Diagnosis and Management of Inhibitors in Hemophilia Glenn Pierce MD PhD WFH Board of Directors La Jolla, California USA World Federation of Hemophilia (WFH) Indonesian Hemophilia Society (IHS) Symposium on Hemophilia Care Sanur, Bali, Indonesia 22 April 2017 Pierce/WFH

Topics to Cover What are inhibitors (neutralizing antibodies)? Assays for inhibitors Genetic and Environmental risk factors How are inhibitors managed? Immune Tolerance Regimens Pierce/WFH Pierce/WFH

Detection and Cause of Inhibitors (Neutralizing Antibodies)

Hemophilia and Inhibitors Inhibitor development is the most severe complication of therapy Development of alloantibodies to replacement therapy Neutralize replacement factor and in moderate/mild hemophilia neutralizes residual factor VIII activity NO EFFECT Epidemiology: Incidence of development of inhibitor Hemophilia A: 25–30% severe; Hemophilia B: 3–5% White GC et al. Thromb Haemost 2001; 85: 560. Hay CR et al. Thromb Haemost 1998; 79: 762-6. Kessler CM. Blood (Am Soc Hematology Educational Program) 2005: 429-35. Pierce/WFH Pierce/WFH

How Is a Factor VIII Inhibitor Identified? Clinical: Bleeding unresponsive to FVIII Patients’ bleeding episodes less well controlled Biochemical: Prolonged aPTT and aPTT mix Anti-Factor VIII detection by Bethesda assay Verbruggen B. The factor VIII inhibitor assays can be standardized: results of a workshop. J Thromb Haemost. 2011; 9:2003-8. Favaloro EJ, Verbruggen B, Miller CH. Laboratory testing for factor inhibitors. Haemophilia. 2014;20 Suppl 4:94-8. Barrowcliffe TW, Hubbard AR, Kitchen S. Standards and monitoring treatment. Haemophilia. 2012; 18 Suppl 4:61-5. Pierce/WFH Pierce/WFH

Diagnostic Laboratory Tools Reduced recovery (recovery <60% suggests inhibitor) Increased clearance (no measurable factor one hour after infusion) Persistently prolonged aPTT following replacement therapy aPTT mixing studies (50% patient plasma + 50% normal plasma) fails to normalize aPTT FVIII: Inhibitor titer >0.6 BU/mL (Bethesda, Nijmegen modification) Lupus anticoagulant test negative (no effect of added phospholipid) Modified assay: Plasma heated to 56°C for 30 min to destroy replacement FVIII Miller CH. Validation of Nijmegen-Bethesda assay modifications to allow inhibitor measurement during replacement therapy and facilitate inhibitor surveillance. J Thromb Haemost. 2012; 10:1055-61. Verbruggen B. Diagnosis and quantification of factor VIII inhibitors. Haemophilia. 2010; 16:20-4. Pierce/WFH Pierce/WFH

aPTT Mix Test aPTT pt plasma + Ca + Tissue TP = Clot aPTT MIX 1:1 Normal + pt plasma + Ca + Tissue TP = Clot Condition aPTT 1:1 Mix Comments Normal 35 sec. 35 sec. No effect Factor Def 60 sec. 35 sec. By providing 50% factor activity, will correct aPTT Inhibitor 60 sec. 59 sec. Cannot dilute effect of inhibitor Pierce/WFH Pierce/WFH

Nijmegen Bethesda Assay Each Bethesda unit of inhibitor consumes a 50% level Modified assay: Plasma heated to 56°C for 30 min to destroy replacement FVIII Pierce/WFH

Classification of Inhibitors Clinically relevant inhibitor development ≥ 2 positive titers + decreased FVIII recovery High-titer inhibitor Inhibitor titer ≥5 BU/mL. Usually strong anamnestic response to re-exposure Low-titer inhibitor Inhibitor titer <5 BU/mL. Usually weak anamnestic response to re-exposure May treat with FVIII at higher doses High Titer (>5 BU BU BU 10 Time 100 Low Titer (<5 BU) 10 Time 100 BU BU 10 Time 100 Transient 10 Time 100 = infusion White GC 2nd et al. Thromb Haemost (2001); 85: 560 Pierce/WFH Pierce/WFH

