Paroxysmal Nocturnal Hemoglobinuria Red Cells Research Articles

Navigating the Complement Pathway to Optimize PNH Treatment with Pegcetacoplan and Other Currently Approved Complement Inhibitors.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare and potentially life-threatening hematologic disorder caused by a somatic mutation in a relevant portion of hematopoietic stem cells. Mutation of the phosphatidylinositol glycan biosynthesis class A (PIGA) gene prevents the expression of cell-surface proteins, including the complement regulatory proteins CD55 and CD59. With decreased or a lack of CD55 and CD59 expression on their membranes, PNH red blood cells become susceptible to complement-mediated hemolysis (symptoms of which include anemia, dysphagia, abdominal pain, and fatigue), leading to thrombosis. State-of-the-art PNH treatments act by inhibiting the dysregulated complement at distinct points in the activation pathway: late at the C5 level (C5 inhibitors, eculizumab, ravulizumab, and crovalimab), centrally at the C3 level (C3/C3b inhibitors and pegcetacoplan), and early at the initiation and amplification of the alternative pathway (factor B inhibitor, iptacopan; factor D inhibitor, danicopan). Through their differing mechanisms of action, these treatments elicit varying profiles of disease control and offer valuable insights into the molecular underpinnings of PNH. This narrative review provides an overview of the mechanisms of action of the six complement inhibitors currently approved for PNH, with a focus on the C3/C3b-targeted therapy, pegcetacoplan.

Ahemolytic PNH: Clinical Features of a Distinct Phenotype of Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem/progenitor cell disorder caused by PIGA mutations. All hematopoietic lineages derived from the affected clone exhibit the PNH phenotype [deficiency of glycosyl phosphatidylinositol linked membrane proteins (GPI-APs)]. Diagnosis is made by flow cytometric analysis of red blood cells (RBC), granulocytes, and monocytes. The percentage of granulocytes and monocytes with absent expression of GPI-APs is a measure of the size of the PNH clone. Typically, the percentage of GPI-AP deficient RBCs is somewhat less than that of granulocytes and monocytes due to selective destruction by complement-mediated intravascular hemolysis, manifested by high LDH, bilirubin, reticulocyte count and low haptoglobin. Herein, we report five patients with large PNH clones based on percentage of GPI-AP deficient neutrophils and monocytes, but with absent or near absent PNH RBCs and no biochemical evidence of hemolysis. We call this unique disease subtype, ahemolytic PNH. Methods: The medical records of PNH patients (174 patients with PNH granulocytes >20%) at the University of Texas Southwestern, Cleveland Clinic Foundation, and National Institutes of Health from 2000 to 2022 were examined. Information pertaining to their clinical, laboratory, and molecular attributes (NGS sequencing) was collected. Five patients with granulocyte clones >50% and RBC clones <5% were identified (Table). Results: Patient 1 is a 63-year-old male with a remote history of non-severe aplastic anemia (AA) diagnosed at 11 years old. The first PNH flow cytometry, obtained at age 61 years, showed 0.02% type II PNH RBC, 0.5% type III PNH RBC, 96% PNH monocyte and 97% PNH granulocytes (Table). There was no biochemical evidence of hemolysis at the time of diagnosis or in the preceding 5 years. Patient 2 is a 50-year-old male who presented with pancytopenia and was diagnosed with acute myeloid leukemia with myelodysplastic-related mutations. Flow cytometry showed 85% PNH monocytes and 59% PNH with no detectable PNH RBCs and no biochemical evidence of hemolysis at diagnosis or in 3 years of follow-up (Table). Patient 3 is a 73-year-old female who presented with pancytopenia and marrow aplasia with unremarkable morphology, and a PNH granulocyte clone of 93.6% with 1.3% type III PNH RBCs and 1.3% and 0.5% type II PNH RBCs, with no evidence of hemolysis (Table). Patient 4 is a 21-year-old female who presented with Budd-Chiari syndrome. Flow cytometry showed 73% PNH granulocytes with 3.6% type III PNH RBCs and 1.8% type II PNH RBCs. There was no biochemical evidence of hemolysis, but because of thrombosis, she was treated complement inhibitor therapy (Table). Patient 5 is a 61-year-old female with history of severe aplastic anemia treated with immunosuppression (IST) and eltrombopag. PNH flow cytometry showed PNH granulocytes of 92.4% and 2.1% PNH type II RBCs 2.1% post IST treatment with no biochemical evidence of hemolysis (Table). Conclusion: Herein, we present five patients with a distinct PNH phenotype that we call ahemolytic PNH. Except for the absence or near absence of PNH erythrocytes, this entity shares clinical and pathophysiological features with classic PNH, including thrombophilia (Budd-Chiari Syndrome, Table). The basis of this phenotype is speculative but may be determined by the genotype of the affected HSPC in which the somatic mutation of PIGA first occurred. According to this hypothesis, somatic mutations other than PIGA skew differentiation toward granulocyte/monocyte lineages. The International PNH Interest Group recognizes the following three categories of PNH: classic PNH, PNH in the setting of another define bone marrow abnormality, and subclinical PNH. Like ahemolytic PNH, subclinical PNH has no biochemical evidence of hemolysis. In this case, however, the absence of hemolysis is due to the small size of the PNH clone (median clone size <1%) as determined by flow cytometry analysis of peripheral blood granulocytes and monocytes. We propose that ahemolytic PNH, defined as patients having less than <5% PNH red cells and >50% PNH granulocytes/monocytes, be recognized as a distinct category of PNH.

