPs

Reptilase time (sec) normal range 18–22 sec.

FGA SNPs

Clauss (mg/mL) normal range 1.5–4.0 mg/mL

FGG SNPs

epistaxis recurrent miscarriages recurrent miscarriages n.d. n.d. n.d. n.d.

c. = cDNA sequence, nucleotide 1 is the A of the ATG-translation initiation codon , n.d. = not determined 1

subjects 1-3 were clinically symptomatic patients sent to our lab for genetic testing

Coagulation Assays Functional fibrinogen levels were determined according to Clauss method. Thrombin and reptilase times were measured using standard coagulation assays.

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Denaturing high Performance Liquid Chromatography Genomic DNA was prepared from EDTA blood leucocytes by standard procedures. Twenty-three exons and their flanking regions of the FGA (GenBank accession number AF361104), FGB (AF388026) and FGG (AF350254) genes were amplified in 27 PCR fragments. Equal volumes of 25µl PCR template from the patient and a wild type control were mixed and heated at 95°C for 10 min followed by incubation at 55°C for 10 min to allow for heteroduplex formation and analyzed by denaturing high performance liquid chromatography (dHPLC, Transgenomic, USA) [3]. In addition, polymorphic sites of the promoters regions (in total 10 fragments, see Table 1) have been analyzed in individuals carrying the gAla82Gly mutation.

Sequencing All aberrant DNA fragments detected by dHPLC were amplified again and purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Sequencing was performed on an automated sequencing system (ABI Prism 310, Applied Biosystems, Weiterstadt, Germany) using BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) according to the manufacturer’es recommendations.

Results and Discussion Screening of 200 blood donors for the presence of polymorphisms in 3 fibrinogen genes (FGA, FGB, FGG), showed a missense mutation in the FGG gene resulting in the substitution of Ala by Gly at amino acid position 82 (c.323C>G). The gAla82Gly mutation (Fibrinogen Dunedin) was previously reported by Brennan et al (2000), and Wyatt et al (2000) as being causative for hypofibrinogenemia and associated with a mild bleeding tendency [4, 5]. Two individuals of the blood donor cohort were found to be heterozygous for the gAla82Gly mutation, corresponding to a frequency of 2 in 400 alleles, or 0.005. The investigation of a further 416 healthy blood donors representing an additional 832 alleles revealed an additional two heterozygous gAla82Gly individuals, resulting in an overall allele frequency of 0.0032 (SD±0.0016). From these data the frequency of homozygous carriers of the gAla82Gly allele in the population is estimated to be 1:95.000, under the assumption of Hardy-Weinberg genotype proportions. We hypothesized that the gAla82Gly mutation may represents a common founder effect. To verify this assumption the 15 most common SNPs within the three fibrinogen genes in the 4 blood donor carriers of the gAla82Gly mutation and additionally in 3 German origin patients with mild hypofibrinogenemia (unpublished results) who were also found to be heterozygous for this variant were analyzed (see Table 1). The resulting FGG-FGA-FGB-haplotype suggested an association with the A allele of the b(-854)G>A polymorphism in the promoter of the FGB gene. This variant has been reported to be associated with increased plasma fibrinogen levels

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[6, 7]. Six of seven carriers of the gAla82Gly mutation were heterozygous for the b(854)G>A polymorphism and 1 homozygous (-854A/A). Analyzing 1232 chromosomes the A allele frequency of the b(-854)G>A polymorphism in the normal population was found to be 0.1437 (SD± 0.0102). The probability that all 7 non related individuals show the b(-854)G>A SNP is calculated to be 0.00001. Thus a strong association should be expected between the b(-854G>A) in the promoter of the bgene and the gAla82Gly mutation of the g-gene. Recently, complete allelic association was reported between the b(-148)C>T, b(-455)G>A, b(-1420)G>A, BbSer159Ser, BbTyr345Tyr and BbArg448Lys polymorphisms. In contrast, the b-854A variant was in complete negative linkage disequilibrium with the b-148T and b-455A alleles [6–8]. Our data represent the first report of a strong association between a variant of the FGG gene and a SNP of the FGB gene. It might be speculated that this association is due to a relatively recent origin of the gAla82Gly mutation. A second missense mutation that we found during the SNP screening was located in the FGB gene and resulted in the substitution of Lys (AAG) by Asp (AAC) at amino acid position 148 (c.534G>A, BbLys148Asp). The BbAsp148Lys mutation was identified in 1 out of 400 chromosomes corresponding to a frequency of 0.0025. Since both, the Bb148Lys and the Bb148Asn variants have been shown to be equally well expressed in plasma with fibrinogen concentrations corresponding to the wild type, the BbLys148Asn variant does not seem to have pathogenetic relevance [9]. The AaArg16Cys), AaArg16His) as well as gArg275Cys) and gArg275His) mutations that occurred at CpG mutation hotspot sites, all causing dysfibrinogenemia, and the most common mutation, aIVS4-1 G>T, associated with afibrinogenemia, were not detected in any of the 200 blood donors screened for the presence of SNPs. So far only few patients with a heterozygous gAla82Gly mutation are known. Wyatt et al (2000) reported two heterozygous carriers of the gAla82Gly mutation with fibrinogen concentrations of 1.7 and 2.2 mg/ml. Two further patients with compound heterozygous mutations (gIVS2+1G>A and gAla82Gly) showed fibrinogen levels of 0.6 and 0.9 mg/ml, suggesting that both mutations have an effect on the fibrinogen concentration [5]. Another patient described by Brennan et al (2000) exhibited a fibrinogen concentration of 0.7 to 1.1 mg/ml [4]. Patients carrying the gAla82Gly mutation were clinically symptomatic with mild bleeding symptoms as epistaxis and menorrhagia. In our study the fibrinogen concentration was only available from the 3 patients but not the blood donors carrying a heterozygous gAla82Gly mutation. Fibrinogen concentrations ranged from 1.4 to 2.4 mg/ml (normal 1.5–4.0 mg/ml). Samples from these patients were sent for genetic testing because they were clinically symptomatic with mild bleeding symptoms e. g. epistaxis and/or recurrent miscarriages. Thus, the data from our group and those published previously, strongly support that gAla82Gly is causal for the clinical symptoms observed in these patients. In conclusion, our results suggest that the gAla82Gly mutation represents the most frequent mutation causing hypofibrinogenemia in central Europe with an estimated frequency for homozygous individuals of about 1:95,000. Heterozygous carriers of the gAla82Gly variant normally do not bleed and have only mildly reduced fibrinogen levels, and thus may not be identified in routine coagulation testing. Our data provide evidence that the gAla82Gly mutation probably originated from a

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common founder and the G-allele of the gAla82Gly variant is associated with the Aallele of b(-854) G>A polymorphism.

References 1. Chung DW, Harris JE, Davie EW. Nucleotide sequences of the three genes coding for human fibrinogen. Adv Exp Med Biol 1990; 281:39–48 2. Hanss M, Biot F. A Database For Human Fibrinogen Variants. Ann NY Acad Sci 2001; 936:89–90 3. Jones AC, Austin J, Hansen N, Hoogendoorn B, Oefner PJ, Cheadle JP, et al. Optimal temperature selection for mutation detection by denaturing HPLC and comparison to singlestranded conformation polymorphism and heteroduplex analysis. Clin Chem 1999; 45:1133–1140 4. Brennan SO, Fellowes AP, Faed JM, George PM. Hypofibrinogenemia in an individual with 2 coding (gamma82 A—>G and Beta235 P—>L) and 2 noncoding mutations. Blood 2000; 95:1709–1713 5. Wyatt J, Brennan SO, May S, George PM. Hypofibrinogenaemia with compound heterozygosity for two gamma chain mutations -gamma 82 Ala—>Gly and an intron two GT—>AT splice site mutation. Thromb Haemost 2000; 84:449–452 6. van ‘t Hooft FM, von Bahr SJ, Silveira A, Iliadou A, Eriksson P, Hamsten A. Two common, functional polymorphisms in the promoter region of the beta-fibrinogen gene contribute to regulation of plasma fibrinogen concentration. Arterioscler Thromb Vasc Biol 1999; 19:3063–3070 7. Behague I, Poirier O, Nicaud V, Evans A, Arveiler D, Luc G, et al. Beta fibrinogen gene polymorphisms are associated with plasma fibrinogen and coronary artery disease in patients with myocardial infarction. The ECTIM Study. Etude Cas-Temoins sur l’Infarctus du Myocarde. Circulation 1996; 93:440–449 8. Fellowes AP, Brennan SO, George PM. Identification and characterization of five new fibrinogen gene polymorphisms. Ann N Y Acad Sci 2001; 936:536–541 9. Maghzal GJ, Brennan SO, Fellowes AP, Spearing R, George PM. Familial hypofibrinogenaemia associated with heterozygous substitution of a conserved arginine residue; Bbeta255 Arg—>His (Fibrinogen Merivale). Biochim Biophys Acta 2003; 1645: 146–151

A Novel Mutation (Asp36Tyr) in the Vitamin K Epoxide Reductase Complex Subunit 1 Gene (VKORC1) Causes Increased Phenprocoumon Requirement C. Geisen, G. Spohn, K. Sittinger, S. Rost, M. Watzka, P. Lages, A. Huth-Kühne, R. Zimmermann, C. R. Müller, E. Seifried, and J. Oldenburg

Introduction Coumarin derivates such as warfarin and phenprocoumon are oral anticoagulants that are prescribed widely for the treatment and prevention of thromboembolic events. It has been well established that other disease states, concomitant medications and dietary vitamin K intake significantly affect coumarin dose requirement. In addition, hereditary warfarin sensitivity has been shown to be caused by two genetic variants of the CYP2C9 gene, Arg144Cys (CYP2C9*2) and Ile359Leu (CYP2C9*3). These alleles were associated with lower warfarin dose requirements, more time to achieve stable dosing and a significantly higher bleeding rate. Recently, we and others have identified the vitamin K epoxide reductase complex subunit 1 gene (VKORC1) [1, 2]. Heterozygous missense mutations of this particular gene were shown to cause hereditary warfarin resistance in four families and in a rat strain. Moreover, homozygous missense mutations in the VKORC1 gene have been found in the phenotype of combined deficiency of vitamin K dependent coagulation factors type 2 (VKCFD2) [1]. In the present study we report on the first heterozygous missense mutation associated with only moderately increased phenprocoumon requirement, indicating that even coumarin doses in the upper normal range may be caused by genetic variations of VKORC1.

