ALBERT

Beschreibung: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by a lack of the terminal complement inhibitor CD59 on erythrocytes that renders these cells susceptible to chronic hemolysis. Eculizumab blocks terminal complement resulting in reductions in hemolysis, thrombotic events, renal impairment and transfusion requirement, as well as improvement in quality of life. The standard dosing regimen for eculizumab is 600 mg/week for 4 weeks (induction); 900 mg one week later; and then 900 mg every 14 ± 2 days (maintenance). This regimen maintains eculizumab levels 〉35 μg/mL, which is sufficient to completely and consistently block complement-mediated hemolysis in patients with PNH. In PNH clinical trials, 900 mg of eculizumab every 14 ± 2 days effectively and consistently blocked complement-mediated hemolysis in 98% of patients (n=195). During the studies, 10–15% of patients experienced an increase in hemolysis (elevation of LDH) near the end of the 14-day dosing interval with a return of pre-eculizumab symptoms such as hemoglobinuria, dysphagia, abdominal pain, or fatigue. The dosing interval was reduced to 12 days, as specified by label, resulting in sustained complement blockade, control of hemolysis and resolution of symptoms in nearly all patients. Three of the original 195 patients (2%) were not consistently blocked with the approved dosing regimen. Alternative eculizumab dosing regimens were investigated in these patients to assess their effectiveness and safety. Two different dosing regimens were employed; both included a maintenance phase with 1200 mg every 14 days. One regimen also included an induction period of 900 mg weekly for 5 doses. LDH, pharmacokinetics (PK), and clinical signs of complement breakthrough were monitored. The time from first eculizumab treatment to initial breakthrough on the 900 mg every 14 days ranged from 2 to 19 mo., and the reduction in the dosing interval to 900 mg every 12 days, as specified in the label, did not adequately control hemolysis in each of these 4 patients. Patient 1 was managed for 6 mo. with 900 mg every 12 days before experiencing additional complement breakthrough episodes (Figure, panel A). LDH levels (closed diamonds) reached 9234 U/L (ULN, 430-450 U/L) and breakthrough symptoms occurred 2 days prior to the next dose. The patient was re-induced with 900 mg eculizumab every 7 days for 5 weeks followed by 1200 mg every 14 days. Trough levels of eculizumab increased (open circles) each week during the induction phase (42.7 – 81.8 μg/ml) resulting in an immediate reduction in LDH to near normal levels. A maintenance dose of 1200 mg every 14 days in this patient resulted in sustained complement blockade. Patient 2 experienced breakthrough hemolysis after 19 mo. of standard dosing. Complement breakthrough occurred during a post-cholecystectomy infective endocarditis. After an adjustment to 900 mg every 12 days did not control complement breakthrough (Figure, panel B), the dose was changed to 1200 mg every 14 days without re-induction. This regimen resulted in sufficient levels of eculizumab to consistently reduce hemolysis to near normal levels. Further episodes of hemoglobinuria and other symptoms of hemolysis were not observed. Two additional patients received 1200 mg every 14 days without re-induction, one following complement breakthrough on the approved dose and the other due to the convenience of the 14 day interval with the 1200 mg dose. Complete complement blockade has been maintained in these patients for 8 mo. and 12 mo. to date, respectively. After 1 year of sustained complement blockade with the 1200 mg maintenance dose, patient 1 again demonstrated a breakthrough. Complement inhibition is now being maintained in this patient by a 1200 mg dose every 14 days with an additional 1200 mg dose in between the 14 day dosing interval every 4–5 doses. There were no reported adverse events in any of the four patients in which the 1200 mg dosing regimens were administered. In summary, these data demonstrate good correlation between eculizumab and LDH levels, suggesting that a breakthrough in complement activity due to insufficient drug levels can be monitored by levels of LDH near the end of the dosing interval. These results illustrate that two alternative-dosing regimens are well tolerated and can be effectively employed in the small percentage of PNH patients in which complement inhibition is not consistently maintained using the standard dose. Figure Figure