Danicopan As Add-on Therapy to Ravulizumab or Eculizumab Versus Placebo in Patients with Paroxysmal Nocturnal Hemoglobinuria and Clinically Significant Extravascular Hemolysis: Phase 3 Long-Term Data

Background C5 inhibitors eculizumab (Ecu) and ravulizumab (Rav) have transformed the natural history of paroxysmal nocturnal hemoglobinuria (PNH). Ecu resulted in patient (pt) survival comparable to that of the general population over 20 y of follow-up under real-world conditions. Overall survival rate at 6 years for pts treated with Rav was 98.4%. Where available, Rav is the standard of care for PNH, inhibiting terminal complement activation to prevent intravascular hemolysis (IVH) and thrombosis. Of pts with PNH treated with Rav/Ecu, 10-20% experience clinically significant extravascular hemolysis (cs-EVH). Efficacy and safety of the first-in-class oral factor D inhibitor danicopan (Dan; ALXN2040) as add-on treatment to Rav or Ecu for pts with PNH and cs-EVH were assessed in a phase 3, randomized, double-blind, placebo (Pbo)-controlled superiority clinical trial (ALPHA, NCT04469465). 12-wk data (double-blind treatment period [TP] 1) showing superiority of Dan vs Pbo on primary and key secondary endpoints were previously reported and represent the final analysis set for the trial. Open-label 24-wk (TP2) and ongoing long-term extension (LTE) data are presented. Methods Pts (≥18 y) with PNH and cs-EVH (hemoglobin [Hgb] ≤9.5 g/dL; absolute reticulocyte count [ARC] ≥120×10 9/L) on Rav/Ecu >6 mos were randomized double-blind 2:1 to Dan or Pbo add-on therapy for 12 wks (TP1). At wk 12, Pbo arm pts switched to Dan (Pbo-Dan) and Dan arm pts continued Dan (Dan-Dan) for another 12 wks (TP2), followed by a 1-y LTE in which all pts received Dan add-on therapy. The initial Dan dose of 150 mg 3 times daily (TID) could be escalated to 200 mg TID based on clinical response at investigator discretion. Primary endpoint was change from baseline (CFB) at wk 12 in Hgb. Other secondary endpoints: proportions of pts with Hgb increase ≥2 g/dL in absence of transfusion and with transfusion avoidance through wk 24; CFB in Hgb at wk 24; and CFB in ARC, lactate dehydrogenase (LDH), and C3 fragment deposition on PNH red blood cells at wks 12 and 24. Safety assessments included treatment-emergent adverse events (TEAEs) and laboratory abnormalities throughout the study. Results As of 20 September 2022, 86 pts were randomized; 60 completed TP2 (Dan n=40; Pbo n=20). Baseline characteristics were similar between arms ( Table). At wk 24 ( Fig), mean Hgb level was maintained in the Dan-Dan arm and increased from wk 12 in Pbo-Dan arm. ARC ( Fig) and other secondary endpoints were maintained in the Dan-Dan arm and improved in Pbo-Dan arm ( Table) at wk 24. The proportion of pts with Hgb increase of ≥2 g/dL in the absence of transfusion was maintained in the Dan-Dan arm and improved in the Pbo-Dan arm from wk 12 (Dan, 59.5%; Pbo, 0%) to wk 24 (Dan-Dan 46.3%; Pbo-Dan, 35.0%). Transfusion avoidance was maintained in the Dan-Dan arm and increased in Dan-Pbo arm from wk 12 (Dan, 83.3%; Pbo, 38.1%) to wk 24 (Dan-Dan 78.0%; Pbo-Dan, 90.0%). Mean LDH levels were maintained from wk 12 to 24 ( Table) and were near normal (<1.5×ULN) in both arms. Transfusions decreased in the Pbo-Dan arm from wk 12 (Pbo; mean [SD], 2.2 [2.3]) through 24 wks (Pbo-Dan; mean [SD], 0.1 [0.5]). The safety analysis included the n=80 pts exposed to Dan during the trial. At wk 24, study drug compliance was 98.8% (10.35) in the Dan-Dan arm and 98.3% (3.95) in Pbo-Dan arm. Escalation to 200 mg TID occurred for 41/57 (71.9%) pts in the Dan-Dan arm and 14/23 (60.9%) in Pbo-Dan arm. There were no deaths, meningococcal infections, or discontinuations due to hemolysis. Through data cut-off, 90% (72/80; 464 events) of pts had ≥1 TEAE after exposure to Dan. Serious AEs related to Dan were reported by 2 pts (gastrointestinal disorders/increased blood bilirubin; headache). 6 events in 4 pts led to withdrawal of study drug. 4 events were reported as breakthrough hemolysis (BTH) based on investigator discretion. Only 1 AE was associated with LDH >2×ULN (actual value 2.2×ULN) and potentially met the BTH definition used in other clinical studies. This AE was related to a complement-amplifying condition, COVID-19. This pt continued the study and BTH was resolved. Conclusions Danicopan as add-on to Rav or Ecu significantly improves Hgb and ARC levels and reduces the need for transfusion by addressing cs-EVH while maintaining control of IVH through 48 wks of treatment. Danicopan demonstrated a favorable benefit-risk profile with no deaths, meningococcal infections, or discontinuations due to hemolysis.

Whole blood transfusion and paroxysmal nocturnal haemoglobinuria meet again: Minor incompatibility, major trouble.

The field of transfusion medicine started out with whole blood transfusion to treat severe anaemia and other deficiencies, and then transitioned to component therapy, largely leaving the practice, and experiences, of whole blood transfusions behind. Currently, the field is circling back and whole blood is gaining ground as an alternative to massive transfusion protocols. Herein we describe a severely anaemic paroxysmal nocturnal haemoglobinuria (PNH) patient initially suspected of suffering from renal haemorrhage, receiving a standard low-titre group O whole blood transfusion during pre-hospital transportation. Following the transfusion, the patient suffered a clinically unmistakable haemolytic transfusion reaction requiring supportive treatment in the intensive care unit. Clinical observations are consistent with an acute haemolytic reaction. The haemolysis was likely due to minor incompatibility between the plasma from the transfused whole blood and the patient's PNH red cells. Recovery was uneventful. This revealed an unappreciated contraindication to minor incompatible whole blood transfusion, and prompted a discussion on the distinction between whole blood and erythrocyte concentrates, the different indications for use and the importance of emphasizing these differences. It also calls attention to patient groups where minor incompatibility can be of major importance.