Methods A 40-year-old patient of Caucasian descent required 40–50 mg phenprocoumon per week to maintain an international normalized ratio (INR) between 2.0 and 3.0, whereas the normal range of weekly phenprocoumon dose is 10–40 mg. Poor compliance was reasonably excluded and no concurrent medication, comorbid disorders of dietary factors could be identified to explain warfarin resistance. All 3 exons and flanking intronic regions of VKORC1 were sequenced on an automated sequencing system (ABI Prism 3100).

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VKOR Vitamin K-Recycling

Coumarin

VK-H2-

CO2

VK-OO22 H2O

Carboxylase FII FVII FIX FX PC PS PZ

ProPeptide

COOH

GLADomaine

Inactive Proenzyme

CR S

COOH

Carboxylation

Binding to PL

Fig. 1. Vitamin K-Cycle. Coumarin derivates exert anticoagulant effect by inhibiting the vitamin K epoxide reductase enzyme complex (VKOR) that recycles vitamin K 2,3-epoxide (VKO) to vitamin K hydroquinone (VK-H2). This cofactor is required by g-glutamyl carboxylase for the post-translational modification of coagulation factors II,VII, IX, X, Protein C, -S, -Z and other peptides

CYP2C9

VKORC1

Warfarin

Vitamin K-Recycling

VK-H2Fig. 2. Warfarin-metabolism and drug-target. Different pharmacokinetic (CYP2C9) and pharmacodynamic (VKORC1) gene variants affect warfarin dose requirement accounting for part of the inter-individual variability

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Arg

Ala

Arg

Asp/Tyr

Arg

Asp

Fig. 3. Analysis of the Asp36Tyr mutation. A heterozygous G to T transversion leads to an amino acid exchange of aspartase to tyrosine

V29L

R58G R58G

V66M V66M

L128R L128R

D36Y

V45A

R98W R98W

Y139C Y139C

Coumarin resistance human , Coumarin resistance rat , VKCFD2 Coumarin resistance human Coumarin resistance rat VKCFD2

VKCFD2, Warfarin-Resistenz, Warfarin-Resistenz Ratte, Warfarin-Sensitivit‰t

Fig. 4. Amino acid sequence alignment of VKORC1. Known mutations of VKORC1 which lead to coumarin resistance are shown in red (human) and orange (rat), respectively. A homozygous missense mutation in VKCFD2 is shown in green. (hs, Homo sapiens; rn, Ratus norvigicus; mm, Mus musculus; fr, Fugu rubripes; xl, Xenopus laevis, ag, Anopheles gambiae)

Results and Discussion Genetic analysis of VKORC1 revealed a heterozygous missense mutation Asp36Tyr in exon 1. Sequencing the coding region in 384 control chromosomes did not detect this variant in a normal cohort, thus indicating that the mutation probably is causal for increased coumarin requirement.

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Previously described mutations in VKORC1 in four patients (Val29Leu, Val45Ala, Arg58Gly, Leu128Arg) led to variable degrees of coumarin resistance. Two of these patients exhibited complete resistance to warfarin while two required 3 to 10 times more warfarin than normal (115–280 mg/week) [1]. Here we describe a novel VKORC1 variant Asp36Tyr leading to increased coumarin requirement of a moderate extend with a phenprocoumon dose close to the upper normal range. The association between an amino acid exchange in VKORC1 and pharmacodynamic coumarin resistance supports the hypothesis, that VKORC1 is the site of action of coumarin derivatives and indicates that VKORC1 sequence is an important determinant of the coumarin dose response. Most interestingly, mutations in VKORC1 causing warfarin resistance are scattered throughout the gene thus indicating that not only one specific epitope seems to be critical for coumarin binding.

Conclusion So far a number of rare genetic variations of VKORC1 have been found to contribute to a broad range of coumarin requirement. It would be of interest, whether future studies may also discover more common haplotypes of the VKORC1 gene that affect coumarin dosing. Such studies will be helpful to understand the anticoagulant response to coumarin derivatives and may improve the safe and effective use by better tailoring of dosage with an individual’s genetic variation. Moreover, novel anticoagulant drugs may be developed based on the knowledge of the structure of VKORC1 and the resulting multiprotein complex.

References 1. Rost S, Fregin A, Ivaskevicius V, Conzelmann E, Hortnagel K, Pelz HJ, Lappegard K, Seifried E, Scharrer I, Tuddenham EG, Muller CR, Strom TM, Oldenburg J (2004) Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2. Nature 427: 537–541 2. Li T, Chang CY, Jin DY, Lin PJ, Khvorova A, Stafford DW (2004) Identification of the gene for vitamin K epoxide reductase. Nature 427: 541–544

Denaturing High Performance Liquid Chromatography Represents an Efficient Technique for Detection of Heterozygous Large Deletions in Antithrombin Gene A. Pavlova, O. El-Maarri, B. Luxembourg, E. Lindhoff-Last, D. Delev, M. Watzka, E. Seifried, and J. Oldenburg

Introduction Antithrombin (AT) is the major physiological inhibitor of blood coagulation. The gene is located on the long arm of chromosome 1 (q23–25) and has 7 exons and 6 introns spanning 13,447 bp of genomic DNA. The gene encoded a glycoprotein with a length of 432 amino acids and a signal peptide of 32 amino acids [1]. The inherited antithrombin deficiency, associated with an increase risk of venous thromboembolic disease is a heterogeneous disorder, showing an autosomal dominant pattern of inheritance. The molecular basis of this disorder relates to single gene substitution (missense, nonsense mutation), small deletions and insertion, and more rarely large deletions and rearrangements [2, 3]. Numerous methods are available for detecting deletions of a few base pairs, but difficulties arise in detecting heterozygous large deletions. Most of PCR based methods cannot be applied, because of the normal signal of the second non deleted allele. Here we present the detection of large deletions in six patients by means of semiquantitative multiplex PCR of the AT gene followed by size-dependent separation of the amplified fragments on a DHPLC system.

Materials and Methods Six AT deficient patients of four unrelated families were investigated. Type I AT deficiency was diagnosed on the basis of parallel reduction in both plasma AT activity and level. All patients suffered thromboembolic events at young age. Amplification and direct sequencing of the coding region and intron/exon boundaries was done for mutation screening. Detection of large deletions was performed by a method combining a quantitative multiplex PCR and DHPLC.

Results and Discussion The AT gene contains ten complete or partial Alu repeat elements which can cause Alu-mediated recombination leading to large intragenic deletions that cannot be detected in heterozygous state by conventional PCR, SSCP analysis, or DNA sequencing. In all families the amplification and sequencing of genomic DNA corresponding to the coding region and intron/exon boundaries of the AT gene failed to idenI. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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tify a causative defect, suggesting major AT gene aberrations. Several different methods exist for detection of large gene deletions but each of these methods has significant shortcomings including expense, time consumption, and limited sensitivity [4]. To overcome this problem we developed a method based on semi-quantitative differential multiplex PCR of all seven exons and flanking regions of AT gene which were examined for the presence of intragenic deletions by DHPLC. Using this approach we identified a complete gene deletion in 3 patients. Two of them belong to one family (Fig. 1). In the second family the deletion found spans over exon 7, Exon 4

Exon 6

Length of 236 253 fragments in bp

Exon 1

273

Exon 7

Exon 3

327

373

Control

Exon 2

Exon 5

580

607

Fig. 1. Large deletion in antithrombin gene spreadover the whole gene (Exon 1–7)

Exon 7

Fig. 2. Deletion of exon 7 in antithrombin gene

Exon 1

Fig. 3. Deletion of exons 1 and 2 in antithrombin gene

Exon 2

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affecting also both members of the family (Fig. 2). A deletion of exon 1 and 2 was seen in the sixth patient (Fig. 3). It is difficult to estimate the proportion of cases of type I AT deficiency due to partial/whole AT gene deletions or rearrangements. The data in literature show great differences (ranging from less than 5% to 13 %) in the reported large deletions as a causal reason for AT deficiency type I. In our cohort of patients with AT deficiency we found that in 11.1% the deficiency type I is due to large deletions. It can be speculated that this quite high frequency of large deletions in AT gene reflects the problem of existing difficulties in detection and characterization of large AT gene defects which leads to under estimation of these aberrations.

Conclusions The detection of large gene deletions and rearrangements in AT gene is essential in order to provide an appropriate genetic counseling and treatment in antithrombin deficient patients. We have developed a DHPLC method combined with a quantitative multiplex PCR as an approach to screen for large heterozygous gene rearrangements in AT gene. This technique turned out to be reliable, easy to perform, cost effective and can be well applied to other genetic disorders with heterozygous large defects.

