Paroxysmal nocturnal hemoglobinuria

Transcription

1 Paroxysmal nocturnal hemoglobinuria Literature review October 2012 B. DEVALET

2 Plan Paroxysmal nocturnal hemoglobinuria history definition diagnosis clinical aspects pathophysiology of disease thrombotic events pathophysiology of thrombosis risk factors for thrombosis treatment Literature treatment of subclinical PNH and AA/PNH treatment of classic PNH prophylaxis of thrombosis treatment of thrombosis treatment during pregnancy

3 PNH: History First description by a german physician Paul Strübing in 1889 More detailed description by Dr Ettore Marchiafava and Dr Alessio Nazari in 1911, then Dr Ferdinando Micheli in 1931 (Marchiafava-Micheli syndrome) Identification of the molecular basis of PNH helped to define the physiology of the complement system in humans. (47)

4 PNH: Definition Rare acquired disorder of hematopoietic stem cells Incidence: not really known but estimated 1-2/ inhab./year Somatic mutation in X-linked gene: the phosphatidylinositol glycan class A (PIG-A) PIG-A si required for synthesis of glycosyl phosphatidylinositol (GPI) anchor Deficiency in GPI anchored proteins (many) Lack of CD59 «MIRL» and CD55 «DAF» (complement regulatory proteins) leads to complement mediated intravascular hemolysis (47)

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6 PNH: Definition Non malignant clonal expansion: 2 steps hypothesis (53) step 1: PIG-A mutation no clonal expansion, asymptomatic step 2: injury to the normal bone marrow T cells and NK cells attack normal hematopoietic cells destroyed but PNH cells escape (absence of GPI anchored targeted peptides or accessory molecules required for T cells attack) bone marrow failure, clonal expansion

7 PNH: Diagnosis (Ham and sucrose tests) Flow cytometry with monoclonal antibodies that bind to specific GPI-anchored proteins (precentage of PNH cells measured)(38) clone size use at least 2 different monoclonal antibodies, directed against 2 different GPI-anchored proteins (rare congenital deficiencies of CD59 or CD55) on at least 2 different cells lineages (risk of falsely negative tests if only red cells are screened recent hemolytic crisis or recent transfusion) phenotypic mosaicism PNH III cells: completely deficient in GPI anchored-proteins PNH II cells: partly deficient «PNH» I cells: express GPI-anchored proteins at normal density

8 PNH: Diagnosis FLAER (fluorescein-labeled proaerolysin)(34) Aerolysin = the principal virulence factor of the bacterium Aeromonas hydrophila binds selectively and with high affinity to the GPI anchor more accurate assessment of the GPI anchor deficit in PNH (PNH clones <1% can be detected) complete blood count, reticulocytes, LDH, bilirubin, haptoglobin, iron stores, BM aspirate and biopsy, cytogenetics

9 PNH: Clinical aspects 3 different forms (clinical polymorphism)(3, 4, 34) Classic PNH young adults intravascular hemolytic anemia (with crisis) Hb <12 g/dl hemoglobinuria (in the morning) jaundice general symptoms (asthenia, malaise) worsening of hemolysis during infection (complement activation above basal level) no evidence of bone marrow failure (neutro >1500/µL, platelets > /µL) dysphagia, abdominal pain, erectile dysfunction (35%) thrombosis reccurent infectious diseases (ORL, lungs): 40%, 2d cause of death risk of MDS/AML :5% / 2,5% at 10y (x100)

10 PNH: Clinical aspects PNH associated with aplastic anemia small PNH clone (majority of patients: <10%) clinical/biochemical evidence of hemolysis 2 or 3 cytopenias (Hb <10 g/dl, neutro <1000/µL, platelets <80 000/µL) BM failure dominates the clinical picture

11 PNH: Clinical aspects Subclinical PNH clone size <1% no hemolysis We find PNH clone in 60% of AA and in 20% of low risk MDS(47) associated to AA: high probability of response to immunosuppressive therapy associated to low risk MDS (refractory anemia) less pronounced morphological abnormalities of the blood cells more severe thrombocytopenia lower rate of karyotypic abnormalities higher incidence of HLA-DR15 lower rate of progression to AML higher probability of reponse to cyclosporine Hypothesis that aplastic anemia and a subgroup of low risk MDS are immune-mediated diseases immunity provides the selection pressure that favors the outgrowth of PNH clone