Beschreibung: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by intravascular hemolysis, often resulting in the need for red blood cell (RBC) transfusions. PNH RBCs lack two complement regulatory molecules - CD59, a terminal complement inhibitor, and CD55, a C3 convertase inhibitor. Eculizumab, a humanized monoclonal antibody that inhibits terminal complement by binding to C5, effectively controls intravascular hemolysis as determined by a dramatic reduction in lactate dehydrogenase (LDH) to levels in or just above the normal range. Control of intravascular hemolysis in these patients led to a reduction in, or cessation of, RBC transfusions. During eculizumab treatment, a majority of patients demonstrate evidence of residual, low-level hemolysis; LDH levels remain slightly elevated, haptoglobin levels are low or undetectable, and bilirubin levels are above normal. We hypothesized that this low-level residual hemolysis may be due to clearance of PNH RBCs through a C3b-mediated mechanism. Therefore we investigated C3 deposition on RBC in PNH patients before and on eculizumab. A direct antiglobulin test (DAT) using monoclonal anti-C3d was positive in 29 out of 39 PNH patients on eculizumab. Of these 29 DAT-positive patients, who were all receiving transfusions, 25 had DAT testing prior to eculizumab therapy and only one of these was positive. DAT was negative in all of 8 normal volunteers. By two-color flow cytometric analysis with anti-CD59 and anti-C3, the majority of patients on eculizumab demonstrated three distinct RBC populations: CD59+/C3− (normal RBCs); CD59-/C3− (PNH RBCs without C3 coating); and CD59-/C3+ (PNH RBCs coated by C3). No CD59+/C3+ RBCs were observed. Of 21 DAT positive eculizumab treated patients tested, the median proportion of total RBCs that were C3b positive was 17.6%. 18 of 29 [62%] eculizumab patients with a positive DAT received at least one transfusion during eculizumab therapy compared with 1 of 10 [10%] for DAT negative patients (p=0.01), although even patients who did not become transfusion independent during eculizumab treatment showed a marked reduction in transfusion requirement. The median hemoglobin value for the 29 DAT positive eculizumab patients was 9.8 g/dL compared with 11.3 g/dL in the 10 DAT negative eculizumab patients (p= 0.08). No apparent relationship between LDH and DAT positivity was observed. It is proposed that resolution of intravascular hemolysis in PNH patients on eculizumab results in deposition of C3b on the surface of PNH RBCs which may explain, at least in part, the residual low level hemolysis occurring in some patients. This appears to be a previously undescribed mechanism of RBC clearance in PNH, most likely obscured by the rapidity of intravascular hemolysis in the absence of eculizumab therapy. Despite the low-level residual hemolysis, patients continue to receive significant benefit from eculizumab treatment.

Beschreibung: Pulmonary hypertension (PHT) is an emerging common complication of hereditary hemolytic anemias. It has been mechanistically and epidemiologically linked to intravascular hemolysis and decreased nitric oxide (NO) bioavailability. While this complication has been described in approximately 30% of adult patients with sickle cell disease and thalassemia, the prevalence of PHT in patients with paroxysmal nocturnal hemoglobinuria (PNH), an acquired disease with the highest levels of intravascular hemolysis observed, has never been determined. PNH patients frequently have symptoms consistent with both hemolysis and PHT including severe fatigue and dyspnea on exertion. Therefore, we examined for the presence of PHT in PNH and explored potential mechanisms associated with its development by measuring the ability of plasma to instantaneously consume NO using ozone-based chemiluminescence. Doppler echocardiography was performed in 24 hemolytic PNH patients to estimate pulmonary artery systolic pressures. Systolic PHT was defined by a tricuspid regurgitant jet velocity (TRV) ≥ 2.5m/s at rest. Eleven (46%) patients had elevated pulmonary artery systolic pressures (mean TRV 2.7m/s ± 0.08) and one (4%) had severely elevated pressures (TRV 3.5m/s). Plasma from PNH patients (n=28) consumed 32.26 ± 8.74μM NO while normal subjects (n=9) consumed 2.42 ± 0.77μM NO (p=0.03). LDH levels correlated with NO consumption (p