References 1. Olds RJ, Lane DA, Chowdhury V, Stefano V, Leone G, Thein SL. (1993) Complete nucleotide sequence of the antithrombin gene: Evidence for homologous recombination causing thrombophilia. Biochemistry 32: 4216–24 2. Lane DA, Bayston T, Ods RJ, Fitches AC, Cooper DN, Millar DS, Jochmans K, Perry DJ, Okajiama K, Thein SL, Emmerich J. (1997) Antithrombin mutation database: 2nd Update. Thromb Haemost 77: 197–211 3. Lane DA, Kunz G, Olds RJ, Thein SL. (1996) Molecular genetics of antithrombin deficiency. Blood Reviews 10:59–74 4. Armour JAL, Barton De, Cockburn Dj, Taylot GR (2002) The detection of large deletions or duplications in genomic DNA. Hum. Mutation 20:325–337

VIIe. Miscellaneous

Recurrent Coronary Stent Thrombosis in a Patient with Combined Aspirin and Clopidogrel Resistance Ch. Templin, A. Schaefer, B. Stumme, H. Drexler, and M. von Depka Prondzinski

Background A 72-year-old woman with an uneventful prior medical history was admitted to our hospital because of an acute ST-elevation myocardial infarction (STEMI). Primary angioplasty with stenting was performed due to a 90% diameter stenosis (Fig. 1) of the proximal right coronary artery (RCA). Prior to intervention she received a loading dose of 300 mg Clopidogrel and 500 mg aspirin (ASA). After intervention 75 mg Clopidogrel and 300 mg ASA once daily were continued. Three days later she developed a recurrent acute STEMI due to stent thrombosis. Two stents were inserted, one proximally and another one distally from the first placed stent in the RCA (Fig. 2). The dose of Clopidogrel and ASA remained unchanged. After 3 further days she suffered a third STEMI due to a restent thrombosis. Therefore another stent was placed within the first stent (Fig. 3).

Fig. 1. Primary angioplasty of the right coronary artery (RCA) due to acute inferior myocardial infarction before (left) and after (right) stent placement

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Fig. 2. Image on the left shows total thrombotic occlusion within the RCA stent three days apart from primary angioplasty. Two additional stents were implanted (right image)

Fig. 3. Illustration of second stent thrombosis 3 days after first stent thrombosis (right image). Coronary flow was restored by an additional stent placement (right)

Method Due to the second stent thrombosis we performed multiple platelet function and aggregation tests. Platelet function analysis (PFA-100™) allows assessing the platelet function in citrated whole blood. By quantifying the time taken for the development of a platelet plug to occlude a capillary coated with platelet agonists collagen and ADP ASA effects are controlled. The time required until full occlusion of the aperture is referred to as closure time (CT). For the platelet aggregation studies, platelet-rich plasma was prepared. Aggregation was induced with ADP detecting Clopidogrel effects and with adrenalin sensitive for ASA but not Clopidogrel.

Recurrent Coronary Stent Thrombosis in a Patient

Clopidogrel 75 mg ASA 300 mg

Clopidogrel 150 mg ASA 500 mg

237

Warfarin ASA 100 mg

120

120

100

100

80

Platelet aggregation 60 [%] 40

80

20

20

60

Closure time [s]

40

0

0 21-Jun

ADP-induced Aggregation

22-Jun

23-Jun

24-Jun

Adrenalin-induced Aggregation

PFAADP

Fig. 4. Course of platelet function (left y-axis) and aggregation (right y-axis) demonstrating combined resistance to dose-independent anti-platelet treatment with ASA and Clopidogrel

Results As shown in Figure 4 platelet function analyses and aggregation studies demonstrated dose independent ASA resistance as PFA-100 showed normal or even shortened CT (normal range: 71–118 s). Furthermore, adrenalin-induced aggregation was normal and even increased over the observation period. ADP-induced aggregation after 1 week of 75 mg Clopidogrel OD was normal (Fig. 4). Therefore, we increased the dose of Clopidogrel to 75 mg BD and ASA to 500 mg OD. Two and three days later respectively, platelet function and aggregation were re-assessed. ADP-induced aggregation showed a short-term platelet inhibition with subsequent rapid normalization, thus suggesting Clopidogrel resistance (Fig.4). As a consequence treatment was switched to Coumadin (target INR 2.5) and ASA 100 mg OD. Since then the patient is clinically stable.

Conclusion ASA resistance is a well known phenomenon [1]. Recent data show a high percentage of Clopidogrel resistance in patients with MI [2]. Our case shows, that there are patients with combined Clopidogrel and ASA resistance in whom even increasing doses of Clopidogrel and ASA were not efficacious. Identification of these patients is required and alternative therapeutic options such as established (e.g. warfarin and heparin) as well as newer anticoagulants (e.g. Ximelagatran) should be considered.

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References 1. Matetzky S, Shenkman B, Guetta V, et al. Clopidogrel resistance is associated with increased risk of recurrent atherothrombotic events in patients with acute myocardial infarction. Circulation. 2004; 109: 3171–3175 2. Gum P, Kottke-Marchant K, Welsh PA, et al. A prospective, blinded determination of the natural history of aspirin resistance among stable patients with cardiovascular disease. J Am Coll Cardiol. 2003;41: 961–965

Coagulation Factor XIII Mutation Profile: Update 2004 V. Ivaskevicius, R. Seitz, H.-H. Brackmann, W. Eberl, K. Kurnik, W. Kreuz, R. Klamroth, H. Rott, R. M. Loreth, F. Herrmann, E. Seifried, and J. Oldenburg

Introduction Congenital factor XIII (FXIII) deficiency (OMIM: +134570, +134580) is known as a rare autosomal recessive disorder, affecting one in 1–3 million individuals. Deleterious mutations occurring in F13A gene or rarely (< 3% of all cases) in F13B gene result in absence or reduction of plasma FXIII activity. Patients suffering from this condition are characterized by life-long bleeding diathesis and/or impaired wound healing, and spontaneous abortions in affected females as well. The F13A gene maps to the short arm of chromosome 6 (p24–25) and spans about 160 kb of genomic DNA. It consists of 15 exons encoding a mature protein of 731 amino acids. The F13B gene is located on the long arm of chromosome 1 (q32–32.1) and contains 12 exons encoding the mature protein of 641 amino acids [1, 2].

Fig. 1. The protein modeling of a novel missense mutation Gly215Arg demonstrated that Arg215 is located at the interface of two beta strands and the barrel 1 and barrel 2 domains. Substitution of the small Gly by the large Arg obviously affects the three dimensional configuration of the FXIII Asubunit I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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V. Ivaskevicius et al. Large Deletions 4% Small Insertions 6%

Normal 4%

Missense 42 %

Small Deletions 16 % Splice Site 28 %

Fig. 2. Mutation Profile in 25 (50 alleles) unrelated FXIIIA deficient patients. Twenty-three patients were homozygous or compound heterozygous for the corresponding mutations, while 2 individuals having residual FXIII activity < 60% were found to be heterozygotes, the second allele in these 2 patients was expected to be normal IVS5-1G > A 26 % Other Mutations 74 %

Fig. 3. Splice site mutation in intron 5 (IVS5-1 G>A) was found to be the most frequent molecular defect responsible for FXIIIA deficiency. This type of mutation (in homozygous or in heterozygous status) was detected in 7 out of 25 patients (13/50 alleles). The haplotype analysis of patients carrying IVS5-1 G>A mutation showed an evidence for a founder effect

Materials and Methods Genomic DNA was prepared from EDTA blood lymphocytes by standard procedures. Fifteen exons and their flanking regions of F13A gene as well as 12 exons of F13B gene were amplified by PCR and analyzed by denaturing high performance liquid chromatography (DHPLC). The abnormal peaks have been further sequenced using ABI 310 Sequencer.

Results and Discussion During the last 4 years we have identified mutations in 28 unrelated FXIII deficient patients. As a screening method denaturing high performance liquid chromatography (DHPLC) was used. In 25 patients FXIII deficiency were caused by molecular

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defects in F13A gene and in 3 patients by molecular defects in F13B gene. In total 27 unique mutations (of them 3 mutations in 13B gene) comprising 13 missense, 8 small deletions/insertions, 4 splice site and 2 large deletions have been identified. The mutations were distributed throughout the F13A gene, although most missense mutations affected the F13A gene part encoding the central core domain.

References 1. Ichinose A, Bottenus RE, Davie EW. Structure of transglutaminases. J Biol Chem 1990 Aug 15; 265 (23): 13411–4 2. Ichinose A. Physiopathology and regulation of factor XIII. Thromb Haemost 2001 Jul; 86 (1): 57–65 3. Takahashi N, Tsukamoto H, Umeyama H, Castaman G, Rodeghiero F, Ichinose A. Molecular mechanisms of type II factor XIII deficiency: novel Gly562-Arg mutation and C-terminal truncation of the A subunit cause factor XIII deficiency as characterized in a mammalian expression system. Blood 1998 Apr 15; 91 (8): 2830–8

Site-Directed Mutagenesis of VKORC1, the Target Protein of Coumarin-Type Anticoagulants S. Rost, A. Fregin, M. Hünerberg, C. R. Müller, and J. Oldenburg

Introduction In Germany, more than 600,000 patients are medicated each year with coumarin drugs for the treatment and prevention of thromboembolic events. Coumarins are antagonists of vitamin K, an essential cofactor for the posttranslational gammaglutamyl carboxylation of vitamin K dependent coagulation factors (FII, FVII, FIX, FX, Protein C, S and Z) [1]. As vitamin K hydroquinone is converted to vitamin K epoxide in every carboxylation step, vitamin K has to be recycled by the vitamin K epoxide reductase complex (VKOR). We have recently identified one component of this enzyme complex, the vitamin K epoxide reductase complex subunit 1 (VKORC1), which is the target protein for coumarin drugs like phenprocoumon (Marcumar) and warfarin (Coumadin) [2]. Mutations in VKORC1 result in two different phenotypes: warfarin resistance (WR) and multiple coagulation factor deficiency type 2 (VKCFD2).