12 PNH: Clinical aspects (3-4) Classic PNH Aplastic anemia-pnh Age 40y 30y Abdominal pain 35% 20% Median clone size 91,6% 16,8% Median survival 18y >22y Thrombotic events (at 10y) 37,9% 27,8% Medullar aplasia 20% at 10y 100%

13 PNH: Pathophysiology of disease (1) Intravascular hemolysis free Hb in the plasma NO fixation to Hb NO depletion smooth muscle dystonias dysphagia abdominal pain erectile dysfunction arterial constriction reduced blood flow to the kidneys arterial hypertension pulmonary hypertension (+undiagnosed thrombosis)

14 PNH: Thrombotic events (1-2) Occurs in 40% of PNH. Cause of death in 40-67% of patients (major cause of death) Arterial (15%) and venous (85%) thrombosis Involve more than one site (20,5%) Usual sites deep veins thrombosis pulmonary embolism myocardial infarction CNS arteries and unusual sites hepatic veins (Budd-Chiari syndrome, the most frequent thrombotic complication of PNH: 40,7%) cavernous sinus CNS veins mesenteric veins dermal veins

15 PNH: Thrombotic events Age of onset lower than in general population (46y vs 73y) Recurrences despite anticoagulant therapy described(3) Thrombosis during pregnancy and post-partum increased risk of thrombosis maternal mortality 20% perinatal mortality 10%

16 PNH: Pathophysiology of thrombosis (1) Unknown - multifactorial Role of hemolysis? controversial no link between severity of hemolysis and thrombosis severity of anemia and thrombosis NO depletion(40) causes arterial constriction increases platelets activation and aggregation enhances TF expression? ADP release induce platelets activation(5) human RBC stroma activate the alternate complement pathway(7) interraction of activated complement components with platelets release of PF-3 activation of coagulation

17 Mechanisms and clinical implications of thrombosis in paroxysmal nocturnal hemoglobinuria Journal of Thrombosis and Haemostasis Volume 10, Issue 1, pages 1-10, 4 JAN 2012 DOI: /j x

18 PNH: Pathophysiology of thrombosis CD59 and CD55-deficient platelets complement can induce platelet activation one study showed increased adherence of patelets to the abdominal vessels(1) PNH platelets, after MAC (membrane attack complex) stimulation, expose more FVa-binding sites and increase thrombin generation more than normal platelets(40) BUT other studies showed impaired function of PNH platelets (reactive downregulation in response to chronic hyperstimulation)

19 PNH: Pathophysiology of thrombosis Endothelial cells(40) direct toxicity of free hemoglobin released endothelial precursor cells seem to be bone marrow-derived cells probable same GPI-anchored proteins deficiency more susceptible to complement injury thrombosis in unusual sites damage resulting from the uptake of monocyte-derived MVs (released from GPI-deficient monocytes upon complement damage) These MVs contain tissue factor, thus increasing TF expression on endothelial cells increased number of EMVs with a prothrombotic and proinflammatory phenotype in PNH increased levels of endothelial cells activation markers (VWF, svcam-1)

20 PNH: Pathophysiology of thrombosis Possible role of C5a protein binds to a receptor on granulocytes recruitement and activation of granulocytes and monocytes (enhanced in GPI-deficient white blood cells?) release of inflammatory molecules damage the endothelium TF expression release of monocyte-derived MVs, containing TF = link between inflammation and thrombosis?

21 PNH: Pathophysiology of thrombosis Tissue factor pathway inhibitor (TFPI) TFPI limits coagulation initiation by inhibiting TF formation mainly produced by the endothelium of the microvasculature bound to GPI-anchored protein (definciency in PNH) loss of TFPI expression has been associated with increased risk of thrombosis

22 PNH: Pathophysiology of thrombosis Neutrophil proteinase-3 (PR-3)(19) membrane bound protein, co-localized with GPIanchored neutrophil antigen 2a absence of neutrophil antigen 2a in PNH leads to loss of expression of neutrophil proteinase-3 PAR-1 (the predominant human platelet thrombin receptor) is a substrate for PR-3 impared down regulation of PAR-1 increased propensity for platelet activation PR-3 also modulates coagulation in various other ways