Inhibitor Development Most inhibitors develop within first 50 exposure days Median age 1.7 – 3.3 years in EU/NA1 Of the ~30% who develop FVIII inhibitors2,3 High titer: ~10-15% (>5 BU) Low titer: ~10% (<5 BU) Transient: ~5% (<5 BU) Bray GL et al. Blood 1994; 83: 2428-35. White GC et al. Thromb Haemost 2001; 85: 560. Astermark J. et al. Haemophilia 2006; 12: 363-71. Pierce/WFH

FIX Inhibitors Associated with anaphylactoid reactions to factor IX1 Reactions may occur prior to identification of inhibitor May preclude treatment with FIX or PCC/APCC May require desensitization ITI may be associated with nephrosis at 6-9 months2 FIX Inhibitors In approximately 3-5% of persons with hemophilia B, inhibitors to FIX develop. Treatment is complicated in these patients by the fact that some experience severe allergic or anaphylactic reactions to FIX infusions simultaneously with the appearance of or before the development of the inhibitor. In addition, the anaphylactoid reactions may preclude treatment with FIX or PCC/aPCC. Use of any FIX-containing product in these patients requires desensitization. Immune tolerance programs have been used in these patients; however, immune tolerance has been associated with nephrotic syndrome that usually occurs 6-9 months into the immune tolerance induction (ITI) regimen. 1Warrier I et al. J Pediatr Hematol Oncol. 1997;19(1):23-27. 2Ewenstein BM et al. Blood. 1997;89:1115,1116. Pierce/WFH Pierce/WFH

Three Components to Inhibitor Risk Immune response genes FVIII-gene mutation Environmental Factors Pierce/WFH Pierce/WFH

Inhibitor Risk Factors INHIBITORS Environmental Factors Nonpatient-related Genetic Factors Patient-related F8 dependent Race Ethnicity Family history Immune Response Genes Severity F8-Gene Mutations MHC IL-10 TNF CTLA4 Treatment regimen Type of Concentrate Danger Signals Age of 1st treatment early vs late Prophyl. vs on demand Intensity of treatment Recombinant vs PD vWF vs non vWF Vaccination Inflammation Severe bleed Tissue damage Oldenburg; Gouw, van den Berg, Oldenburg, et al. F8 gene mutation type and inhibitor development in patients with severe hemophilia A: systematic review and meta-analysis. Blood 2012 119:2922-2934; doi:10.1182/blood-2011-09-379453 Pierce/WFH

Mutation Type and Inhibitor Prevalence 100 75 50 25 Single Domain 25% Multi 88% Light chain 40% Heavy chain 17% Non A-Run 21% A-Run 3% C1-C2 10% Non C1-C2 Large Deletions 41% Nonsense 31% Small Deletions 16% Intron 22/1 Inversions 21%/17% Missense 5% Splice site 3% Pierce/WFH Schwaab R. et al. Thromb Haemost (1995);74:1402-1406, Oldenburg et al. Haematologica (2000);85:7-13

Immune Response Genes and Inhibitor Development Schematic model of anti-FVIII inhibitor formation. The causative FVIII mutation and HLA class II will be the main contributors to the risk of development of antibodies; from very low risk (green) unlikely to experience any antibodies with commercially available FVIII concentrates to very high risk (red). The final immune response and outcome will then be fine-tuned by T-regulatory cells and a variety of immune regulatory molecules, the activity of which will be defined genetically by therapy-related factors and immune system challenges. Astermark J. FVIII inhibitors: pathogenesis and avoidance. Blood. 2015; 125:2045-51. Pierce/WFH

Prophylaxis vs on-demand treatment During first 50 days of exposure Environmental Risk Factors for High-Titer Inhibitors in PUPs: CANAL Study First FVIII exposure Duration of treatment (>5 days) RR=4.1 Surgical procedures RR=3.2 Prophylaxis vs on-demand treatment 60% lower risk with prophylaxis RR=0.4 During first 50 days of exposure Treatment intensity (>50 IU/kg) RR=3.0 Positive family history, high risk FVIII gene mutations, and intensive treatment at initial bleed result in 57% inhibitor development Gouw SC, van der Bom JG, van den Berg MH, Treatment-related risk factors of inhibitor development in previously untreated patients with hemophilia A: the CANAL cohort study. Blood 2007; 109: 4648-4654; ter Avest. Risk stratification for inhibitor development at first treatment for severe hemophilia A: a tool for clinical practice. J Thromb Haemost 2008; 6:2048-2054. Pierce/WFH Pierce/WFH