Results and Discussion We have investigated the role of individual amino acids of the VKORC1 protein after site-directed mutagenesis and recombinant expression in HEK 293 cells. All seven cysteine residues at positions 16, 43, 51, 85, 96, 132 and 135 of VKORC1 which may be involved in the formation of disulfide bonds or the provision of reducing equivalents were substituted by serine residues. Furthermore, a highly conserved Ser/Thr residue at position 57 was substituted by alanine. All resulting recombinant protein variants showed a varying decrease of VKOR activity (Table 1). In particular, mutations of the supposed thioredoxin motif Cys132-X-X-Cys135 destroyed Table 1. VKOR activity of recombinant VKORC1 proteins after successive substitution of all seven cysteine residues by serine and of serine 57 by alanine. Replacement of cysteines 51, 132 and 135 results in an almost complete loss of VKOR activity Mutation

C16S

C43S

C51S

C85S

C96S

C132S

C135S

S57A

Activity [%]

18.40

20.34

6.07

24.57

30.80

6.12

5.35

9.97

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120

VKO R activity [%]

100 wildtype

80

Tyr139Cys Tyr139Gly

60

Tyr139Ser

40

Tyr139Phe

20 0

0

5

10

war

20

farin

40

[µM

]

Tyr139Phe Tyr139Ser Tyr139Gly Tyr139Cys wildtype

60 80

100

Fig. 1. VKOR activity after mutagenesis of tyrosine 139, the supposed warfarin binding site. Substitution of Tyr139 results in varying degrees of resistance to warfarin.VKORC1 containing a phenylalanine at position 139 shows highest VKOR activity and almost complete warfarin resistance

wildtype

140

Arg98Lys

VKOR activity [%]

120

Arg98Asn Arg98Gln

100

Arg98Glu 80

Arg98Asp

60

Arg98Trp

40 20 0 0

5

10

20 40 warfarin [µM]

60

80

100

Fig. 2. Comparison of VKORC1 proteins mutated at arginine 98. Substitution of arginine 98 by lysine stimulates VKOR activity to higher than wildtype levels. Replacement by structurally unrelated amino acids (asparagine, glutamine, aspartate or glutamate) leads to an incremental decrease of VKOR activity. Lowest VKOR activity is obtained by the substitution of arginine 98 by tryptophan which is the only known mutation in VKCFD2 patients so far

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VKOR activity completely (Table 1). Mutations at tyrosine 139 which were detected in resistant rats retained good VKOR activity and conferred insensitivity towards warfarin (Fig. 1). Tyrosine 139 is embedded in a hydrophobic sequence context TYA and may be part of the warfarin binding site. Mutation Arg98Trp which had been found in patients with the VKCFD2 phenotype abolished VKOR activity completely. However, when arginine 98 was substituted by structurally related amino acids the proteins retained 20 to 100 % of wildtype activity and warfarin sensitivity (Fig. 2).

Conclusion Natural and site-directed mutagenesis experiments are important tools for studying the role of individual amino acids of the VKORC1 protein and their involvement in the binding sites for the substrate vitamin K and the antagonist warfarin. Our study supports the hypothesis of different binding sites for vitamin K epoxide and Coumarins and underlines the crucial roles of the coumarin binding motif TYA and the thioredoxin motif CXXC. Understanding the structure and function of the VKORC1 protein is the basis for the development of new anticoagulants with an improved efficacy/side effect profile.

References 1. Furie et al. Vitamin K-Dependent Biosynthesis of g-Carboxyglutamic Acid. Blood 1999; 93: 1798–1808 2. Rost et al. Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2. Nature 2004; 427 (6974): 537–41

Treatment of Dilution Coagulopathy by Fibrinogen and Platelet Concentrates C. De Lorenzo, M. Spannagl, and B. Heindl

Severe bleeding, e.g. after trauma or during surgery often induces a coagulopathy. Causes are consumption, loss and dilution of both clotting factors and platelets. Dilution is mainly caused by crystalloid and colloid solutions as well as red blood cells applied to maintain normovolemia and oxygen transport capacity. Colloids are accused to interact with platelets and clotting factors and may therefore worsen the coagulopathy. The purpose of our in-vitro study was to characterize the effect of hemodilution on clot firmness using a step by step dilution technique. Additionally, we examined whether the application of fibrinogen and/or platelets offers a reasonable therapeutic option in such a situation. For this purpose samples of citrated whole blood were freshly drawn from healthy volunteers and immediately diluted with either hydroxyethyl starch (HES 6% 130/0.4) (Voluven, Fresenius, Bad Homburg) or with sodium chloride 0.9% (NaCl 0.9%). Both solutions were mixed with sodium citrate in a 1:10 proportion before use. Whole blood samples were diluted 20%, 40%, 60% and 80 % of the total volume, respectively. At a dilution of 60% fibrinogen (Haemocomplettan, ZLB Behring, Bern) or platelets or a combination of both were added. The chosen dose corresponded to an application of 6 g fibrinogen or one platelet concentrate of 150 ml based on a total blood volume of 5 l. The samples were measured for clot firmness using thrombelastography (ROTEM, Pentapharm, Munich). The INTEM test reflects the intrinsic activation cascade plus the interaction of platelets and fibrin, the FIBTEM test represents solely fibrin formation and polymerization by blocking platelet function. Using HES 6 % there was a clearly reduced maximum clot firmness (MCF), starting with a dilution of 40 % in the INTEM (MCF < 40 mm) and of 20% in the FIBTEM (MCF < 8 mm). Using NaCl 0,9 %, MCF only started to be impaired at a dilution of 60%. Dilution coagulopathy induced by HES 6% did not respond adequately to application of either fibrinogen or platelet concentrate. In contrast, clot firmness of samples diluted by NaCl 0.9 % returned to normal values after application of fibrinogen concentrate. This effect was not achieved, however, by using platelet concentrate.

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HES 6%

NaCl 0.9%

(mean ± SD)

MCF (mm) INTEM

MCF (mm) FIBTEM

MCF (mm) INTEM

MCF (mm) FIBTEM

whole blood dilution 60% +fibrinogen +platelets (PL) +fibrinogen + PL

56 ± 4 23 ± 5 28 ± 4 29 ± 3 34 ± 4

12 ± 2 1±2 1±2 0±0 3±3

58 ± 5 36 ± 5 47 ± 6 40 ± 4 51 ± 3

13 ± 4 6±2 20 ± 4 8±2 19 ± 3

In-vitro the onset of coagulopathy caused by dilution develops more rapidly using the colloid solution HES 6 % than using the crystalloid solution NaCl 0.9 %. The application of high-dose fibrinogen is an effective therapeutic option to correct clot firmness impaired by dilution with crystalloid solutions but not with colloid solutions, as their molecule structure obstructs the polymerization of fibrinogen. The combined application of both fibrinogen and platelet concentrate is not superior to the exclusive use of fibrinogen concentrate.

Pathogenesis of Hepatic Veno-Occlusive Disease in Patients Undergoing Hematopoietic Stem Cell Transplantation M. Dávid, O. Tóth, Á. Nagy, B. Meng, J. Tábori, and H. Losonczy

Introduction Hepatic veno-occlusive disease (VOD) is a common complication following hematopoietic stem cell transplantation (HSCT), it is reported to occur in 5–70% of patients undergoing HSCT. The different incidence of VOD between series may be related to the number of patients included, the different selection criteria, the difference in the definition of VOD and the variable incidence of risk factors [1–3]. Clinically VOD is characterized by painful hepatomegaly, jaundice and fluid retention which is usually ascites. The development of hepatic VOD is the consequence of the injury of hepatocytes surrounding the central veins in zone 3 of the liver acinus. Endothelial damage, subendothelial edema, activation of the blood coagulation system and fibrogenesis, and finally occlusion of the hepatic venules and hepatocyte necrosis develops. As many as half of the patients with VOD die, usually of multi-organ failure [4]. Risk factors for VOD include: virus hepatitis in the disease history, elevated liver function tests at the time of high-dose chemotherapy and HSCT, high dose chemoor radiotherapy (especially abdominal irradiation), unrelated or mismatched stem cell transplantation, administration of norethisteron, vancomycin, acyclovir or amphotericin B. Different prophylactic strategies have been explored to prevent the injury and the subsequent thrombosis of the hepatic venules. They include the administration of prostaglandin E1, pentoxyphyllin, ursodeoxycholic acid, unfractionated and low molecular weight heparins and defibrotide. The results of the clinical trials have been variable therefore the strategy for VOD prophylaxis remains controversial [5–12]. Research in VOD pathogenesis has concentrated in four different areas, like the determination of the degree of glutathione depletion, the importance of inflammatory mediators in the process, the activation of fibrogenesis and of blood coagulation system [13]. Our aims were a) to perform thrombophilia studies before stem cell transplantation, b) to examine endothelial function and fibrogenesis before and 10 days after hematopoietic stem cell transplantation and c) compare the data of patients with and without hepatic VOD. I. Scharrer/W. Schramm (Ed.) 35th Hemophilia Symposium Hamburg 2004 ” Springer Medizin Verlag Heidelberg 2006

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Materials and Methods Patients 64 patients undergoing high dose chemotherapy and autologous hematopoietic stem cell transplantation between January 2002 to November 2004 at the Ist Department of Internal Medicine, University of Pécs (Hungary) were included into the study. Patients’ characteristics are listed in Table 1. Table 1. Patients’ characteristics No. of patients: Mean age (range): Sex (male/female):

64 39.77 ± 14.68 years 35/29

(19–66 years)

Indications for HSCT: Multiple myeloma: Non-Hodgkin’s lymphoma: Hodgkin’s disease: Ewing sarcoma:

14 patients 31 patients 18 patients 1 patient

Median time from diagnosis to HSCT (range): 18 months (6–24) Conditioning regimens:

Prior liver disease:

BEAM CY-TBI MEL 200 BU-CY BU-MEL

39 patients 4 patients 14 patients 6 patients 1 patient

CMV+:

1 patient

Abbreviations: BU: busulfan, CY: cyclophosphamide, MEL: melphalan, TBI: total body irradiation, BEAM: BCNU, etoposide, cytosar, melphalan

Conditioning Regimens The majority of patients received BEAM conditioning regimen (BCNU: 300 mg/m2, etoposide 400 mg/m2 x 4, ARA–C 400 mg/m2 x 4, melphalan 140 mg/m2) (n: 39), in patients with multiple myeloma melphalan 200 mg/m2 was administered (n: 14), the other regimens included: busulfan 4 mg/kg x 4 and cyclophophamide 60 mg/kg x 2 (n: 6), cyclophosphamide 60 mg/kg x 2 and fractionated TBI 200 cGy x 6 (n: 4), and busulfan 4 mg/ kg x 4 and melphalan 140 mg/m2 (n: 1).