23 PNH: Pathophysiology of thrombosis Urokinase-type plasminogen activator receptor (u-par)(10,13,15,17) central role in the regulation of haemostatic processes on cell surface binding of u-pa (urokinase plasminogen activator) to u-par converts plasminogen into plasmin involved in fibrinolysis, make it localized pericellularly GPI-anchored protein underexpressed on cellular surface in PNH (granulocytes and monocytes) increased concentrations of soluble upar (released from PNH hematopoietic cells) impaired and displaced fibrinolytic system? Other fibrinolysis parameters conflicting results

24 PNH: Pathophysiology of thrombosis Increased circulating MVs in PNH patients(1,8) procoagulant properties of MVs (exposing PS, containing TF) have been demonstrated in vitro complement activation may stimulate the release of procoagulant MVs no link between clone size and number of MVs majority of them come from platelets(8) EMVs levels are also increased in PNH patients, with prothrombotic and proinflammatory phenotypes(9) similar level of erythrocyte MVs in vivo but PNH erythrocytes release higher amounts of procoagulant MVs upon complement stimulation in vitro (may suggest rapid clearance from the circulation)(40)

25 PNH: Pathophysiology of thrombosis Increase of procoagulant activity (fact V, fibrinogen, fact VIII, fact IX, fact X and vwf)(10) positive correlation with clone size Frequency of congenital thrombophila factors is not increased in PNH(40) testing for such factors may identify PNH patients at additional risk its value for treatment decision in PNH is unknown authors do not recommend routine testing

26 PNH: Risk factors for thrombosis (1-2) Large WBC clone(4) clone >50%: 10y cumulative incidence of thrombosis = 34,5% clone <50%: 10y cumulative incidence of thrombosis = 5,3% Previous history of thrombosis Old age (>55y) Use of transfusions(3) Other??? Elevation of D-dimers? Indications of prophylactic anticoagulation?

27 PNH: Treatment of subclinical PNH and AA/PNH (47) Subclinical PNH no symtoms, no treatment close monitoring (every 6-12 months) because expansion of the clone may occur PNH associated with aplastic anemia treatment of the underlying bone marrow failure syndrome (allogenic transplantation, immunosuppressive therapy)

28 PNH: Treatment of classic PNH Eculizumab (Soliris ; Alexion Pharmaceuticals)(6, 18, 31) humanized monoclonal antibody

29 PNH: Treatment of classic PNH/ eculizumab blocks the activation of terminal complement C5 and prevents the formation of C5a and C5b-9

30 PNH: Treatment of classic PNH/ eculizumab administration schedule min IV infusion induction dose of 600 mg every 7 days for 4 doses then 900 mg 7 days later followed by a maintenance dose of 900 mg every 14 days vaccination against Neisseria meningitidis at least 14 days prior to receiving eculizumab 0,5%/y risk of neisserial sepis (even after vaccination)(34) revaccinate every 3 to 5 years medical attention if patient develops signs of neisserial infection

31 PNH: Treatment of classic PNH/ eculizumab TRIUMPH (randomized placebo-controlled phase 3 study) in transfusion-dependant PNH patients with good bone marrow reserve. reduces hemolysis, transfusion requirements, improves anemia, fatigue and measures of quality of life SHEPHERD (open-label phase 3 study)(6) to investigate the long-term safety and efficacy of eculizumab 97 patients enrolled, median age 41 y, follow up 52 weeks 89/97 pat. got a fast response and maintained complete inhibition of hemolysis efficient most common adverse events: headache, nasopharyngitis, upper respiratory tract infection 8,3% of infections possibly related to treatment 44 SAEs (none was considered probably or definely related to treatment) well tolerated

32 PNH: Treatment of classic PNH/ eculizumab Impact on overall survival? Survival for patients on eculizumab appears to be similar to that of the normal population(50)

33 PNH: Treatment of classic PNH/ eculizumab Reasons for suboptimal response(47-48) breakthrough intravascular hemolysis near the end of a treatment cycle cycles of 13 or 12 days increase dose of eculizumab extravascular hemolysis Frequently, persistant elevation of reticulocytes, bilirubin and LDH levels, presistant reduction of haptoglobin A small group of eculizumab-treated patients: little or no improvement in either anemia or constitutional symptoms inhibition of intravascular lysis by eculizumab increase of PNH red blood cells, loaded with early components of the complement system (C3 opsonins) eculizumab does not block the C3 convertase (not down regulated by CD55) recognized by reticuloendothelal cells hemolysis in the spleen (and the liver)

34 PNH: Treatment of classic PNH/ eculizumab

35 PNH: Treatment of classic PNH/ eculizumab What to do? No treatment if no constitutional or anemia symptoms or transfusion dependance Corticosteroids or splenectomy?