Summary of Inhibitor Studies with pd-FVIII and rFVIII Iorio A, Halimeh S, Holzhauer S, et al. Rate of inhibitor development in previously untreated hemophilia A patients treated with plasma-derived or recombinant factor VIII concentrates: a systematic review. J Thromb Haemost 2010;8:1256-65. Incidence rate and 95% confidence interval of all inhibitors in PUPs with HA. Study author and year of publication are indicated on the y-axis and the incidence rate (0–50%) on the x-axis. Studies ordered by pdFVIII and rFVIII usage and by year of publication. The pooled estimate is based on random effects, and shown for subgroups and for the whole population. (R) or (PD) are indicated in brackets when only data of the relevant subgroup of patients are considered Confounders: Study period, frequency of inhibitor testing, and median follow up accounted for most variability Pierce/WFH Pierce/WFH

Plasma-derived FVIII versus recombinant FVIII No Increased Risk of Inhibitors for pdFVIII vs rFVIII in previously transfused Patients Who Switch Product Plasma-derived FVIII versus recombinant FVIII Risk of inhibitor RR=0.8 Risk of high titer inhibitor RR=0.9 PD FVIII/VWF versus recombinant FVIII Risk of inhibitor RR=1.0 Risk of high titer inhibitor RR=1.1 Switch between FVIII products Risk of inhibitor RR=1.1 Gouw SC et al. Blood (2007); 109(6): 4693-4697; Xi M, Makris M, Marcucci M, Santagostino E, Mannucci PM, Iorio A, J Thromb Haemost. 2013 Sep;11(9):1655-62 Pierce/WFH Pierce/WFH

How Are Inhibitor Patients Tolerized?

History of Immune Tolerance Induction: First Observation 1974: Young boy with FVIII deficiency and high titer inhibitor with life-threatening bleed Treatment Standard therapy: Prothrombin complex concentrate Added high dose FVIII Outcome Child recovered Inhibitor titer after 3 weeks of therapy 256  38 BU1 Treatment discontinuation: inhibitor rebounded Reinstituted daily high dose FVIII/PCC  disappearance of inhibitor2,3 Brackmann HH, Gormsen J. Lancet 1977; 2: 933 Brackmann HH et al. Vox Sang 1996; 70 (Suppl 1): 30-5 Brackmann HH et al. Prog Clin Biol Res 1984; 150: 181-95 Pierce/WFH

Treatment Plan for New Inhibitor Patients 1. Eradicate inhibitor Immune Tolerance: FVIII 50-200 U/kg/d or qod 2. Stop bleeding If >5 BU: FEIBA* 50-75 U/kg q 8-12h (< 200 U/kg/day) FVIIa 90 mcg/kg q 2-3h) If <5 BU: FVIII to achieve > 0.50 IU/ml (50%) *Factor VIIII Inhibitor Bypass Activity Pierce/WFH

Immune Tolerance – Clinical Results Prior immune tolerance therapy options Bonn protocol (high dose FVIII + activated PCC) (FVIII 200 IU/kg/day) Van Creveld (Dutch) protocol (low dose FVIII 50 IU/kg/day or qweekly) Malmö protocol (immunoadsorption +/- high-dose FVIII IVIG + immunosuppression (cyclophosphamide) *One Turkish study used 25 IU/kg tiweek, 4/7 tolerized over 1-4 months Randomized controlled trial, International-ITI study, evaluated Daily FVIII 200 IU/kg vs FVIII 50 IU/kg x 3 weekly No difference in inhibitor clearance, but less bleeding and shorter time to inhibitor disappearance in high dose cohort DiMichele DM, Br J Haematol. 2012;159:123-34; Hay CR1, DiMichele DM, Blood. 2012;119: 1335-44; Athale AH, Marcucci M, Iorio A, Cochrane Database Syst Rev. 2014 Apr 24;4 Oldenburg J et al. Vox Sang 1999; 77 (Suppl 1): 49-54; Mauser-Bunschoten EP et al. Vox Sang 1996; 70 (Suppl 1): 66-7; Freiburghaus C et al. Haemophilia 1999; 5: 32-9; *Kavakli K et al, Haemophilia 1998;4:104-8 Pierce/WFH Pierce/WFH