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Prophylaxis of Hepatic VOD 100 IU/kg bw/24 hours infusion Na-Heparine was administered in all cases from day -8 to day +30 for the prevention of VOD according to Attal et al. [9]. Clinical Criteria for VOD (Seattle Criteria) The diagnosis of VOD was established according to previously described clinical criteria: occurrence of two or more of the following events before day 21 after stem cell support: 1. hyperbilirubinemia (> 34.2 µmol/l) 2. ascites or sudden weight gain (> 5% of baseline body weight) 3. painful hepatomegaly. No other explanation for these signs and symptoms, like hepatitis, septicemia, heart failure etc. could be present at the time of diagnosis. Clinical Course and Outcome of VOD (according to Bearman et al.) [14] Mild hepatic dysfunction: serum Bilirubin: 80 ± 50 µmol/l, weight gain 2.5–5%, GOT elevation: 2–5 x the normal Moderately severe hepatic dysfunction: serum Bilirubin: 130 ± 113 µmol/l, development of ascites or weight gain > 5%, GOT elevation: > 5 x the normal Severe hepatic dysfunction: serum Bilirubin: 440 ± 260 µmol/l, hepatic encephalopathy, ascites causing respiratory insufficiency Methods The methods that we have applied were able to cover most of the known etiological factors of hepatic VOD, like blood coagulation, endothelial dysfunction, inflammatory mediators and fibrogenesis. Before high dose Chemotherapy Blood coagulation laboratory tests for hereditary and acquired thrombophilia Fibrinogen (Prothrombin-Fibrinogen Recombinant, IL), APC resistance, nAPC-SR (APC Resistance V, IL), antithrombin activity (Antithrombin kit, IL), antithrombin antigen (Laurell rocket electrophoresis), protein C activity (ProClot kit, IL), protein C antigen (Protein C ELISA, Helena), protein S activity (Protein S kit, IL), protein S free antigen (COALIZA Protein S free, Chromogenix), Lupus anticoagulant (APTTLA, Stago), plasma homocystein level (IMx immunoassay, in collaboration with the Institute for Clinical Chemistry).

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Molecular biological studies for hereditary thrombophilia DNA isolation with Chelex 100 (according to Walsh et al.) [15], FII 20210 A allele (according to Poort et al.) [16], FV:Q506 (Leiden mutation) (according to Zöller et al.) [17] and MTHFR C677T polymorphism (according to Arruda et al.) [18]. TNF-a gene d allele polymorphism A microsatellite method (Genescan) according to Udalova et al. [19] performed in collaboration with the Institute for Medical Genetics and Child Development. Breefly polymorphism of the TNFd microsatellite was detected by electrophoresis of fluorescently labeled PCR product through an ABI PRISM 310 genetic analyzer (PE Biosystems, Cheshire, UK). PCR for the TNFd microsatellite loci was performed in a thermocycler with a 13 µl reaction mixture containing DNA (50–200 ng), 1.22 mM MgCl2, 9.8 mM NH4SO4, 171.4 mM Tris, 150 µM of dNTPs, 1.15 µM of each primer (Udalova et al.) and 1 U Taq polymerase. Cycling conditions were the following: 3 min at 94 ºC and 30 cycles of 30 s at 94 ºC (denaturation), 60 s at 64 ºC (annealing) and 60 s at 72 ºC (elongation). Prior to loading onto the ABI PRISM 310, PCR products were diluted 1:100 with distilled water. 1 µl of each diluted PCR product was mixed with 12 µl of deionized formamide and 0.5 µl of Genescan 500 size standard (PE Biosystems). Each sample was denatured in a thermocycler for 3 minutes at 95 ºC and than snap-cooled using an ice-water bath.

Before and 10 days after high dose chemotherapy Endothelial dysfunction Plasminogen activator inhibitor-1 (PAI-1) antigen (COALIZA PAI-1, Chromogenix) and von-Willebrand Factor antigen (VWF Antigen ELISA, Helena). Fibrogenesis N-terminal propeptide of type III procollagen (PIIINP) (I125 Intact PIIINP RIA Kit, Orion Diagnostica) according to Rio et al. [20] in collaboration with the Institute for Human Anatomy. With the kit it is possible to measure the PIIINP concentration in human serum with a competitive RIA technique. A mixture is prepared from the radioactive antigen and antibodies against that antigen. Than known amounts of unlabeled antigen are added to samples of the mixture. These compete for the binding sites of the antibodies. At increasing concentrations of unlabeled antigen, an increasing amount of radioactive antigen is displaced from the antibody molecules. The antibody-bound antigen is separated from the free antigen in the supernatant fluid and the radioactivity of each is measured. From these data a standard binding curve can be drawn. The samples are run in parallel. After determining the ratio of bound to free antigen, the antigen concentrations can be read directly from the standard curve.

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Statistics Data were given as mean ± standard deviation (SD). Differences in the means of continuous measurements were tested with student’s t-test. All tests were two-sided. Patients have given written informed consent for the blood collections.

Results 4 moderately severe cases (6%) of hepatic VOD developed, in all cases Bu-Cy protocol was administered as conditioning regimen, they were successfully treated with the combination of defibrotide, high-dose N-acetylcystein, high-dose thiogamma and diuretics. In 6 patients (9%) venous thromboembolic complications could be detected during the transplant procedure: in 1 patient subtotal pulmonary embolism developed from a proximal femoral vein thrombosis, both were successfully treated with local thrombolysis (streptokinase), in 5 patients venous thrombosis of the central line developed, in 3 cases the internal jugular vein thrombosis was treated with low molecular weight heparin, in 2 cases local thrombolysis with urokinase has been performed successfully.

Thrombophilia studies In 19 patients (30%) there was no abnormality during the thrombophilia studies. In 23 patients (36%) single and 22 patients (34%) combined defects could be detected. So altogether 70% of the cases we could prove abnormality. In 8 patients (12 %) decreased protein C activity with normal antigen levels, in 6 cases (9%) decreased protein S activity with normal antigen levels, in 3 patients (4%) antithrombinopathy were detected. APC resistance and FV:Q506 Leiden mutation could be found in 6 patients (9%), the presence of FII 20210A allele in 2 patients (3%). In 13 patients (20%) elevated fibrinogen levels, in 7 patients (11%) the presence of lupus anticoagulant could be detected. In 30/64 patients – which means 46.8% of the total patient population – elevated plasma homocystein levels were found (Mean: 28.16 µmol/l, range: 14.9–64 µmol/l). According to the literature the cutoff value for elevated plasma homocystein level was 12 µmol/l. Genetic alterations were proved in only 11/30 (36%) of the hyperhomocysteinemia cases, in 7 patients heterozygosity, in 4 cases homozygosity for MTHFR C677T mutation could be detected. So in most of the hyperhomocysteinemia cases there was no genetic abnormality in the background. Because of this unexpected result, we have decided to measure the patients’ folic acid level, but it was normal in 28/30 patients (data are not shown here). In most of patients where VOD developed, combined defects could be detected (Table 2).

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Table 2. Results of thrombophilia studies in patients with hepatic VOD (only the abnormalities are shown) Patient 1: PC activity: 48% PC antigen: 89%

MTHFR C677T: HO HC: 19.1 µmol/l

Patient 3: fibrinogen: 12.5 g/l

HC: 21.2 µmol/l

Patient 2: PS activity: 38% PS free antigen: 96%

HC: 17.1 µmol/l

Patient 4: PC activity: 56% PC antigen: 106%

HC: 14.2 µmol/l

Abbreviations: PC: protein C, PS: protein S, HC: plasma homocystein level

Endothelial dysfunction Von-Willebrand factor antigen (VWF Ag) levels were elevated already before the transplantation (128.6 ± 37%) and it increased significantly after the stem cell support (177.4 ± 32.5%) (p < 0.0002) in patients where there was no detectable hepatic VOD (Fig. 1). In patients with VOD the same tendency could be found (Fig. 2). Plasminogen activator inhibitor-1 antigen (PAI-1 Ag) levels did not change significantly before and after HSCT in patients without hepatic VOD (67.9 ± 37.6 vs. 58.5 ± 34.6 ng/ml) (Fig. 3), but it increased markedly in patients where VOD existed (Fig. 4). vWF Ag (%) 180 160

p<0.0002 177.4 ± 32.5

140 120 100 80

128.6 ± 37

Before SCT After SCT Normal control

103 ± 25

60 40 20 0 Before SC T

After SCT

Fig. 1. VWF Ag results in patients without VOD

Normal control

vWF Ag (%) 210 190 210

170

195

150

204

180

130

Before SCT After SCT

174

110

154 103

90

1. pt

2. pt

3. pt

106

4. pt

Fig. 2. VWF Ag results in patients with VOD

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253

p= 0.98

60 50 40

67.9 ±37.6

58.5 ± 34.6

54 ± 28.5 Before SCT After SCT Normal range

30 20 10

Fig. 3. PAI-1 Ag results in patients without VOD

0 Before SCT

After SCT

Normal range

PAI-1 Ag (ng/ml)