Lactate dehydrogenase versus haemoglobin: which one is the better marker in paroxysmal nocturnal haemoglobinuria?

Lactate dehydrogenase versus haemoglobin: which one is the better marker in paroxysmal nocturnal haemoglobinuria?

Extravascular Hemolysis Due to C3-Loading in Patients with PNH Treated with Eculizumab: Defining the Clinical Syndrome

IntroductionComplement-mediated intravascular hemolysis in patients with paroxysmal nocturnal hemoglobinuria (PNH) is effectively prevented by the terminal-complement inhibitor eculizumab, reducing symptoms and protecting against the clinical consequences of the disease, such as thrombosis and renal failure. Due to the continued activation of the proximal complement cascade upstream of C5, PNH red cells become opsonized with fragments of C3, leading to extravascular hemolysis and a continued transfusion requirement in a proportion of patients. Lactate dehydrogenase (LDH) is utilised as the main indicator of hemolysis in PNH but is particularly a measure of intravascular rather than extravascular hemolysis and therefore is not necessarily elevated in proportion to transfusion requirement due to C3-loading. We therefore conducted data analysis to determine whether other parameters were more indicative of extravascular hemolysis.MethodsData from patients with PNH treated in the Leeds centre of the UK PNH National Service was reviewed retrospectively. Criteria for inclusion in the analysis were treatment with eculizumab for ≥13 months, a mean platelet count >50 x 109/L and mean absolute neutrophil count >0.5 x 109/L (to exclude patients with significant bone marrow failure). Mean values for hemoglobin, absolute reticulocyte count, LDH (adjusted for upper limit of normal), total bilirubin and the proportion of PNH red cells loaded with C3 were collated covering a 48-month period. C3 loading of red cells was assessed by flow cytometry (Sutherland et al., Curr Protoc Cytom. 2015). Red cell transfusion requirements for the latest 12 months and latest eculizumab dosage were also collected.ResultsOne hundred and forty-one patients were identified for inclusion by the above criteria; 70 males and 71 females. The median hemoglobin level for all patients was 109g/l. Seventy-two percent had a mean hemoglobin below 120g/l despite treatment with eculizumab and all patients had variable amounts of C3 on their red cells demonstrating that some degree of extravascular hemolysis is always seen. During the latest 12 months, 51 (36%) patients received at least one transfusion, with 23 (16%) requiring 3 or more transfusions. Thirty (21%) patients were receiving a higher dose of eculizumab (1200mg or more every 2 weeks) due to either sub-therapeutic eculizumab levels and/or measureable terminal complement levels, assessed by the CH50, at the standard dose of 900mg every two weeks.Patients were stratified by both reticulocyte count and LDH and the means for each parameter calculated as shown in Table 1. Bilirubin, C3 loading, transfusion requirement and requirement for an increased dose of eculizumab all correlated better when stratifying by reticulocytes rather than by LDH.When patients were scored on the severity of C3 loading, bilirubin values and reticulocyte count, those with the worst (highest) joint scores were 3 times more likely to be transfused. 72/141 (51%) patients had all three of the following: reticulocyte count <230 x 109/l, bilirubin <30umol/l and less than 30% of their PNH red cells staining for C3. 55 of these 72 (76%) were not transfused in the last 12 months. In contrast 9 patients had reticulocytes >230, bilirubin ≥30 and C3-loading ≥30% and 7 of these 9 continued to require transfusions.ConclusionReticulocyte count appears to be a better indicator of extravascular hemolysis than LDH, correlating more strongly with raised bilirubin, increased C3 loading of PNH red cells and increased transfusion requirement. Using these parameters patients who are more likely to continue to require transfusion support on eculizumab can be identified. Such an approach may be used to identify patients who are more likely to benefit from inhibition of the complement cascade that could reduce the level of C3-associated extravascular hemolysis. [Display omitted] DisclosuresRichards:Alexion Pharmaceuticals, Inc.: Honoraria. Munir:Alexion Pharmaceuticals, Inc.: Honoraria; Gilled: Honoraria; AbbVie: Honoraria; Janssen: Honoraria; Roche: Honoraria. Griffin:Alexion Pharmaceuticals, Inc.: Honoraria. Arnold:Alexion Pharmaceuticals, Inc.: Honoraria. Hill:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Hillmen:Roche: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding.