International ITI Study: No difference between treatment arms Success: 66 of 115 patients reached endpoint 46 (69.7%) complete success 3 (4.5%) partial success 17 (25.8%) failure 24 of 58 Low Dose (50 IU/kg tiweekly) vs. 22 of 57 High Dose (200 IU/kg/day) (P = 0.909) Low Dose subjects bled more often (odds ratio, 2.2; P = .0019) No bleeding in 8 of 58 Low Dose subjects vs 21 of 57 High Dose subjects Hay & DiMichele, Blood, 2012; 119: 1335-1344 Pierce/WFH

Differences Between Hemophilia A and B Factor VIII inhibitors Inhibitors develop in ~ 30% severe patients 50% high titer Infusion reactions uncommon Success rate with immune tolerance ~75% Immune tolerance not complicated by nephrosis Factor IX inhibitors Inhibitors develop in <5% of severe patients 80% high titer Infusion reactions common Anaphylaxis, angioedema May require desensitization, steroids, diphenhydramine Success rate with immune tolerance ~25% Immune tolerance may be complicated by nephrosis membranous glomerulonephritis edema, decreased albumin, proteinuria @7-9 months Reactions may preclude use of any product containing factor IX APCC, pdFIX, rFIX Ewenstein BM, et al. Blood 89:1115-6,1997; Dioun AF et al. J Allergy Clin Immunol 192:113-7, 1998 Pierce/WFH

How Are Inhibitor Patients Treated?

Treatment Options for Inhibitors Factor VIII & Inhibitors High dose factor VIII Bypassing products NovoSeven FEIBA Factor IX & Inhibitors High dose factor IX Bypassing products NovoSeven FEIBA in some circumstances Hedner, U. Recombinant activated factor VII: 30 years of research and innovation. Blood Reviews 2015;29:S4 - S8. Santagostino, E et al. Recombinant activated factor VII in the treatment of bleeds and for the prevention of surgery-related bleeding in congenital haemophilia with inhibitors. Blood Reviews 2015; 29:S9 - S18. Kempton, Meeks. Toward optimal therapy for inhibitors in hemophilia. Blood 2014 124:3365-3372. Pierce/WFH

FEIBA mechanism of action FEIBA contains Factors II, VII, IX, X and variable amounts of their activated forms; small amounts of FVIII too Pierce/WFH Pierce/WFH

Rfactor viia mechanism of action rFVIIa activates FIXa to activate FX to Fxa to produce thrombin This thrombin burst is less than the thrombin burst triggered by the intrinsic pathway via Tenase (FIXaFVIIIaFXa) Pierce/WFH

Treatment Options/Regimens and Safety NovoSeven® - Single dose 270 µg/kg 90 µg/kg at 2-3 hour interval until hemostasis is achieved Works via activation of FX to FXa= thrombin burst FEIBA® (aPCC) 50-100 U/ kg recommended at 6-12-hour intervals 200 U/kg/day should not be exceeded Produces anamnestic response in up to 31% of hemophilia patients Contains “mainly non-activated” FII, FIX, FX, FVIIa. Activates FXa Both drugs: good safety profile in inhibitor patients except thrombosis risk rFVIIa 2.29 / 105 infusions pd-aPCC 8.24 / 105 infusions van Veen JJ. Major surgery in severe haemophilia A with inhibitors using a recombinant factor VIIa and activated prothrombin complex concentrate hybrid regimen. Haemophilia. 2014 Jul;20(4):587-92. Antunes SV Randomized comparison of prophylaxis and on-demand regimens with FEIBA NF in the treatment of haemophilia A and B with inhibitors. Haemophilia 2014;20(1):65-72. Pierce/WFH Pierce/WFH

Treatment with rFVIIa and FEIBA (aPCC) Black Box warnings: Serious arterial and venous thrombotic events following administration of NovoSeven® RT have been reported. Monitor patients for signs or symptoms of activation of the coagulation system and for thrombosis Risk of thrombotic and thromboembolic events following infusion of FEIBA VH or FEIBA NF, particularly following the administration of high doses and/or in patients with thrombotic risk factors. Recombinant factor VIIa (rFVIIa; NovoSeven®) is approved for the treatment of bleeding episodes in patients with haemophilia A or B and inhibitors.1 Until recently, the only recommended dose was 90 µg/kg bodyweight, repeated at 2–3-hour intervals to obtain haemostasis. With this regimen, an average of 2.2 injections are required to achieve haemostasis.2 NovoSeven® prescribing information, 2007 Key et al. Thromb Haemost 1998; 80:912―918 Kempton, Meeks. Toward optimal therapy for inhibitors in hemophilia. Blood 2014 124:3365-3372. Pierce/WFH Pierce/WFH