Fig. 4. PAI-1 Ag results in patients with VOD

110 100 90 80 70 60 50 40 30 20 10 0

86

1. pt

104

108

92 90

2. pt

76

3. pt

102 68

Before BMT After BMT

4. pt

Results of the Examinations for Activated Fibrogenesis There was no significant difference between the N-terminal propeptide of type III procollagen (PIIINP) results before and after HSCT in patients without hepatic VOD (6.8 ± 3.7 µg/l vs. 7.4 ± 4.3 µg/l, p = 0.24) (Fig. 5), but a 5-fold increase could be detected in patients where hepatic VOD developed (Fig. 6). Results of the TNFd Microsatellite allele Polymorphism Studies The analysis was performed in the samples of 42 patients (in 2 cases the evaluation of the results were not possible because of technical difficulties), so the results of 40 patients are available (Table 3). Table 3. The results of microsatellite analysis of TNFd allele polymorphism HO for d3 allele: HZ for d3 allele: NO d3 allele:

8 patients 24 patients 8 patients

Abbreviations: HO: homozygous, HZ: heterozygous, TNF: tumor-necrosis factor

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PIIINP µg/l 20

15

p = 0.24 Before SCT After SCT

10

5

6.8 ± 3.7

7.4 ± 4.3

Before SCT

After SCT

Fig. 5. Results of PIIINP in patients without VOD

0

PIIINP (µg/l) 30 25 20

21

29

15

32

26

Before BMT After BMT

10 5

6.1

5.3

4.9

7.5

0 1. pt

2. pt

3. pt

4. pt

Fig. 6. Results of PIIINP in patients with hepatic VOD

The allele frequency in the general population is 47%, as it is known from the literature, the allele frequency among our HSCT patients was 54%, the difference is not statistically significant (p= 0.54). All the patients with hepatic VOD were heterozygous for the presence of TNFd3 allele.

Discussion Hepatic VOD is one of the most common life-threatening complications of high dose chemotherapy and HSCT. Recent data indicate that endothelial damage and repair processes are ongoing during the transplant procedure, as it is proved by elevated levels of vWF Ag, PIIINP etc. Recently research in VOD pathogenesis has concentrated in four different areas, like glutathione depletion, inflammatory mediators, fibrogenesis and blood coagulation, but these areas frequently overlap. The strategies for the prevention and treatment of the disease have been based on the observations coming from pathogenetical studies. There is strong evidence that glutation depletion results in hepatocyte necrosis. The major cause of glutathione depletion is the high-dose chemo- and radiotherapy, especially the administration of cyclophosphamide and busulphan. N-acetylcystein (NAC), a thiol antioxidant is the precursor of intracellular reduced glutathio-

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ne synthesis. The administration of NAC together with tissue plasminogen activator caused a complete resolution of hepatic VOD in three patients. Glutamine is utilized by hepatocytes to replenish intracellular glutathione. The administration of Lglutamine was effective in two patients with hepatic VOD [21, 22]. Alterations in the plasma level of some inflammatory mediators have been reported. These are included elevations in TNF-alpha levels after HSCT, increased expression of genes for IL-1-beta and TNF-alpha as well as increased levels of circulating IL-2 receptor [23–25]. In the literature Haire et al. followed 42 patients for the development of hepatic VOD, CNS and pulmonary dysfunction. The TNFd genotype was determined by PCR method. Organ toxicity occurred in 57% of patients with at least one TNFd3 allele and 17% of patients with no TNFd3 allele [26]. Fibrosis is a very classical histologic feature of VOD. Hepatic stellate cells are the most important mediators of liver fibrosis. Activation of these cells results in the expression of smooth muscle cell specific alpha actine and type III and IV collagen. In patients with hepatic VOD significantly more stellate cells are activated resulting the production of N-terminal propeptide of type III procollagen (PIIINP), which has been reported to be elevated in patients with hepatic VOD [20, 27, 28]. The deposition of fibrinogen and factor VIII in the vessel walls of patients with hepatic VOD was the first observation that proved the role of blood coagulation in the pathogenesis. Hypercoagulability, included the deficiencies of antithrombin, protein C and S and elevation of D-dimer, prothrombin fragment F1+2 could be detected. Elevated plasminogen activator inhibitor-1 (PAI-1) and von-Willebrand factor (VWF) antigen levels were also proved as characteristic markers of hepatic VOD [29–31]. From hereditary thrombophilias FV Leiden mutation and the presence of FII 20210A allele were reported to increase the incidence of the development of VOD [32, 33]. In 23 patients (36%) single and 22 patients (34%) combined defects could be detected in our thrombophilia studies. So altogether in 70% of the cases we could prove abnormality. The most surprising result was that in 30/64 patients – which means 46.8% of the total patient population – elevated plasma homocystein levels were found (Mean: 28.16 µmol/l, range: 14.9–64 µmol/l) with genetic alterations in only 11/30 (36%) of the hyperhomocysteinemia cases. So the hyperhomocysteinemia could be attributed to acquired causes. There was no folic acid deficiency in the background. In 3/4 of the patients where VOD developed, combined defects could be detected. The von-Willebrand factor antigen (VWF Ag) levels were elevated already before the transplantation (128.6 ± 37%) and increased significantly after the stem cell support (177.4 ± 32.5%) (p < 0.0002) in patients where there was no detectable hepatic VOD. In patients where VOD developed the same tendency could be found. Plasminogen activator inhibitor-1 antigen (PAI-1 Ag) levels did not change significantly before and after HSCT in patients without hepatic VOD (67.9 ± 37.6 vs. 58.5 ± 34.6 ng/ml), but it increased markedly in patients where VOD existed. There was no significant difference between the N-terminal propeptide of type III procollagen (PIIINP) results before and after HSCT in patients without hepatic VOD (6.8 ± 3.7 µg/l vs. 7.4 ± 4.3 µg/l, p = 0.24), but a 5-fold increase could be detected in patients where hepatic VOD developed.

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The results of the TNFd microsatellite allele polymorphism studies are available in 40 patients. The allele frequency in the general population is 47%, as it is known from the literature, the allele frequency among our HSCT patients was 54%, the difference is not statistically significant (p = 0.54). All the patients with hepatic VOD were heterozygous for the presence of TNFd3 allele. In our survey it was possible to combine the examinations for hereditary and acquired thrombophilia, the studies for endothelial dysfunction, the examinations of the increased fibrogenesis and of inflammatory mediators as the most important etiological factors in the development of hepatic VOD. 4 moderately severe cases (6%) of hepatic VOD developed in our series, all patients were successfully treated with the combination of defibrotide, high-dose Nacetylcystein, high-dose thiogamma and diuretics. This is in accordance with the incidence reported in other large prospective surveys, like the EBMT study performed in 1,652 patients (VOD incidence was 5.3%). In 6 patients (9%) venous thromboembolic complications could be detected during the transplant procedure, which were treated successfully with low molecular weight heparin or thrombolysis depending on the severity of the venous thromboembolism. In conclusion, our survey shows that the incidence of hepatic VOD is not as high as it was previously expected, the type of high dose therapy has an impact on the development of hepatic VOD, it is more common in patients with combined thrombophilia, especially in patients with hyperhomocysteinemia. The examination of endothelial dysfunction (von-Willebrand factor antigen, PAI-1 antigen) and increased fibrogenesis (N-terminal propeptide of type III procollagen) can be essential to predict the development of hepatic VOD following HSCT.

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9. Attal, M., Huguet, F., Rubie, H. et al.: Prevention of hepatic veno-occlusive disease after bone marrow transplantation by continuous infusion of low-dose heparin: A prospective randomized trial. Blood 1992. 79 (11): 2834–2840. 10. Simon, M., Hahn, T., Ford, L.A. et al: Retrospective multivariate analysis of hepatic venoocclusive disease after blood or marrow transplantation: possible benefit from the use of low molecular weight heparin. Bone Marrow Transplant 2001. 27 (6): 627–633. 11. Forrest, D.L., Thompson, K., Dorcas, V.G. et al.: Low molecular weight heparin for the prevention of hepatic veno-occlusive disease (VOD) after hematopoietic stem cell transplantation: a prospective phase II study. Bone Marrow Transplant 2003. 31: 1143–1149. 12. Chalandon, Y., Roosnek, E., Mermillod, B. et al.: Prevention of veno-occlusive disease with defibrotide after allogeneic stem cell transplantation. Biol Blood Marrow Transplant 2004. 10 (5): 347–354. 13. Bearman, S.I.: Avoiding hepatic veno-occlusive disease: what do we know and where are we going? Bone Marrow Transplant 2001. 27: 1113–1120. 14. Bearman, S.I.: The syndrome of veno-occlusive disease after marrow transplantation Blood 1995. 85 (11): 3005–3020. 15. Walsh, P.S., Metzger, D.A., Higuchi, R.: Chelex 100 as a medium for simple extraction of DNA for PCR based typing from forensic material. Biotechniques 1991. 10: 506–513. 16. Poort, S.R., Rosendaal, F.R., Reitsma, P.H. et al.: A common genetic variation in the 3’untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996. 88: 3698–3703. 17. Zöller, B., Dahlbäck, B.: Linkage between inherited resistance to activated protein C and factor V gene mutation in venous thrombosis. Lancet 1994. 343: 1362–1363. 18. Arruda, V.R., von Zueben, P.M., Chiaparini, L.C. et al.: The mutation Ala677-Val in the methylene tetrahydrofolate reductase gene: a risk factor for arterial and venous thrombosis. Thrombosis and Haemostas 1997. 77: 818–821. 19. Udalova, I., Nedospasov, S.A., Webb, G.C. et al.: Highly informative typing of the human TNF locus using six adjacent polymorphic markers. Genomics 1993. 16: 180–186. 20. Rio, B., Bauduer, F., Arrago, J.P. et al.: N-terminal peptide of type III procollagen: A marker for the development of hepatic veno-occlusive disease after BMT and basis for determining the timing of prophylactic heparin. Bone Marrow Transplant 1993. 11: 471–472. 21. DeLeve, L.D.: Cellular target for cyclophosphamide toxicity in murine liver: role of glutathione and site of metabolic activation. Hepatol 1996. 24: 830–837. 22. Ringden, O., Remberger, M., Lehnmann, S.: N-acetylcysteine for hepatic veno-occlusive disease after allogeneic stem cell transplantation. Bone Marrow Transplant 2000. 25: 993–996. 23. Holler, E., Kolb, H-J., Moller, A.: Increased serum levels of tumor necrosis factor alpha precede major complications of bone marrow transplantation. Blood 1990. 75: 1011–1016. 24. Tanaka, J., Imamura, M., Kasai, M.: Cytokine gene expression in peripheral blood mononuclear cells during graft-versus-host disease after allogeneic bone marrow transplantation. Br J Haematol 1993. 85: 558–565. 25. Khoury, H., Adkins, D., Brown, R., Trinkaus, K., Vij, R., Miller, G., Goodnoug, L.T., DiPersio, J.: Does early treatment with high-dose methylprednisolone alter the course of hepatic regimen-related toxicity? Bone Marrow Transplant 2000. 25: 737–743. 26. Haire, W.D., Cavet, J., Pavletic, S.Z.: Tumor necrosis factor d3 allele predicts for organ dysfunction after allogeneic blood stem cell transplantation. Blood 2000. 96 (Suppl.1.): 584a. 27. Eltumi, M., Trivedi, M., Hobbs, J.R. et al.: Monitoring of veno-occlusive disease after bone marrow transplantation by serum amino-propeptide of type III procollagen. Lancet 1993. 342: 518–521. 28. Heikinheimo, M., Halili, R., Fasth, A.: Serum procollagen type III is an early and sensitive marker for veno-occlusive disease of the liver in children undergoing bone marrow transplantation. Blood 1994. 83: 3036–3040. 29. Shulman, H.M., Gown, A.M., Nugent, D.J.: Hepatic veno-occlusive disease after bone marrow transplantation. Immunohistochemical identification of the material within occluded central venules. Am J Pathol 1987. 127: 549–558.