36 PNH: Treatment of classic PNH/ eculizumab Reduction of thombosis with eculizumab Number of TE events and incidence rates determined in patients from 3 clinical studies with eculizumab(31) 87-94% reduction of the thrombotic events rate during eculizumab treatment Decrease in plasma D-dimers levels with eculizumab(44) suppression of thrombin generation Study of markers of thrombin generation and fibrinolysis in 23 PNH patients(18) before and after 5 and 11 w of eculizumab treatment eculizumab reduces the activation of the plasma hemostatic system and the vascular endothelium role of the endothelial cell activation in the pathogenesis of thrombosis in PNH? no effect of eculizumab on the number of EMVs, on MVs procoagulant activity or on the number of ICAM-1 expressing EMVs.

37 PNH: Treatment of classic PNH/ eculizumab Rapid and sustained decrease in the markers of thrombin generation and inflammation during eculizumab therapy(44) significant reduction in D-dimers, thrombinantithrombin complex, IL-6, soluble P-selectin (expressed by activated platelets and endothelial cells), TFMP (tissue factor bearing microparticles) no link between hemolysis (LDH) and markers of thrombingenerationand inflammation the decrease of TFMP didn t correlate with changes in markers of thrombin generation and inflammation

38 PNH: Treatment of classic PNH/ eculizumab What happens if we stop eculizumab? risk of hemolytic crisis increased number of PNH red blood cells during eculizumab treatment risk of thrombosis? one case described of a first thrombotic event 3 weeks after the last eculizumab dose (women died) during anticoagulant prophylaxy (therapeutically dosed phenprocoumon/marcoumar ) no hemolysis mechanism?

39 PNH: Treatment of classic PNH (2) Bone marrow transplantation the only curative option important graft-versus-pnh effect lack of suitable donnors high rates of treatment-related morbidity/mortality 2-year survival probability: 56%(34) acute and chronic graft versus host disease: 15% and 20% only for patients with life threatning cytopenias or disabling hemolysis or thrombosis that is not controlled with eculizumab Supportive care transfusions if needed (efficient, may lessen hemolysis by suppressing erythropoiesis) folate supplements iron supplements if needed (hemoglobinuria, hemosiderinuria), rare during eculizumab therapy (sometimes iron depletion therapy needed) EPO: increases hemolysis symptoms, may be used when patients remain anemic with eculizumab and have low EPO level corticosteroids: long term toxicity, use for acute hemolysis crises androgens (Danazol)? no data

40 PNH: Prophylaxis of thrombosis (1-2) Primary prophylactic anticoagulation? no randomized trials some data suggest that warfarin effectively reduces thrombotic risk anticoagulation in PNH patients is difficult thrombocytopenia (aplasia, liver failure) risk of bleeding difficult to maintain a therapeutic INR with warfarin oesophagal spasm mesenteric insufficiency irregular feeding recommendation of the international PNH interest group: consider vitamin K antagonist prophylaxis in patients with PNH granulocyte clone >50% and no contraindications for prophylaxis other authors say: No proven benefit for primary prophylactic anticoagulation (except pregnancy)

41 PNH: Prophylaxis of thrombosis (1-2) aspirin? glycoprotein IIb-IIIa receptor antagonists? platelets may have a role in the pathophysiology of thrombosis in PNH most thrombotic events occur in the venous system LMWH? New oral anticoagulants (rivaroxaban, dabigatran, )? Immunosuppressive therapy? need to define subgroups of PNH patients who could benefit from anticoagulation (risk factors) Eculizumab is the best preventive treatment for thrombosis

42 PNH: Treatment of thrombosis (1-2) If life-threatening thrombosis thrombolytic therapy Anticoagulation therapy foundation of treatement warfarin? LMWH? NOAC? for how long? lifelong?