Elucidation of Autoinflammatory Mechanism in Pigt-PNH

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder caused by somatic mutations in PIGA gene (PIGA-PNH). PNH red blood cells are susceptible to complement-mediated intravascular hemolysis because of a loss of the glycosylphosphatidyl-inositol (GPI) anchored proteins including complement regulatory proteins. Recently, a PNH patient caused by the PIGT mutations was reported (PIGT-PNH) and had unusual symptoms similar to autoinflammatory syndrome. Whereas PIGA is essential for the first step in the biosynthesis of GPI anchors, PIGT is involved in transfer of GPI to proteins; thus its defect causes accumulation of free GPI, non-protein linked GPI, in affected cells. It is likely that the free GPI accumulated in affected cells together with activated complement contribute to inflammasome activation. Here, we aimed to investigate whether free GPI together with complement activation can induce inflammasome activation in human monocytic cells in PIGT-PNH.We generated THP-1 human monocytic cells in which PIGT or PIGA gene was deleted using the CRISPR/Cas9 system. Each knockout cell was rescued with a corresponding wild-type cDNA. Cells were differentiated into macrophages under RPMI-1640 medium containing PMA (100 ng/ml, 3h) in the presence of 10% fetal bovine serum. For induction of inflammasome activation, both of the priming signal and the stimulating signal are necessary. PMA-differentiated cells were primed for 5hr with Pam3CSK4 (200 ng/ml), which specifically activates the Toll-like receptor 2 (TLR 2), followed by ATP stimulation (5 mM, 1h), or cells were stimulated by acidified serum (21:1 of 0.4M HCl) alone, which activates alternative complement pathway. Heat-inactivated (56°C for 30 min) serum was used as a negative control. ELISA kits were used to measure the interleukin (IL) -1β levels in supernatants according to the manufacturers' instructions. Total RNA was extracted from cultured cells and quantitative Real-Time PCR (qRT-PCR) was performed for mRNA level of IL-1β and NLRP3.Results are expressed as means ± SD of three independent experiments. Priming of PMA-differentiated WT, PIGT deficient (PIGTKO), and PIGA deficient (PIGAKO) THP-1 cells with Pam3CSK4, followed by ATP stimulation induced similar levels of IL-1β secretion (167.9±61.5, 144.3±19.7, 200.3±48.8 pg/1.6x105 cells). In contrast, when cells were stimulated with acidified serum alone, PIGTKO cells significantly enhanced IL-1β secretion compared to WT and PIGAKO cells, in which PIGTKO cells showed about 50- and 2-fold increase, respectively (23.7±2.2, 1221.8±91.6, 568.2±101 pg/1.6x105 cells; P<0.01 PIGTKO compered to PIGAKO). This elevated levels of IL-1β were decreased comparably to WT cells by the treatments with heat-inactivated acidified serum or anti-C5 monoclonal antibody, indicating IL-1β secretion was enhanced due to complement activation in PIGTKO and PIGAKO cells. PIGT and PIGA rescued cells showed similar levels of IL-1β to WT cells. Interestingly, qPCR revealed that PMA treatment alone induced transcription of proIL-1β and NLRP3 and further incubation of PIGTKO cells with acidified serum showed similar elevation in transcription compered to WT and PIGAKO cells.Thus, our data demonstrated that complement activation works as both a priming signal and a stimulating signal for inflammasome activation in PIGAKO and PIGTKO THP-1 cells. The facts that the PIGT-PNH patients, but not PIGA-PNH patients, showed autoinflammatory symptoms and that PIGTKO THP-1 secreted higher level of L-1β than PIGAKO THP-1 cells upon stimulation with acidified serum, indicate that the free GPI accumulated in PIGTKO cells contribute to inflammasome activation together with complement activation. DisclosuresOsato:Alexion Pharmaceuticals: Honoraria, Research Funding. Nishimura:Alexion Pharma GK: Honoraria, Research Funding. Ueda:Alexion Pharma GK: Honoraria, Research Funding. Kinoshita:Alexion Pharmaceuticals: Honoraria. Kanakura:Kyowa Hakko Kirin: Research Funding; Fujimotoseiyaku: Research Funding; Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Toyama Chemical: Research Funding; Bristol Myers: Research Funding; Eisai: Research Funding; Astellas: Research Funding; Pfizer: Research Funding; Nippon Shinyaku: Research Funding; Shionogi: Research Funding; Chugai Pharmaceutical: Research Funding.

BCX9930, a Potent, Selective, Oral Factor D Inhibitor, Demonstrates Proof-of-Concept As Monotherapy in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

BCX9930, a Potent, Selective, Oral Factor D Inhibitor, Demonstrates Proof-of-Concept As Monotherapy in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

Dysphagia with Blood Disorder? an Unusual Presentation for Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired hemolytic disorder of the multipotent myeloid stem cells, due to underexpression of surface complement inhibitory proteins CD55 and CD59, affecting 0.13/100,000 patients per year. Besides anemia and thrombosis, PNH can also cause smooth muscle dystonias owing to various mechanisms in the body. Studies have described smooth muscle dystonias presenting as recurrent abdominal pain, lethargy and erectile dysfunction, but intermittent dysphagia is rare with PNH. We describe a case of dysphagia with negative work up as initial presentation of PNH. 16-year-old female with no significant past medical history presented to the clinic with 1 week of ongoing fatigue and difficulty swallowing. She never had any similar complaints in the past nor in her family. The dysphagia was progressively worsening, initially for solid, then with liquids, accompanied by nausea, vomiting and globus sensation in the middle chest. No prior psychiatric history, binging-purging behavior, chest pain, shortness of breath, heat/cold intolerance, weight changes, bladder or bowel dysfunction. She also denied any recent travels, allergies, transfusions, sick contacts, or over-the-counter medication use. Initial labwork showed Hb 11.2 g/dL, MCV 80 fL, WBC 9 x103 K/uL, and platelet 219,000 K/uL. Metabolic panel, troponin, BNP, ANA, rheumatoid factor, lyme serology, vitamin B12, folate and iron studies were within normal limits. Mentzer index &amp;lt;13, serum protein electrophoresis ruled out Thalassemia. LDH was elevated and Haptoglobin decreased. Direct and indirect Coomb's antibody test were negative. Peripheral smear showed normocytic normochromic anemia with normal morphology of RBCs. Flow cytometry showed lack of CD55 and CD59. Esophagogastroduodenoscopy, done for dysphagia, to rule out luminal obstruction was unremarkable. CT chest with contrast ruled out external compression on esophagus. Videofluoroscopic swallow study revealed segmental, dynamic obstruction to both solids and liquids in the lower third of the esophagus. Dysphagia was attributed to PNH and patient was started on eculizumab. She responded to the medication in 3 weeks, during which outpatient symptomatic therapy was given for her anemia along with soft semisolid diet. In PNH, the RBCs may have varying degree of CD59 and glycosyl phosphatidylinositol (GPI) anchor protein deficiency, with unaffected cells described as type I, partially affected cells called type II, and complete lack of the proteins deemed type III red cells. PNH red cell type III and type II red cells are 15 to 25 times and 3 to 5 times more sensitive to complement-mediated lysis than the type I normal red cells. This variability in the severity of the deficiency, as well as in the proportion of the cell population affected, defines the extent of the clinical manifestations of the disease. Approximately 40% of patients have a combination of types I, II and III PNH cells With hemolysis, after exceeding haptoglobin's binding capacity, the free hemoglobin binds any available nitrous oxide (NO) in the blood. Hemolysis also reduces endogenous NO generation by conversion of l-arginine, which is a component in NO generation, to ornithine, thereby further reducing the systemic availability of NO. NO is endogenous smooth muscle relaxor via activation of cGMP. Decreases in NO levels are hypothesized to cause smooth muscle contraction, manifesting as abdominal pain, erectile dysfunction and rarely, dysphagia in the lower two-third of the esophagus which is predominantly smooth muscle. These symptoms occur more commonly in patients with large PNH clone sizes and, therefore, higher hemolytic rates. In this case study, our focus was to recognize PNH among differentials for new onset dysphagia due to complement-mediated intravascular hemolysis causing gastrointestinal dysmotility. In doing so, invasive investigations may be avoided. Advance studies may be needed to completely understand the causes and correlate between PNH, intravascular hemolysis and dysphagia. Disclosures No relevant conflicts of interest to declare.