Beschreibung: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder of hemopoietic stem cells (HSC) that is characterized by intravascular hemolysis and venous thrombosis. The PNH HSC’s (and their progeny) have a somatic mutation of the X-linked phosphatidylinositol glycan class A gene (PIG-A) which causes a complete or partial deficiency of glycophosphatidylinositol (GPI) anchored proteins, which in turn produces the symptoms of the disease. PIG-A mutations account for all of classical acquired PNH cases thus far reported as a single acquired PIG-A mutation on the only active X-chromosome results in GPI-deficiency. A novel mutation in the promoter of the phosphatidylinositol glycan class M gene (PIG-M) has recently been reported in two unrelated families exhibiting autosomal recessive inheritance of congenital GPI-deficiency. This point mutation reduces PIG-M transcription and causes partial deficiency of GPI-anchored proteins. It is possible that this point mutation in PIG-M occurs at low frequency in the population but predisposes to mutation of the other PIG-M allele and could result in acquired PNH. To determine the prevalence of the PIG-M in classical acquired PNH the PIG-M promoter region spanning the proposed mutation was sequenced to look for heterozygotes with the C → G substitution at position −270. This mutation was not identified in 36 patients with PNH. There is convincing evidence that GPI-deficient HSC’s have no intrinsic proliferative advantage over normal HSC’s suggesting that other factors underlie the clonal expansion of PNH cells. The most likely explanation appears to be due to factors which are extrinsic to the PNH clone such as immune attack on normal HSC. Recently secondary molecular events within the PNH clone have been suggested to provide the growth advantage allowing expansion of the PNH clone. One such proposed event is a translocation affecting the HMGA2 gene, which has been described in 2 cases of PNH. HMGA2 is a member of the high motility group of proteins and acts as an architectural transcription factor. HMGA2 proteins are associated with gene activation and are mainly expressed during embryonic development. Rearrangements of HMGA2 commonly occur in benign mesenchymal tumours, but have also been identified as infrequent events in myeloid malignancies. To gain insight into the potential role of HMGA2 deregulation in the pathogenesis of PNH we evaluated the expression of HMGA2 mRNA by means of quantitative RT-PCR in peripheral blood samples of 42 PNH patients (median age: 42, range: 18–81) and ten normal controls. All PNH samples had large granulocyte clones (median size 97.81%) but showed no increase in HMGA2 mRNA. In nine of the 42 PNH patients blood samples underwent additional enrichment for CD15 positive cells to maximize the proportion of PNH granulocytes present. This again showed no aberrant expression of HMGA2 mRNA. This is the largest reported group with PNH evaluated for HMGA2 expression. Despite two case reports suggesting that the deregulation of HMGA2 may be a pathophysiological factor in occasional cases of PNH our data indicates that this mechanism accounts for the growth advantage of PNH cells in, at most, only a small minority of patients. Similarly, the PIG-M promoter mutation observed in congenital GPI-deficiency was not found in any of our patients with classical acquired PNH indicating that it has no or little role in the pathophysiology of PNH.

Beschreibung: Paroxysmal nocturnal hemoglobinuria (PNH) is a hematologic disorder characterized by clonal expansion of red blood cells (RBCs) lacking the ability to inhibit complement-mediated hemolysis. Eculizumab, a humanized monoclonal antibody that binds the C5 complement protein, blocks serum hemolytic activity. This study evaluated the long-term safety and efficacy of eculizumab in 11 patients with PNH during an open-label extension trial. After completion of an initial 12-week study, all patients chose to participate in the 52-week extension study. Eculizumab, administered at 900 mg every 12 to 14 days, was sufficient to completely and consistently block complement activity in all patients. A dramatic reduction in hemolysis was maintained throughout the study, with a decrease in lactate dehydrogenase (LDH) levels from 3110.7 IU/L before treatment to 622.4 IU/L (P = .002). The proportion of PNH type III RBCs increased from 36.7% at baseline to 58.4% (P = .005). The paroxysm rate of days with gross evidence of hemoglobinuria per patient each month decreased from 3.0 during screening to 0.2 (P 〈 .001) during treatment. The median transfusion rate decreased from 1.8 U per patient each month before eculizumab treatment to 0.3 U per patient each month (P = .001) during treatment. Statistically significant improvements in quality-of-life measures were also maintained during the extension study. Eculizumab continued to be safe and well tolerated, and all patients completed the study. The close relationship between sustained terminal complement inhibition, hemolysis, and symptoms was demonstrated. (Blood. 2005; 106:2559-2565)