Thank You

Inhibitor Risk Relative to F8 Genotype 5883 patients with severe hemophilia A Pierce/WFH Gouw SC, van den Berg HM, Oldenburg J et al. Blood 2012 Pierce/WFH

Immune Response Genes Immune response gene Odds ratio 95% CI P value MHC class-II DRB11501 1.91 0.8–4.11 — DQB10602 2.72 ns2 IL-10 1.23 1.01–1.53 0.043 Allele 134 positive 5.44 2.1–13.74 <0.0014 A>G -1082 1.64 TNF- Genotype A2 4.85 1.0–22.55 0.03105 Homozygous 196 CTLA-4 SNPS -318 0.37 0.1–0.87 0.0127 CT60 Hay et al. Thromb Haemost 1997;77:234–7; Oldenburg et al. Thromb Haemost 1997;77:238–42; Lozier et al. Haemophilia 2011;17:641–9; Astermark et al. Blood 2006;107:3167–72; Pavlova et al. J Thromb Haemost 2009;7:2006–15; Astermark Blood 2006;108:3739–45; Astermark et al. J Thromb Haemost 2007;5:263–5; Astermark et al, Blood. 2013;121(8): 1446-1454 Pierce/WFH

SIPPET Study Results Survey of Inhibitors in Plasma-Products Exposed Toddlers In PUPs, does recombinant FVIII result in more inhibitor formation than plasma-derived FVIIII? 251 patients analyzed from 42 sites, 14 countries Randomized, prospective trial 4 rFVIII vs 4 plasma-derived FVIII containing VWF utilized Variety of regimens: on demand, different prophylaxis schedules Cumulative incidence 26.8% (CI95 18.4-35.2) and 44.5% (CI95 34.7-54.3), respectively rFVIII was associated with an 87% higher incidence of inhibitor development than pdFVIII (HR 1.87, CI95 1.17- 2.96) Multiple methodologic differences from previous studies Conflicting results, however this is the only prospective, randomized study Bethesda positive >0.4 BU; study terminated prematurely due to 50% usage of Gen 2 FVIII; 1-50 EDs analyzed; ~75% of subjects from India, Egypt, Iran Peyvandi, Mannucci et al, N Engl J Med 2016;374:2054-64 Pierce/WFH Pierce/WFH

The relative risk for regular prophylaxis was 1 The relative risk for regular prophylaxis was 1.0 in the period from 1 to 10 exposure days, 0.95 from 11 to 20 exposure days, 0.22 from 21 to 30 exposure days, 0.27 from 31 to 40 exposure days, and 0.32 from 41 to 75 exposure days 50 ED Pierce/WFH

Mild/Moderate- Less at 50ED but continued increase Cumulative Inhibitor Incidence in 1112 Non- severe Hemophilia A Patients vs. Cumulative Exposure Days to Factor VIII Concentrates 50 ED Mild/Moderate- Less at 50ED but continued increase Cumulative inhibitor incidence in 1112 nonsevere hemophiliaA patients, according to cumulative exposure days to factor VIII concentrates. Pierce/WFH Eckhardt CL et al. Blood 2013;122:1954–1962 96 Pierce/WFH

ITI – Success or Failure Defined When to stop Time to ITI success is variable: 3 months - 2+ years Criteria for successful ITI Undetectable inhibitor level (<0.6 or 1.0 BU/mL) FVIII plasma recovery >66% FVIII T1/2 >6h after 72 h washout Absence of anamnesis upon further FVIII exposure Criteria for failure of ITI Failure to achieve full or partial success within 33 months Less than a 20% reduction in the inhibitor titer for any 6-month period during ITI after the first 3 months of treatment Astermark J et al. Haemophilia 2006; 12: 363-71; DiMichele DM et al. Haemophilia 2007; 13 (Suppl 1): 1-22. Pierce/WFH Pierce/WFH