A review of the treatment landscape in paroxysmal nocturnal haemoglobinuria: where are we now and where are we going?

Review of current and future treatments for patients with Paroxysmal Nocturnal Haemoglobinuria What is Paroxysmal Nocturnal Haemoglobinuria? Paroxysmal nocturnal haemoglobinuria (PNH) is a very rare disease. It arises from PNH stem cells in the bone marrow. In a normal bone marrow these are inactive; however, if there has been a problem in the bone marrow, the PNH stem cells can expand and make PNH red blood cells, white blood cells and platelets. The problem with these cells is that they lack the cell surface markers that usually protect them. Red blood cells are broken down in the circulation rather than the spleen, which gives rise to PNH symptoms such as abdominal pain, difficulty swallowing, erectile dysfunction and red or black urine (known as haemoglobinuria). The white blood cells and platelets are 'stickier' increasing the risk of blood clots. Previously life expectancy was reduced as there were limited treatment options available. What was the aim of this review? To provide an overview of current and future treatment options for PNH Which treatments are available? • Eculizumab is an treatment given through a vein (intravenous) every week for 5 weeks then every 2 weeks after this, and has been available for 13 years, improving life expectancy to near normal.• Ravulizumab is a newer intravenous treatment similar to eculizumab but is given every 8 weeks instead of every 2 weeks. In clinical studies it was comparable with eculizumab.• Future Treatments - There is new research looking at different methods of treatment delivery, including injections under the skin (subcutaneous) that patients can give themselves, treatments taken by mouth (oral) or a combination of an intravenous and oral treatment for those patients who are not optimally controlled on eculizumab or ravulizumab. What does this mean? PNH is now treatable. For years, the only drug available was eculizumab, but now different targets and drug trials are available. Ravulizumab is currently the only second licenced product available, in USA and Europe, there are other medications active in clinical trials. Why is this important? The benefit for patients, from treatment every 2 weeks to every 8 weeks is likely to be improved further with the development of these new treatments, providing patients with improved disease control and independence.As we move into an era of more patient-friendly treatment options, the PNH community both physicians and patients look forward to new developments as discussed in this article.

Analysis of platelets by flow cytometry in patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

The marked pro-thrombotic tendency in PNH is likely to be at least partly due to the population of platelets derived from the abnormal stem cell clone. However, identification of GPI (−) platelets by flow cytometry can be technically difficult. Here we describe a technique that involves the addition of aspirin immediately after the separation of platelet rich plasma and the use of gel filtration to isolate platelets away from plasma proteins and other blood cells. In a study of 92 analyses of samples from 50 patients, we have demonstrated that the percentage of PNH platelets correlates well with the percentage of PNH granulocytes. We also provide data on several cases where there was an extreme discrepancy between the proportion of PNH granulocytes and red cells; in these cases, the demonstration of abnormal platelets suggests that the patient is likely to be at risk of thrombosis. We believe this test will be potentially useful in the evaluation of samples from such patients and may serve as a tool to investigate the causes of hypercoagulability in PNH.

Eculizumab treatment: stochastic occurrence of C3 binding to individual PNH erythrocytes

BackgroundC5 blockade by eculizumab prevents complement-mediated intravascular hemolysis in paroxysmal nocturnal hemoglobinuria (PNH). However, C3-bound PNH red blood cells (RBCs), arising in almost all treated patients, may undergo extravascular hemolysis reducing clinical benefits. Despite the uniform deficiency of CD55 and of CD59, there are always two distinct populations of PNH RBCs, with (C3+) and without (C3−) C3 binding.MethodsTo investigate this paradox, the phenomenon has been modeled in vitro by incubating RBCs from eculizumab untreated PNH patients with compatible sera containing eculizumab, and by assessing the C3 binding after activation of complement alternative pathway.ResultsWhen RBCs from untreated patients were exposed in vitro to activated complement in the context of C5-blockade, there was the prompt appearance of a distinct C3+ PNH RBC population whose size increased with time and also with the rate of complement activation. Eventually, all PNH RBCs become C3+ to the same extent, without differences between old and young (reticulocytes) PNH RBCs.ConclusionsThis study indicates that the distinct (C3+ and C3−) PNH RBC populations are not intrinsically different; rather, they result from a stochastic all-or-nothing phenomenon linked to the time-dependent cumulative probability of each individual PNH red cell to be exposed to levels of complement activation able to trigger C3 binding. These findings may envision novel approaches to reduce C3 opsonization and the subsequent extravascular hemolysis in PNH patients on eculizumab.