Beschreibung: The 2007 IWCLL guidelines indicate that a diagnosis of Chronic Lymphocytic Leukemia (CLL) requires a B-cell count above 5,000/μL in the absence of other features; below this level the diagnosis is Monoclonal B-cell Lymphocytosis (MBL). There is little outcome data for MBL patients and it is not clear whether the detection of low levels of CLL cells, seen in 3% of the general population, is of clinical relevance. We have therefore investigated two hospital populations: the first with normal blood counts and no history of cancer; and the second MBL patients referred for investigation of a current or prior lymphocytosis. Blood samples from 1520 outpatients aged 60–80 with a normal blood count were screened: CLL cells were detected in 78/1520 (5.1%) with a median CLL cell count of 140/μL (range 15 – 1,248). Chromosomal abnormalities were frequently detected in purified CLL-phenotype cells (deletion 13q14 in 15/38, trisomy 12 in 4/22) although poor-risk abnormalities (deletion 11q or 17p) were not detected. The median IgVH mutation was 6.6% (range 0.5 – 13.7%) with 85% of cases showing 〉2% mutation from germline. The IgVH gene usage was heavily biased with a similar profile to mutated CLL. Detection of CLL cells in individuals with a normal count was not associated with increased mortality (estimated yearly rate 6.2% vs. 8.9% for matched controls, P=0.76) or risk of developing CLL as subsequent lymphocyte counts remained normal in all cases. A diagnosis of MBL was established in 309 of 2228 referrals for investigation of lymphocytosis between 1995 and 2000. A cohort of 185 MBL patients was monitored for a median 6.7 years (range 0.2 – 11.8): the presenting B-cell count was a median 3,100/μL (range 30 – 5,000), age 73 years (range 42 – 96); IgVH mutation rate was 7.1% (range 1.3 – 9.3%) with 96% of cases showing 〉2% mutation from germline. Progression to a lymphocyte count above 30,000/μL occurred in 15% of cases (28/185) and chemotherapy for progressive CLL was required in 7% (13/185). The absolute B-cell count was the only independent risk factor for an increasing disease levels. Neither IgVH mutation status nor CD38 expression predicted risk of disease progression or requirement for treatment. During follow-up 33% died: age above 70, hemoglobin concentration below 11 g/dL and T-lymphopenia (CD3+ 2,400μL) had significantly longer survival. Development of progressive disease did not predict overall survival: 7/13 patients requiring therapy remain alive at a median 1.9 years (range 0–8.6 years) after initiation of treatment. The total lymphocyte count had no impact on the risk of disease progression, time to treatment or overall survival. CLL-phenotype cells are genetically equivalent to CLL even when detected in the general population but are not associated with increased mortality or risk of progression to CLL when present below 1,500/μL. MBL patients with higher levels of CLL cells show a steady increase in disease levels over time with 1–2% per year requiring chemotherapy for progressive disease. As such, periodic monitoring is indicated but this should have a minimal impact on lifestyle as MBL patients are often elderly with multiple health issues. MBL is a newly described disorder which is related to CLL in a similar way that MGUS is related to myeloma.

Beschreibung: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by a lack of the terminal complement inhibitor CD59 on erythrocytes that renders these cells susceptible to chronic hemolysis resulting in anemia, fatigue, abdominal pain, kidney dysfunction, pulmonary hypertension, and life-threatening thromboembolism (TE). During pregnancy, hemolysis frequently worsens and the incidence of TE increases, posing a high risk of both fetal and maternal mortality. Eculizumab is a terminal complement inhibitor that reduces hemolysis, transfusion requirements, and TE while improving fatigue and quality of life in patients with PNH. Eculizumab contains a hybrid constant region with components of both IgG2 and IgG4. Whether eculizumab crosses the placenta is not known. The eculizumab clinical trial protocols specified withdrawal of patients who became pregnant during the study. There were 5 reported pregnancies during the PNH clinical trials (n=195) prior to withdrawal from the study. To evaluate the safety and efficacy of eculizumab in the management of PNH during pregnancy, we reviewed the physician-reported adverse events (AEs), PK/PD, and distribution of eculizumab, duration of drug exposure during gestation, complications during and after pregnancy, and general health of the newborn in patients with PNH who became pregnant during the eculizumab clinical trials. Of the 5 reported pregnancies, one patient elected termination and 3 patients withdrew from eculizumab therapy between 4 to 16 weeks of gestation and continued pregnancy through term. There were no apparent complications or AEs related to drug and all 3 patients delivered healthy newborns. One