43 PNH: Treatment during pregnancy (34) greater need for folate and iron supplementation prophylactic full anticoagulation with LMWH if no contraindication example: enoxaparin 1mg/kg/12h SC starting when pregnancy is confirmed monitor anti-factor Xa levels sometimes need to switch to unfractionated heparin if a cesarean section is planned anticoagulation has to be continued during the post-partum period eculizumab? listed as category C pharmaceuticals (drug with little or no information available) important potential benefit of eculizumab in reducing thrombosis IgG2 isotypes do not cross the placenta, probably little impact on the fetus. single case report: eculizumab was administered to a pregnant PNH patient in week 30 of her pregnancy (twins). No complications were reported.

44 Literature 1) Weitz IC. Thrombosis in patients with paroxysmal nocturnal hemoglobinuria. Seminars in Thrombosis and Hemostasis. 2011; 37(3): ) Ziakas PD, Poulou LS, Rokas GI, Bartzoudis D, Voulgarelis M. Thrombosis in paroxysmal nocturnal hemoglobinuria: sites, risks, outcome. An overview. J Thromb Haemost. 2007;5(3): ) Peffault de Latour R, Mary JY, Salanoubat C, Terriou L, Etienne G, Mohty M, et al. Paroxysmal nocturnal hemoglobinuria: natural history of disease subcategories. Blood. 2008;112(8): ) Moyo VM, Mukhina GL, Garrett ES, Brodsky RA. Natural history of paroxysmal nocturnal haemoglobinuria using modern diagnostic assays. Br J Haematol. 2004;126(1): ) Hall C, Richards S, Hillmen P. Primary prophylaxis with warfarin prevents thrombosis in paroxysmal nocturnal hemoglobinuria (PNH). Blood. 2003;102(10): ) Brodsky RA, Young NS, Antonioli E, Risitano AM, Schrezenmeier H, Schubert J, et al. Multicenter phase 3 study of the complement inhibitor eculizumab for the treatment of patients with paroxysmal nocturnal hemoglobinuria. Blood. 2008;111(4): ) Poskitt TR, Fortwengler HP Jr., Lunskis BJ. Activation of the alternate complement pathway by autologous red cell stroma. J Exp Med. 1973;138(3): ) Hugel B, Socie G, Vu T, Toti F, Gluckman E, Freyssinet JM, et al. Elevated levels of circulating procoagulant microparticles in patients with paroxysmal nocturnal hemoglobinuria and aplastic anemia. Blood. 1999;93(10): ) Simak J, Holada K, Risitano AM, Zivny JH, Young NS, Vostal JG. Elevated circulating endothelial membrane microparticles in paroxysmal nocturnal haemoglobinuria. Br J Haematol. 2004;125(6): ) Grunewald M, Siegemund A, Grunewald A, Schmid A, Koksch M, Schopflin C, et al. Plasmatic coagulation and fibrinolytic system alterations in PNH: relation to clone size. Blood Coagul Fibrinolysis. 2003;14(7): ) Wiedmer T, Hall SE, Ortel TL, Kane WH, Rosse WF, Sims PJ. Complement-induced vesiculation and exposure of membrane prothrombinase sites in platelets of paroxysmal nocturnal hemoglobinuria. Blood. 1993;82(4): ) Gralnick HR, Vail M, McKeown LP, Merryman P, Wilson O, Chu I, et al. Activated platelets in paroxysmal nocturnal haemoglobinuria. Br J Haematol. 1995;91(3): ) Ploug M, Eriksen J, Plesner T, Hansen NE, Dano K. A soluble form of the glycolipid-anchored receptor for urokinase-type plasminogen activator is secreted from peripheral blood leukocytes from patients with paroxysmal nocturnal hemoglobinuria. Eur J Biochem. 1992;208(2): ) Ronne E, Pappot H, Grondahl-Hansen J, Hoyer-Hansen G, Plesner T, Hansen NE, et al. The receptor for urokinase plasminogen activator is present in plasma from healthy donors and elevated in patients with paroxysmal nocturnal haemoglobinuria. Br J Haematol. 1995;89(3): ) Plesner T, Behrendt N, Ploug M. Structure, function and expression on blood and bone marrow cells of the urokinase-type plasminogen activator receptor, upar. Stem Cells. 1997;15(6): ) Ninomiya H, Hasegawa Y, Nagasawa T, Abe T. Excess soluble urokinase-type plasminogen activator receptor in the plasma of patients with paroxysmal nocturnal hemoglobinuria inhibits cellassociated fibrinolytic activity. Int J Hematol. 1997;65(3):