CD59 Deficiency Is Critical for C3 Binding on Red Blood Cells of Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) during Anti-C5 Treatment (eculizumab)

C5-blockade with eculizumab prevents complement-mediated intravascular hemolysis in PNH patients and its clinical consequences. However, a distinct population of PNH red blood cells bound with C3 fragments appears in almost all treated patients. This C3 binding results in extravascular hemolysis that in some patients reduces the clinical benefit from eculizumab. In each PNH patients on eculizumab there are always two distinct populations of PNH red blood cells, one with (C3+) and one without (C3-) C3 binding. This phenomenon is somehow paradoxical since the glycosylphosphatidylinositol (GPI)-linked complement regulators, CD55 and CD59, are uniformly deficient on the surface of PNH red cells.To investigate this phenomenon, we have modeled in vitro the C3 binding in the context of C5 blockade by incubating red blood cells from PNH patients with AB0-matched sera from patients on eculizumab. Complement alternative pathway has been activated by mild acidification (in presence of Mg/EGTA to prevent the activation of complement classical pathway) and C3 binding has been assessed by flow cytometry at serial time points. In these experimental conditions a fraction of PNH red blood cells, similar to what happens in vivo, become promptly C3+ and its size increases with the time: from 9.4±2.7% after 5 minutes to 21.2±9.5% after 24 hours.The membrane defects of PNH cells suggested that the deficiency of CD55, which regulates the formation and accelerates the dissociation of C3 convertases, should be responsible for C3 binding to PNH red blood cells in presence of eculizumab (Parker CJ. Hematology Am Soc Hematol Educ Program. 2011;2011:21-29).In order to verify experimentally this hypothesis we have inactivated CD55 or CD59 on normal red blood cells by using blocking monoclonal antibodies (moAb - listed in the figure legend), and we have tested them in vitro upon activation of complement alternative pathway by mild acidification in presence or absence of C5 blockade.We found that CD55 inactivation on normal red blood cells results neither in hemolysis (without C5 blockade) nor in any C3 binding (with C5 blockade). As expected without C5 blockade CD59-inactivated normal red blood cells undergo hemolysis but, surprisingly, we found that in presence of C5 blockade they become bound with C3 fragments (Figure 1), just as it occurs in vivo in PNH patients on eculizumab. The simultaneous inactivation of both CD55 and CD59 further increased the level of C3 binding. Thus, at variance with the starting hypothesis, the deficiency of CD59, not that of CD55, plays the major role in C3 binding to PNH red cells of patients on eculizumab.Therapeutic C5 blockade in PNH patients has unmasked a novel function of CD59: in addition to prevent MAC formation, it plays a central role also in the regulation of C3 activation on cell surface through molecular mechanisms not elucidated yet. It remains to be established the physiological role, if any, of this novel function of CD59 and whether it play a role in determining the pleomorphic clinical features of the congenital CD59 deficiency. Finally, these findings may lead to investigate innovative approaches to reduce C3 binding and extravascular hemolysis in PNH patients on eculizumab and, in a broader context, to modulate complement activity. [Display omitted] DisclosuresRisitano:Novartis: Research Funding; Alexion Pharmaceuticals: Other: lecture fees, Research Funding; Rapharma: Research Funding; Alnylam: Research Funding.

Analysis of Platelets By Flow Cytometry in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

Paroxysmal Nocturnal Hemoglobinuria (PNH) is characterized by a clonal population of hematopoietic stem cells with an acquired somatic mutation in the PIG-A gene, giving rise to populations of circulating mature cells that are unable to synthesize glycosylphosphatidylinositol (GPI). The disease is most readily diagnosed by flow cytometry analysis of red blood cells, using antibodies specific for the GPI-linked protein CD59, or analysis of granulocytes, using antibodies specific for the GPI-linked protein CD24, along with the FLAER reagent, a fluorescent protein that binds to the GPI structure and which is detected only on the surface of GPI (+) cells. However, other mature blood lineages can be derived from the PNH clone. Notably, thrombosis is a major life threatening complication of PNH and may be triggered by complement activation on platelets that belong to the GPI-negative stem cell clone. The PNH clone size generally predicts thrombosis, but sometimes the proportion of PNH red cells and granulocytes are highly discordant, in which case there might be a role for the determination of the proportion of PNH platelets. Historically, flow cytometry analysis of platelets in patients with PNH has been technically difficult. Here is described a method to do this that avoids technical challenges by using aspirin and sepharose gel filtration of platelets to prevent their activation as well as simultaneous determination of CD59 expression and uptake of the FLAER reagent. Red cells were analyzed based on CD59 expression and granulocytes based on CD24 and FLAER. We analyzed blood samples from 48 patients with PNH and or AA/PNH who provided informed consent, 16 of whom had a prior history of thrombosis. To separate platelet rich plasma (PRP), whole blood collected in EDTA tubes was centrifuged at 200g for 7 minutes at room temperature with the brake turned off. After this step, there was no further centrifugation or vortexing of the platelets. A solution of aspirin was made up immediately prior to use and was added to the PRP at a final concentration of 0.5mMolar. Aspirinated PRP was then loaded on top of a sepharose-2B column using Tyrode's buffer. The platelet-rich turbid drops were collected, to isolate platelets from red cells and coagulation proteins. 50 ul of platelet rich buffer was then incubated with FLAER-Alexa-488 (Pinewood, 1:20 dilution) and CD59-PE (Serotec, 1:10 dilution) in the dark for 30' at room temperature. To prevent doublet events from confounding the analysis, the platelet suspension was diluted 1:200 in Hanks with 0.1% BSA. The sample was passed through a 35 uM Falcon cell strainer, and platelets were identified by forward/side scatter acquired on a log-log scale on a BD Facscan. The median proportion of PNH red cells, granulocytes and platelets was 24%, 86%, 76% respectively in the group without a history of thrombosis and 23% ,82%, and 65% in the group with a history of thrombosis. The proportion of PNH platelets was highly correlated with the proportion of PNH granulocytes (r=0.84). In two patients with almost undetectable PNH red cells and over 90% PNH granulocytes, the proportion of PNH platelets was over 90%; both were on prophylaxis and neither had thrombosis. It is predicted that this technique may be useful for determining thrombosis risk, particularly when the results from the analysis of rbc's and granulocytes are discordant. DisclosuresNo relevant conflicts of interest to declare.