patient was treated with low molecular weight heparin (LMWH) from the time she discontinued eculizumab to three months after delivery. She developed hyperpyrexia and was diagnosed with fever of unknown origin shortly after delivery. She was treated and then discharged. There have been no reported postpartum complications in any of these deliveries to date. The fifth patient withdrew from the study but continued on eculizumab treatment throughout the whole pregnancy and post-partum. She initiated eculizumab treatment in 2002 reducing her LDH from 10,300U/L to 490 U/L (normal range 150 to 480 U/L). Her transfusion requirement reduced from 4 units every 6 weeks to 2 units per year in order to maintain her hemoglobin above 8 g/dl. She was commenced on LMWH at week 8 of gestation and she remained on it for the duration of her pregnancy and postpartum. During pregnancy, she was transfused more frequently to maintain her hemoglobin above 9 g/dl. At week 28 of gestation, she experienced an episode of hemoglobinuria and abdominal pain for 3–4 days prior to her next dose of eculizumab and therefore the dosing interval was adjusted from 14 to 12 days (11 days to avoid weekends) as per the approved 900mg dose. She had no further episodes of breakthrough and her LDH levels were maintained below 450 U/L. The patient was induced at term and delivered a healthy newborn. On the day of delivery, there was no detectable eculizumab in the cord blood (see table). At day 1 and 9 postpartum, there was no detectable eculizumab in breast milk samples. To date, the patient shows no clinical signs of thrombosis or other morbidities typically associated with PNH. The first evaluation of eculizumab treatment from conception to delivery in a patient with PNH treated with eculizumab demonstrated that the drug was tolerated and the pregnancy was successful. There was no evidence of thromboses or other morbidities during the postpartum period. There have been no reported events of congenital anomaly/birth defects in the offspring of any patient with PNH who became pregnant during participation in our clinical studies. Further, there are currently two patients being followed who received eculizumab treatment either during the last trimester (started eculizumab at week 26) or throughout gestation to term. Pregnancy in PNH is associated with an extremely high maternal risk. A review of the 5 clinical trial cases demonstrated that patients receiving eculizumab during pregnancy had no obvious complications through the post-partum period and that a slightly higher dose of eculizumab may be required during pregnancy than in non-pregnant patients with PNH. In addition, eculizumab does not appear to cross the placenta or to be secreted into breast milk. Eculizumab therapy may play a significant role in the management of pregnancy in patients with PNH. Table. Eculizumab levels at delivery Eculizumab Levels (μg/ml)* Post Partum Day 0 Day1 Day 9** NM. Not Measured *, a level of 35 μg/ml or more has been shown to completely block terminal complement activation **An additional dose was given between Day 1 and Day 9 Eculizumab serum levels in mother 116.1 81.3 146 Eculizumab serum levels in cord blood 0 NM NM Eculizumab in breast milk 0 0 0

Beschreibung: The depletion of nitric oxide (NO) by cell-free plasma hemoglobin and arginase during intravascular hemolysis has been implicated in the dysregulation of vasomotor tone and the enhancement of procoagulant and prothrombotic activities. Pulmonary hypertension (PHT), an emerging common complication of hereditary hemolytic anemias, has been mechanistically and epidemiologically linked to intravascular hemolysis and NO depletion. Paroxysmal nocturnal hemoglobinuria (PNH) is a disease characterized by chronic and brisk hemolysis, as well as elevated cell-free plasma hemoglobin. We have previously reported that approximately 50% of PNH patients have PHT as measured by doppler echocardiograpy. Further, the level of N-terminal pro-B-type natriuretic peptide (NT-proBNP) has been generally demonstrated to be a sensitive and specific measure of right-sided cardiac stress due to PHT and has more recently been shown to be a strong predictor of pulmonary hypertension (PHT) and mortality in patients with hemolytic anemias (defined as NT-proBNP ≥ 160 pg/ml). Eculizumab, a terminal complement inhibitor, has been demonstrated to significantly and rapidly reduce hemolysis, thereby providing beneficial effects on regulation of smooth muscle tone and thrombosis in patients with PNH. To evaluate the efficacy of eculizumab in the regulation of cell-free plasma hemoglobin levels, nitric oxide depletion, and subsequent cardiovascular morbidities in patients with PNH. Levels of