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30. Faioni, E.M., Krachmalnikoff, A., Bearman, S.I. et al.: Naturally occurring anticoagulants and bone marrow transplantation: Plasma protein C predicts development of venocclusive disease of the liver. Blood 1993. 81: 3458–3462. 31. Gordon, B.G., Haire, W., Kessinger, A.: High frequency of antithrombin 3 and protein C deficiency following autologous bone marrow transplantation for lymphoma. Bone Marrow Transplant 1991. 8: 497–502. 32. Duggan, C., Schmidt, M., Lawler, M., et al.: The prothrombin gene variant G20210A, but not factor V Leiden may be associated with veno-occlusive disease following BMT. Bone Marrow Transplant 1999. 24: 693–694. 33. Ertem, M., Akar, N.: Factor V Leiden mutation as a predisposing factor for veno-occlusive disease following BMT. Bone Marrow Transplant 2000. 26:1110–1111.

Subject Index

Abciximab 109–113, 115–116 accident 6, 9–11 acetylic salicylic acid [ASA] 235–237 Acquired Immune Deficiency Syndrome [AIDS] 5–6, 8, 173–174 ADAMTS-13 123, 165 – activity 124 – deficiency 124 – gene(s) 123–125 – inhibitor 124 – mutation(s) 124, 127 Adenosinediphosphate [ADP] 111–117, 236–237 ADVATE 38–40, 68, 153–156 AIDS [Æ Acquired Immune Deficiency Syndrome) anticardiolipin antibodies 102, 104–105 anticoagulants 130–131, 226, 242, 244 antiphospholipid antibodies 131 antithrombin [AT] 89 – activity 230, 249 – antigen 249 – deficiency 210, 230, 232, 255 – gene 230–232 APC [activatedÆ protein C] aPTT [activated Æ partial thromboplastin time] 153–155 arachidonic acid 112, 116 arthrodesis 205 arthropathy 49, 51, 57, 92, 138, 183–184 ASA [Æ acetylic salicylic acid] AT [Æ antithrombin] BeneFIX 16 bleeding(s) XXXI, 6, 8–12, 17–18, 55, 58, 67, 70, 87–88, 90–92, 95–98, 109–110, 115, 117–118, 127–132, 142, 159, 165, 179–181, 183, 192, 194, 199, 202, 205, 219, 239, 245 – complication(s) 51, 58, 109, 136, 205 – disorder(s) 8, 29, 42, 49, 165, 167, 183–184, 215

– episode(s) 42, 45, 89–92, 142–143, 159, 197, 219 – tendency 42, 128, 136, 142–143, 161, 179–182, 223 – Æ joint bleeding blood – coagulation 66, 136, 154, 230, 249, 255 – product(s) 11, 25, 29, 45 – transfusion 21–22, 24, 26, 29, 99, 201 Bovine Spongiform Encephalopathy [BSE] 23, 29–30, 32 BSE [Æ Bovine Spongiform Encephalopathy] cancer 6–11, 17, 123, 168, 187 CD – CD4+ cell(s) 6, 168, 170–171, 174 – CD5- cell(s) 166 – CD23- cell(s) 166 – CD19+ cell(s) 69, 76–78, 166 – CD20+ cell(s) 70, 78–79, 166 – CD138+ cell(s) 69, 76–78 cell(s) – red cell(s) 123, 131 – stem cell(s) 252 – – transplantation 247–248, 252–254, 256 Ciprofloxacin 130, 132 cirrhosis [Æ liver cirrhosis] CJD [Æ Creutzfeldt-Jacob disease] Clopidogrel 235–237 clotting – disorders XXXI – factor(s) 21–22, 29, 42, 49, 65, 102, 105, 136, 150, 157, 245 – time 127–127 coagulation 98, 166, 197 – activation 65 – cascade 221 – disorder(s) 98, 142 – factor(s) 30, 61, 242

260

Subject Index

– – deficiency 242 – system 61, 65, 102, 247 coagulopathy 14–15, 245–246 collagen 111–117, 236 Coumadin 237, 242 Coumarin 211, 226–229, 242, 244 Creutzfeldt-Jacob disease [CJD] 8 – variant Creutzfeldt Jacob disease [vCJD] 21, 23–26, 29–32 – new variant Creutzfeldt Jacob disease [nvCJD] 29 cryoprecipitate(s) 157–158, 162–163 cyclophosphamide 131, 248, 254 D-Dimer 65, 255 DNA 68, 82–83, 121, 215, 223, 230, 240, 250 – cDNA XXXII, 215 – ssDNA 67–68 domain – A1 66, 216–218, – A2 66, 216–218 – A3 66, 216 – B 66, 72, 216, 218 – C1 216 – C2 66, 77, 216 drug(s) 6, 9–11 DVT [deep vein Æ thrombosis] EACH-Registry [European registry for acquired hemophilia Æ hemophilia registry] ELISA 21, 67–69, 71, 74, 102, 249 Endogenous Thrombin Potential [ETP] 61–65 Epinephrine 111–117 epistaxis 159, 222, 224 epitope(s) 66, 68, 77–79 – mapping 35, 66, 68, 71–72 Eptifibatide 109–116 ESCHQoL-study 178 ETP [Æ Endogenous Thrombin Potential] Factor – concentrate(s) 17, 95–97, 163, 199, 201–202, 219 FII 61, 87, 92, 117, 227, 242 – gene 89 – mutation(s) 87, 90–91, 142–143, 251 FV 87, 92, 117 – gene(s) 24, 89 – Leiden 61, 96, 209–211, 219, 251, 255 – mutation 87–88, 90–91, 142–143

FVII 227, 242 – deficiency 14, 110 – – recombinant FVIIa 44–45, 109–118, 131–132, 158, 191, 194, 196–197, 205 FVIII 14, 25–26, 30–31, 33, 38, 40, 42, 50, 57, 61–65, 66–71, 73, 75, 77–79, 81, 87, 89–90, 92, 95–102, 104, 110, 136, 143, 144, 150–151, 154–155, 158, 163, 167, 179–181, 184, 187, 190–191, 193–194, 197–201, 205, 255 – activity 42, 81, 88, 136, 142, 150–151, 153–156, 179–181, 187–188, 192 – antibody/ies 35, 44 – antigen 81 – concentrate(s) 35, 49, 90–92, 96, 100–101, 153, 156, 179, 181 – gene(s) 33, 66, 81–82, 87, 91, 136, 179, 215–218 – inhibitor(s) 66, 68, 71, 73, 75–78, 96, 102, 187–188, 190, 192, 194, 216 – molecule(s) 66, 69, 71–72 – plasma-derived FVIII [pd FVIII] 16, 35, 50, 66, 96, 190–192, 203 – recombinant FVIII [rFVIII] 14, 16, 29, 34, 35, 50, 66, 96, 153, 191–192, 203, 216 – FVIIIa 66 FIX 14, 25, 30–31, 61–63, 66, 71–72, 87, 102, 104, 110, 136, 158, 194, 197, 201, 205, 227, 242 – activity 66 – concentrate(s) 35 – gene 34 – plasma-derived FIX [pd FIX] 16 – recombinant FIX [rFIX] 16 – FIXa 179 FX 66, 117, 179, 227, 242 FXa 179 FXI 31–32, 61, 102, 104, 120 FXII 102, 104, 112 – activity 105 – FXIIa 112 FXIII 130–132, 240 – activity 130–132, 239, 240 – concentrate 131 – deficiency 14, 131–132, 239, 240 – inhibitor 130–132 – mutation 239 Feiba 44–45, 65, 191, 205 FFP [Æ fresh frozen plasma] fibrin 102, 116–118, 128, 153, 179, 245 fibrinogen 65, 102, 112, 116–117, 126–129, 136, 221–224, 245–246, 249, 251–252, 255