Progress Towards Creation of a Rhesus Macaque Animal Model for PNH Disease Via Crispr/Cas9 Technology to Knock out the PIG-a Gene

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem/progenitor cell (HSPC) disease characterized by severe intravascular hemolysis, bone marrow failure, and propensity to thrombosis, causing early death in untreated patients. PNH has been linked to acquired somatic loss-of-function mutations in the X-linked PIG-A gene in HSPC, with resultant disruption of the first step of the biosynthesis of GPI anchors and loss of cell-surface expression of GPI-linked proteins such as CD55 or CD59. PNH red cells are sensitive to complement-mediated lysis due to loss of these GPI-linked proteins. Even though the molecular mechanism of PNH has been well-characterized, the apparent clonal dominance of PIG-A mutant HSPC over residual unmutated HSPC is still poorly understood. Murine models for PNH generated via conditional knockout of PigA do not recapitulate the hemolysis, thrombotic phenotype, or clonal dominance. We took advantage of the newly-described CRISPR-Cas9 gene editing technology to disrupt the PIG-A gene via non-homologous end joining DNA repair and generate a relevant animal model for PNH, in order to investigate these important pathophysiologic questions in rhesus macaques, a species phylogenetically closely related to humans. We constructed a lentiviral vector expressing GFP, Cas9, and one of a series of five guide RNAs manually designed to target the rhesus macaque PIG-A gene at several sites within exon 2, where most human missense mutations are clustered. Two of these five gRNAs also 100% matched homologous sequences in the human PIG-A gene, while three of the gRNAs had 1-2 mismatches with the human sequence. We characterized the efficiency of each guide at the genotypic level using the SURVEYOR assay for locus disruption and direct sequencing, and at the phenotypic level by flow cytometry for GPI-linked proteins. We used human K562 or rhesus macaque FRhK-4 cell lines for proof of concept in vitro studies. The majority of human K562 cells transduced with vectors expressing guide RNAs targeting human PIG-A sequences in exon 2 gradually lost expression of cell surface CD55 and CD59, approximately 70% of double negative for CD55 and CD59 markers by 3 weeks following transduction, in contrast to K562 cells transduced with control vectors not expressing gRNA. It is notable that of the 5 guides tested in K562 cells, the 2 guides resulting in efficient knockdown of CD55 and CD59 were targeted to regions with 100% homology between the rhesus and the human sequence of PIG-A. The other 3 guides with only 1 or 2 mismatches to the human sequence were very inefficient at gene disruption in human cells, suggesting high fidelity and limited off-target effects The SURVEYOR assay confirmed disruption of the K562 PIG-A gene by these two gRNAs (see Figure). Upon sequencing, we demonstrated a variety of indels consisting of insertions or deletions of 1 to 40 nucleotides at the target site in the PIG-A gene. Analysis for off-target indels is ongoing. Similar experiments were carried out in the FRhK4 rhesus cell line, with the SURVEYOR assay demonstrating gene disruption with all five gRNA targeting sequences, all 100% homologous to the rhesus PIG-A gene targets (see Figure) . In FRhK-4 rhesus cells all the PIG-A gRNAs, knocked down CD55 and CD59 expression. gRNAs #4.1, #6.2 and #9.1 showed higher efficiency than the others, with approximately 78% of double negative cells for CD55 and CD59 markers 3 weeks following transduction. We have validated the lentiCRISPR-/Cas9 approach for use in rhesus cells for PIG-A knockout in vitro, and the guide-RNAs have shown effectiveness and specificity. We are in the process of implementing the technique in primary rhesus macaque CD34+ HSPC prior to proceeding to transplantation of these cells in an autologous rhesus model, allowing investigation of the mechanisms of clonal dominance and thrombosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical and Immunological Characterisation of Coversin, a Novel Small Protein Inhibitor of Complement C5 with Potential As a Therapeutic Agent in PNH and Other Complement Mediated Disorders