45 Literature 17) Sloand EM, Pfannes L, Scheinberg P, More K, Wu CO, Horne M, et al. Increased soluble urokinase plasminogen activator receptor (supar) is associated with thrombosis and inhibition of plasmin generation in paroxysmal nocturnal hemoglobinuria (PNH) patients. Exp Hematol. 2008;36(12): ) Helley D, Peffault de Latour R, Porcher R, Rodrigues CA, Galy-Fauroux I, Matheron J, Duval A, Schved J-F, Fischer A-M, and Socié G on behalf of the French Society of Hematology. Evaluation of hemostasis and endothelial function in patients with paroxysmal nocturnal hemoglobinuria receiving eculizumab. Haematologica. 2010;95: ) Jankowska AM, Szpurka H, Calabro M, Mohan S, Schade AE, Clemente M, Silverstein RL, Maciejewski JP. Loss of expression of neutrophil proteinase-3: a contributory factor in thrombotic risk in paroxysmal nocturnal hemoglobinuria. Haematologica Jul;96(7): ) Burnier L, Fontana P, Kwak BR, Angelillo-Scherrer A. Cell-derived microparticles in haemostasis and vascular medicine. Thromb Haemost Mar;101(3): ) Williams JC, Mackman N. MPs or ICs? Blood Jan 27;117(4): ) Beyer C, Pisetsky DS. The role of microparticles in the pathogenesis of rheumatic diseases. Nat Rev Rheumatol Jan;6(1):21-9. Epub 2009 Dec 1. 23) Owens AP 3rd, Mackman N. Microparticles in hemostasis and thrombosis. Circ Res May 13;108(10): ) van der Pol E, Hoekstra AG, Sturk A, Otto C, van Leeuwen TG, Nieuwland R. Optical and non-optical methods for detection and characterization of microparticles and exosomes. J Thromb Haemost Dec;8(12): ) Lacroix R, Robert S, Poncelet P, Dignat-George F. Overcoming limitations of microparticle measurement by flow cytometry. Semin Thromb Hemost Nov;36(8): Epub 2010 Nov 3. Review. 26) Yuana Y, Oosterkamp TH, Bahatyrova S, Ashcroft B, Garcia Rodriguez P, Bertina RM, Osanto S. Atomic force microscopy: a novel approach to the detection of nanosized blood microparticles. J Thromb Haemost Feb;8(2): Epub 2009 Oct ) Freyssinet JM, Toti F. Membrane microparticle determination: at least seeing what's being sized! J Thromb Haemost Feb;8(2): Epub 2009 Oct ) Lacroix R, Robert S, Poncelet P, Kasthuri RS, Key NS, Dignat-George F; ISTH SSC Workshop. Standardization of plateletderived microparticle enumeration by flow cytometry with calibrated beads: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop. J Thromb Haemost Nov;8(11): ) Mullier F, Bailly N, Chatelain C, Dogné JM, Chatelain B. Needs, interests, and limitations of calibrated polystyrene beads for flow cytometry-based quantification of biological microparticles. J Thromb Haemost Jun 4. 30) Jy W, Ricci M, Shariatmadar S, Gomez-Marin O, Horstman LH, Ahn YS. Microparticles in stored red blood cells as potential mediators of transfusion complications. Transfusion Apr;51(4): ) Hillmen P, Muus P, Duhrsen U, Risitano AM, Schubert J, Luzzatto L, et al. Effect of the complement inhibitor eculizumab on thromboembolism in patients with paroxysmal nocturnal hemoglobinuria. Blood. 2007;110(12): ) Ziakas PD, Poulou LS, Rokas GI, Bartzoudis D, Voulgarelis M. Thrombosis in paroxysmal nocturnal hemoglobinuria: sites, risks, outcome. An overview. J Thromb Haemost 2007;5: ) Borowitz MJ, Craig FE, Digiuseppe JA, Illingworth AJ, Rosse W, Sutherland DR, Wittwer CT, Richards SJ; Clinical Cytometry Society. Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytometry B Clin Cytom Jul;78(4):