hemoglobinemia, arginase and nitric oxide depletion were assessed in 73 evaluable eculizumab- and placebo-treated PNH in the phase III randomized, placebo-controlled trial (TRIUMPH). In addition, levels of N-terminal pro-B-type natriuretic peptide (NT-proBNP) were assessed as a measure of PHT, and systolic and diastolic systemic arterial pressures were examined in eculizumab- and placebo-treated patients. At baseline, levels of lactate dehydrogenase (LDH), cell-free plasma hemoglobin, arginase 1 and arginase 1 enzyme activity were highly elevated compared to normal values. Levels of hemolysis and NO consumption were shown to be much greater in PNH (more than 6- and 10-fold, respectively) than in patients with other hemolytic diseases. There were substantial correlations between cell-free plasma hemoglobin levels and both LDH (R = 0.5094) and plasma consumption of nitric oxide (NO) (R = 0.9529). Strong correlations between arginase 1 and both cell-free plasma hemoglobin (R = 0.9367) and arginase 1 enzyme activity (R = 0.9081) were also demonstrated. Following eculizumab therapy, measures of hemolysis were significantly reduced from baseline, including LDH (2200 ± 158 to 327 ± 68 U/L) and cell-free plasma hemoglobin (98.8 ± 23.24 to 15.2 ± 5.05 mg/dL), while levels in placebo-treated patients remained unchanged; a concomitant reduction in NO consumption was also observed (see Figure). In addition, at baseline, 46.6% (34/73) of PNH patients in the TRIUMPH study had levels of NT-proBNP ≥ 160 pg/ml, indicating PHT in these patients. Eculizumab-treated patients showed a 50% reduction in the incidence of PHT over the course of the 26-week treatment period from 52.5% to 26.3%, while PHT did not change with placebo (39.4% to 43.8%; P

Beschreibung: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by clonal expansion of PNH red cells that are highly sensitive to lysis by terminal complement. The primary lesion in PNH is bone marrow failure in the form of immune-mediated aplastic anemia and peripheral blood cytopenias of varying severity. We have previously reported the successful control of hemolysis and transfusion in 11 patients with the terminal complement inhibitor eculizumab. Ten of these 11 patients remain on eculizumab therapy after approximately 3 years with maintained reductions in intravascular hemolysis and transfusion. The effectiveness of eculizumab therapy in these patients is through the protection of the PNH red cell from complement-mediated lysis and the expansion of this cell population. Flow cytometry studies have shown that the percentage of PNH red cells increased significantly from a mean of 36.7% before treatment to 58.4% at week 64 of therapy. Importantly, granulocyte, monocyte and platelet PNH clone sizes were 〉90% before treatment and remained stable for all patients throughout the trial suggesting that the majority of hematopoiesis is derived from PNH stem cells. We hypothesize that the PNH red cell clone should approach the clone size of other myeloid hematopoietic cells in a given patient when hemolysis is prevented by eculizumab therapy as this more accurately depicts PNH stem cell activity. Furthermore, the magnitude and rapidity of response in terms of absolute red cell counts is unknown and could potentially provide important insights into the pathophysiology of PNH. In all patients hemolysis was substantially reduced by 21 days. In 9/11 patients, there was a rapid rise in PNH red cell count with the mean absolute number of PNH red cells increasing from 1.37 x 1012/L before treatment to 1.50 x 1012/L at 2 weeks (P=0.21), 1.74 x 1012/L at 4 weeks (P=0.002), and 2.11 x 1012/L at 12 weeks (P=0.001) of eculizumab treatment. The maximum theoretical red cell response was achieved in a mean of 178 days (range 49 – 419 days). The mean absolute number of PNH red cells increased to 2.37 x 1012/L at maximum response (P=0.001), an increase of 73% (range 36% – 207%). All patients achieved a maximum response prior to 18 months of treatment and clone size was subsequently stable. In 2 patients, despite the effectiveness of eculizumab in resolving hemolysis, there was no change in absolute numbers of PNH red cells pre and post-treatment. This is likely due to a combination of a lower degree of hemolysis and more profound bone marrow insufficiency in these patients. The determination of absolute PNH red cell counts during the first 12 months of eculizumab therapy may identify which patients will become transfusion independent and which patients may benefit from additional growth factor support to boost erythropoiesis. Furthermore, long-term eculizumab therapy appeared to be associated with a stable PNH red cell clone size in this initial clinical study.