Why Does ITI Work? Hypothetical Mechanisms Generation of peripheral tolerance Burn out FVIII-specific B-cell clones Add immunoregulatory molecules to high dose FVIII (eg, rituximab) Rituximab some evidence of efficacy in ~40-50% of refractory inhibitor patients Ongoing prevention research: Oral tolerance with rFVIII made in plants IgG induces T-reg mediated tolerance through amino acid sequences in Fc- role of this mechanism in Fc fusions rFVIII and rFIX? Transplacental transfer of rFVIIIFc in nonclinical studies to induce tolerance in offspring Gupta N, Sci Transl Med. 2015 Feb 18;7(275):275ra21. doi: 10.1126/scitranslmed.aaa1957; Scott DW, Haemophilia 2014 May;20 Suppl 4:80-6;Wang X...Herzog R, Blood 2015 Apr 9;125:2418-27 Pierce/WFH

Santagostino Study: Main Efficacy Results 25% 50% 64% 31% 53% 66% 10 20 30 40 50 60 70 9 hours 24 hours 48 hours % episodes controlled successfully VAS 270 μg/kg single dose 3  90 μg/kg multiple dose Santagostino conducted a randomised clinical trial in patients with haemophilia A and inhibitors to assess the efficacy and safety of two different regimens of rFVIIa in the treatment of haemarthroses: 90 μg/kg repeated as necessary every 3 ± 1 hours single dose of 270 μg/kg. Treatment response was assessed by patients and caregivers. Improvement in signs and symptoms (swelling, pain and functional limitation) from baseline was recorded on a visual analogue scale (VAS) at 1, 3, 6, 9, 12, 24 and 48 hours after the onset of treatment. A score of 0 meant “no improvement versus baseline” while a score of 100 meant “complete relief”. Additionally, patients and caregivers rated the efficacy of treatment at 9, 24 and 48 hours as “effective” (definite relief of signs and symptoms), “partially effective” (modest improvement) or “ineffective” (no improvement). Successful therapy was defined as a rating of “effective” plus a VAS score of at least 70. Failure was defined as a rating of “ineffective” and a VAS score of 30 or less. Combinations not meeting these criteria were considered partial responses. As shown here, rates of treatment success were similar for standard and high dose therapy at 9, 24 and 48 hours. However, the median number of injections needed was significantly greater for standard dose therapy (3) than for high dose therapy (1; P=0.0001). The total amount of rFVIIa required was the same for both dosage regimens (270 μg/kg). Santagostino et al. J Thromb Haemost 2006; 4:367–371 Santagostino et al. J Thromb Haemost 2006; 4:367–371. Pierce/WFH Pierce/WFH

Treatment of Bleeds High titer inhibitors (>5 BU/mL) Bypassing agents Recombinant factor VIIa (NovoSeven®) Plasma derived activated prothrombin complex concentrate (aPCC, FEIBA™) Recombinant Porcine factor VIII Concomitant medication Tranexamic acid (25 mg/kg po q6h) as adjunctive therapy together with rFVIIa Tranexamic acid use not documented together with FEIBA Leissinger C, Thromb Haemost. 2014;112:445-58; Kempton CL, Haemophilia. 2012; 18:798-804; Leissinger CA, Konkle BA, Antunes SV, Expert Rev Hematol. 2015; 8:375-82 Pierce/WFH Pierce/WFH

Efficacy – plasma derived aPCC /PCC DiMichele D and Négrier C. Haemophilia. 2006;12(4):352-62 Négrier C et al. Thromb Haemost (1997); 77: 1113-1119 Hilgartner MW et al. Transfusion (1990); 30: 626-630 Hilgartner MW et al. Blood (1983); 61(1): 36-40 Sjamsoedin LJM et al. NEJM (1981); 305: 717-721 Astermark J et al. Blood (2007) 109: 546-551 Pierce/WFH Pierce/WFH

Efficacy – rFVIIa Key NS et al. Thromb Haemost (1998);80:912-918 Santagostino E et al. J. Thromb Haemost (2006);4: 367-371 Kavakli IH et al. Thromb Haemost (2006); 95: 600-605 Ingerslev J et al. Blood Coag Fibrin (1998); 9(S1): S107-S110 Parameswaran MA et al. Haemophilia (2005); 11:100-106 Astermark J et al. Blood (2007) 109: 546-551 Pierce/WFH Pierce/WFH