Subject Index

– afibrinogenemia 14, 224 – dysfibrinogenemia 126–129, 221, 224 – hypofibrinogenemia 221–224 fibrinolysis 117 Fibrogammin 131–132 fresh frozen plasma [FFP] 131, 157–158, 162–163 Glycoprotein 66, 230 – Ia 166 – Ib 166 – IIa 166 – IIb 109–110, 112–113, 115–118, 166 – IIIa 109–110, 112–113, 115–118, 166 – IX 166 GTH [= Gesellschaft für Thrombose- und Hämostaseforschung Æ Society for Thrombosis and Hemostasis Research] Haemate HS 26, 150–151, 191 Haemocomplettan 245 HBV [Æ hepatitis B virus] HCV [Æ hepatitis C virus] Hemofil 191 hemophilia XXXI, 3, 8–9, 11, 13–14, 30–31, 34, 38–39, 44–45, 51–52, 57, 61, 65, 87–88, 92, 95–96, 99, 100, 135, 137–138, 140, 144, 157, 159–161, 163, 168, 171, 173–174, 194, 197–198, 200–202, 204–205, 219–220 – A 3–6, 14–17, 33–37, 40, 49, 57, 61, 63–64, 66–67, 76, 78, 81, 84, 87–92, 95–96, 136–138, 142, 153, 157–158, 162–163, 170, 179, 181–182, 190, 194, 197, 199, 205, 215–219 – B 3–6, 14–17, 35–36, 61, 63, 87, 136, 138, 157–158, 162, 168, 199, 219 – center(s) / (treatment centers) 3–4, 29–31, 34, 44, 49, 52, 61, 87, 138, 142, 157, 163, 198, 200 – registry (register) 13, 204 – – European registry for acquired hemophilia [EACH-Registry] 42–43 – treatment 157, 163 – acquired hemophilia 42–43, 110, 187 – acquired hemophilia A 132 – congenital (inherited) hemophilia 42, 110 hemorrhage(s) 50, 89, 126–127, 138, 165–166 hemostasis 38, 49, 98, 100–101, 110, 116, 129, 136, 197

261

heparin 65, 102, 109–110, 131, 237 – low molecular weight heparin 50, 111–113, 115–116, 131, 150, 209, 211, 247 – unfractionated heparin 209, 247 hepatitis 151, 247, 249 – B virus [HBV] 21 – C 49, 57, 202 – – virus [HCV] 9, 11, 21, 57, 168–174, 178 hirudin 102, 109–110, 113, 116 HIV [Æ human immunodeficiency virus] homocysteine 251–252, 255 – hyperhomocysteinemia 210, 251, 255–256 human immunodeficiency virus [HIV] 5–11, 17–18, 21, 25, 49, 57, 68, 169–178, 181 – infection 3, 168 IFN [Æ Interferon] IgG 25, 66–69 IgM 21 immune– tolerance 66, 79, 138, 190 – – therapy [ITT] 70, 79, 190–191, 193, 199, 292 immuno– globulin(s) 31, 76, 78, 166–167 – suppression 131 – intravenous immunoglobulins [IVIG] 131 infarction – myocardial 109–110, 127, 136, 235 inhibitor(s) 4–5, 16–17, 33–36, 39–40, 44–45, 57, 61, 67–71, 73–79, 81, 96, 100, 102–105, 118, 120, 122, 130, 132, 138, 158, 160, 187–189, 191, 197–202, 205, 216, 218, 219, 230 – elimination 42, 66, 79, 189, 190, 193 – development 34, 36–40, 50, 91, 95, 99, 100, 190, 197–202 Interferon 168 ITP [idiopathic thrombocytopenic Æ purpura] ITT [Æ immune tolerance therapy] IVIG [intravenous Æ immunoglobulin] joint 44, 52, 54, 57, 89, 91–92, 138–139, 140, 144–146, 183 – bleeding 49, 91, 159, 169, 191, 194 – destruction 205 – replacement 49, 51, 205

262

Subject Index

Kallikrein 120 Kogenate 191

NovoSeven 16, 65, 110, 112, 116–117, 151, 191, 205

leucocyte(s) 24, 118, 223 lipoprotein (a) 96 liver – cirrhosis 9, 11, 17, 126, 168, 172 – disease 5–11, 17–18, 168–169, 173, 248 LMWH [low molecular weight Æ heparin] Lp(a) [Æ lipoprotein (a)] lupus anticoagulant(s) 97, 98, 101–105, 237, 249, 251 lymphocyte(s) 77, 81–82, 215, 240

Octa SD plus 191

magnetic resonance imaging [MRI] 58, 89, 166 Mann-Whitney U test 89, 95 Marcumar 242 methylentetrahydrofolate reductase [MTHFR] 96, 219, 250–252 Monoclate 67 MRI [Æ magnetic resonance imaging] MTHFR [Æ methylentetrahydrofolate reductase] murder 6, 9–11 mutation(s) 13, 33, 66, 81, 89, 121–125, 136, 202, 210, 215–222, 229, 240–241, 244 – deletion(s) – – big deletion 203, 218 – – large 33, 90, 121–122, 215, 230–231, 232, 240–241 – – small 33, 121–122, 179, 199, 203, 216, 218, 230, 240–241 – frameshift 90, 216 – intron-1-inversion 33, 81, 96, 215, 217 – intron-22-inversion 33, 81, 90, 96, 179, 191, 193, 199–200, 203, 215–217 – insertions 121–122, 216, 218, 230 – – small 33, 121–122, 240–241 – inversion-22 90 – missense mutation 33, 90, 96, 121–122, 136, 199, 203, 215, 216, 221, 223–224, 228, 230, 239–241 – nonsense mutation 33, 90, 121–122, 215, 216, 230 – point mutation 81, 215, 216 – splice site mutation 33, 96, 121–122, 215, 216, 240–241 – stop mutation 96, 203 myocardial Æ infarction

PAI [Æ plasminogen activator inhibitor] partial thromboplastin time – activated partial thromboplastin time [aPTT] 88, 96, 103, 105, 136, 151, 166–167, parvovirus B19 25 Petterson score 87, 89–91 Phenprocoumon 226, 229, 242 physiotherapy 53–54, 144, 146 prion(s) 23–27 plasminogen activator inhibitor [PAI] 136–137, 250–253, 255–256 platelet(s) XXXII, 109–118, 166–167, 236, 245–246 – concentrate(s) 245–246 – count 115–116 – poor plasma [PPP] 61–63, 111 – rich plasma [PRP] 61–63, 65, 111–112, 115–116, 118 PPP [Æ platelet poor plasma] prednisolone 167 prednisone 187 previously treated patient [PTP] 38–40, 199–202 previously untreated patient [PUP] 34, 37–40, 88, 90–92, 199 protein – C 227, 242, 255 – – activity 89, 249, 251–252 – – antigen 89, 249, 252 – – deficiency 90, 210 – activated protein C [APC] – – resistance 249, 251 – S 227, 242, 255 – – activity 89, 249, 251–252 – – antigen 249 – – deficiency 210 – free protein S 89 – – antigen 249, 252 – Z 227, 242 prothrombin – mutation(s) 61, 63–64, 96, 219 – time [PT] 323 – activated prothrombin complex concentrate 44 protocol – Bonn protocol 190

Subject Index

PRP [Æ platelet rich plasma] PT [Æ prothrombin time] PTP [Æ previously treated patient] pulmonary embolism 209–210, 219 PUP [Æ previously untreated patient] purpura – idiopathic thrombocytopenic purpura [ITP] 110, 115 PVB19 [Æ Parvovirus B19] quality of life 51, 172, 174, 184, 191, 193 Rabies virus 22 Recombinate 38–40, 153 replacement therapy 42, 49–50, 61, 65, 89, 143, 150–151, 158, 197 Ribavirin 168 Ristocetin cofactor 4–5, 14 Rituximab 66–67, 69–70, 74–76, 78, 131–132, 166–167, 187–189 RNA 82 – mRNA [messenger RNA] 81, 84 Scrapie 23 septin(s) XXXI–XXXII Society for Thrombosis and Hemostasis Research [GTH] 34 Spearman rank 89 Streptokinase 251 stroke 127 Suicide 6, 9–11 Thrombin 61, 111–112, 116, 118, 128, 179 – activation 117 – activity 117 – generation 45, 61–65, 111, 115–118 – inhibitor 109, 111, 113, 115–116, 118 – time 223 thrombocyte(s) XXXI–XXXIII, 131, 136 thrombocytemia 131 thrombocytopathy XXXIII, 117 thrombocytopenia 110, 115–117, 123, 125 thromboembolism 44, 219 – venous thromboembolism 50, 209–211, 256

263

thrombophilia 90, 92, 96, 98–99, 211, 247, 251, 256 – acquired thrombophilia 249, 256 – inherited (hereditary) thrombophilia 209, 249–250, 255 thrombosis 117, 126–129, 130–131, 150, 192, 209, 219–220, 235–236, 251 – arterial thrombosis 128 – deep vein thrombosis [DVT] 127, 210 thrombotic-thrombocytopenic purpura [TTP] 123–125 Tirofiban 110, 116 tranexamic acid 196, 205 treatment on demand 37, 90–91, 157, 191, 219 TTP [Æ thrombotic-thrombocytopenic purpura / Moschkowitz] UFH [unfractionated Æ heparin] Urokinase 251 Vitamin K 226–227, 242, 244 von-Willebrand 15, 165 – disease [VWD] 3–6, 14, 96, 117, 150, 181 – – type I 14 – – type II 14 – – type III 14 – factor [VWF] XXXII, 38, 61, 64, 66, 77, 123–124, 150–151, 165, 167, 190 – – antigen 81, 166–167, 250–252, 254–256 – – multimer(s) 123, 165 – syndrome [VWS] 64, 137, 166 – acquired VWS 150–151, 165, 167 – inherited (congenital) VWS 165 Warfarin 130, 226–227, 229, 237, 242–244 West Nile Fever Virus [WNV] 21–22 WFH [World Federation of Hemophilia] 31, 44, 135 Ximelagatran 237