46 Literature 34) Brodsky RA. How I treat paroxysmal nocturnal hemoglobinuria. Blood. 2009;113: ) Hardij J, Mejia J, Gheldof D, Varin E, Michiels C, Chatelain B, Lucas S, Mullier F, Dogné JM. The CPS disc centrifuge: a new original method to detect microparticles (MPs). International Society of Thrombosis and Hemostasis. July 2011, Abstract ) Kozuma Y, Sawahata Y, Takei Y, Chiba S, Ninomiva H. Procoagulant properties of micrparticles released from red blood cells in paroxysmal nocturnal haemoglobinuria. Br J Haematol Mar;152(5): Epub 2011 Jan ) Piccin A, Murphy WG, Smith OP. Circulating microparticles: pathophysiology and clinical implications. Blood Rev May;21(3): ) Peffault de Latour R, Amoura Z, Socié G. L hémoglobinurie paroxystique nocturne. La revue de médecine interne 31(2010) ) Weitz IC. Thrombosis in paroxysmal nocturnal hemoglobinuria- insights into the role of complement in thrombosis. Thromb Res Apr;125 Suppl2:S ) Van Bijnen ST, Van Heerde WL, Muus P. Mechanisms and clinical implications of thrombosis in paroxysmal nocturnal hemoglobinuria. J Thromb Haemost 2012;10: ) Raghupathy R, Derman O. Response of paroxysmal nocturnal hemoglobinuria clone with aplastic anemia to rituximab. Case Report Hematol. 2012;2012: Epub 2012 May 7. 42) Bellido M, van der Velden VH, Leebeek FW, Te Boekhorst PA. Thrombotic complications without evidence of hemolysis in paroxysmal nocturnal hemoglobinuria: is eculizumab indicated?ann Hematol Jul 4 43) Peffault de Latour R, Schrezenmeier H, Bacigalupo A, Blaise D, de Souza CA, Vigouroux S, Willemze R, Terriou L, Tichelli A, Mohty M, de Guibert S, Marsh J, Passweg J, Mary JY, Socie G.Allogeneic stem cell transplantation in paroxysmal nocturnalhemoglobinuria. Haematologica Jun ) Weitz IC, Razavi P, Rochanda L, Zwicker J, Furie B, Manly D, Mackman N, Green R, Liebman HA.Eculizumab therapy results in rapid and sustained decreases in markers of thrombin generation and inflammation in patients with PNH independent of its effects on hemolysis and microparticle formation. Thromb Res Sep;130(3): Epub 2012 Apr ) Liebman HA, Feinstein DI. Thrombosis in patients with paroxysmal noctural hemoglobinuria is associated with markedly elevated plasma levels of leukocyte-derived tissue factor. Thromb Res. 2003;111(4-5): ) van Bijnen ST, van Rijn RS, Koljenovic S, te Boekhorst P, de Witte T, Muus P.Possible high risk of thrombotic events in patients with paroxysmal nocturnal haemoglobinuria after discontinuation of eculizumab. Br J Haematol Jun;157(6): Epub 2012 Feb 29 47) Parker CJ. Management of paroxysmal nocturnal hemoglobinuria in the era of complement inhibitory therapy. Hematology Am Soc Hematol Educ Program. 2011;2011:21-9. Review. 48) Röth A, Hock C, Konik A, Christoph S, Dührsen U. Chronic treatment of paroxysmal nocturnal hemoglobinuria patients with eculizumab: safety, efficacy, and unexpected laboratory phenomena. Int J Hematol Jun;93(6): Epub 2011 May 25.

47 Literature 50) Kelly RJ, Hill A, Arnold LM, Brooksbank GL, Richards SJ, Cullen M, Mitchell LD, Cohen DR, Gregory WM, Hillmen P.Longterm treatment with eculizumab in paroxysmal nocturnal hemoglobinuria: sustained efficacy and improved survival. Blood Jun 23;117(25): Epub 2011 Apr 1. 51) Pu JJ, Brodsky RA. Paroxysmal nocturnal hemoglobinuria from bench to bedside. Clin Transl Sci Jun;4(3):