▪Coversin is a clinical stage recombinant protein molecule (16.7kDa) derived from a salivary molecule from the Ornithodros moubata tick where it assists the parasite to feed without provoking a host immunological response. It prevents activation of complement C5 and binds leukotriene B4 (LTB4) with high affinity. It is not an antibody and can therefore be manufactured in prokaryotic cells such as E. coli leading to potential savings in manufacturing costs. As its mode of action on C5 is similar to that of the monoclonal antibody eculizumab (Soliris®), it is considered to be a promising alternative therapeutic approach to thrombotic and hemolytic diseases mediated by complement activation including atypical hemolytic uremic syndrome (aHUS), catastrophic anti-phospholipid syndrome (APS) and paroxysmal nocturnal hemoglobinuria (PNH).In order to test this hypothesis, experiments were undertaken to determine whether Coversin could inhibit the hemolysis of PNH red cells in vitro using a modified Ham test combined with visualisation of the results by flow cytometric measurement of CD59 expression. Initial experiments showed maximum inhibition of hemolysis compared to control occurred at a concentration of approximately 10mcg/mL. Further experiments using PNH red blood cells also demonstrated the effectiveness of Coversin in blocking in vitro hemolysis of PNH red cells that had both type III (complete GPI-deficiency) and type II (partial GPI deficiency) red cells and also the deposition and accumulation of C3d on both types of PNH red cells. In a comparative experiment Coversin 10mcg/mL was found to be as effective as eculizumab 50mcg/mL, a molar equivalent dose.As Coversin is a non-humanised xenologous protein a mouse study was performed in order to assess immunogenicity and its potential to induce the formation of neutralising antibodies. Daily repeat subcutaneous doses of Coversin (0.57mg/kg) or vehicle were administered to 5 groups of 6 BALB/c mice and blood samples taken after 7, 14, and 28 days administration with an additional group where samples were taken 14 days following cessation of 28 days dosing. Immunoblots showed that by Day 14 33% of mice had developed low titre IgG antibodies to Coversin. By 28 days this had risen to 75% of mice receiving the active compound. Using CH50 lytic assays the antibodies were shown to be non-neutralising and no animals exhibited clinical signs of allergy or injection site reactions. The group of 6 mice that received vehicle showed no evidence of antibodies at 28 days.A heterozygous C5 mutation leading to a p.Arg885His polymorphism and interfering with the binding of eculizumab was recently reported in Japanese PNH patients and this was associated with a poor response to therapy. This was subsequently found to affect 3.5% of the general Japanese and Han Chinese population. A similar polymorphism has now been shown in a 4 year old Caucasian child with persistent thrombocytopenia following a stem cell transplant for chronic granulomatous disease. Prolonged eculizumab treatment was ineffective leading to discovery of the mutation. Serum from the patient was spiked with ascending concentrations of Coversin and CH50 activity was measured by ELISA. This produced a dose response curve corresponding to that found in serum from normal subjects. Coversin, which binds to a slightly different site on the C5 molecule, seems therefore to be unaffected by the polymorphism and to achieve superior inhibition of C5 activation compared to eculizumab.A Phase I, single ascending dose clinical trial was performed in 24 healthy volunteers. In the top dose cohort total blockade of complement C5 was achieved following subcutaneous injection at the predicted therapeutic loading dose of 0.57mg/kg. Activity remained below 50% for 48 hours suggesting that dosing once a day is feasible at steady state (see figure). The drug was well tolerated and no drug-related side effects were seen. Phase II clinical trials in PNH and other thrombotic micro-angiopathies (TMAs) are planned.Coversin appears to be a promising alternative to eculizumab for patients with PNH and other TMAs including those with polymorphisms which interfere with optimal binding of eculizumab to complement C5. [Display omitted] DisclosuresWeston-Davies:Volution Immuno Pharmaceuticals (VIP) SA: Consultancy. Nunn:Volution Immuno Pharm: Consultancy. Prudo:Volution Immuno Pharmaceuticals (VIP) SA: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Rapidly evolving skin manifestations due to progressive thrombosis in a patient with paroxysmal nocturnal haemoglobinuria resolved with prompt initiation of eculizumab

Funding sources: none. Conflicts of interests: P.M. has sat on advisory boards for Alexion. Dear Editor, Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired clonal disorder of the haematopoietic stem cell. PNH arises from mutations in the PIGA gene, which encodes glycosylphosphatidylinositol (GPI)‐anchored proteins. Consequently, these mutations result in a deficiency of GPI‐anchored proteins in cell membranes.1 This deficiency causes PNH red blood cells to be susceptible to complement‐induced intravascular haemolysis.2 However, PNH is also characterized by a high rate of thrombosis.3 4 Venous and arterial thrombosis can be the first symptom of PNH. PNH‐induced thrombosis often occurs at unusual sites and may progress in days to weeks.5 Moreover, thromboembolic complications and consequences are the leading cause of morbidity and death in PNH.4 The pathogenesis of thrombosis in PNH is complex and not completely elucidated at this time.3 The clinical picture of PNH is highly variable and depends on the PNH clone size, factors of complement activation and whether or not there is an underlying bone marrow disease. Complications of PNH occur spontaneously or can be triggered by bacterial or viral infections, for example by highly contagious parvovirus B19.6 7 Cutaneous manifestations of PNH comprise rapidly evolving purpura, haemorrhagic bullae and ulcerations presumably due to necrosis, secondary to intravascular clotting.8 9 10

Polymorphism of the complement receptor 1 gene correlates with the hematologic response to eculizumab in patients with paroxysmal nocturnal hemoglobinuria

Complement blockade by eculizumab is clinically effective in hemolytic paroxysmal nocturnal hemoglobinuria. However, the response is variable and some patients remain dependent on red blood cell transfusions. In 72 patients with hemolytic paroxysmal nocturnal hemoglobinuria on eculizumab we tested the hypothesis that response may depend on genetic polymorphisms of complement-related genes. We found no correlation between the complement component C3 genotypes and the need for blood transfusions. On the other hand, we found a significant correlation with the HindIII polymorphism of a complement regulatory gene, the complement receptor 1 (CR1) gene. At this locus two co-dominant alleles are known, of which H (common) is associated with high expression, whereas L (rare) is associated with low expression of CR1 on red blood cells. Patients who still needed blood transfusion on eculizumab accounted for 18% of the H/H homozygotes, 33% of the H/L heterozygotes and 68% of the L/L homozygotes (P=0.016). Thus, patients with paroxysmal nocturnal hemoglobinuria who have the L/L genotype are seven times more likely to be sub-optimal responders to eculizumab. Both in vitro and in vivo we found that the CR1 HindIII genotype correlates with the abundance of paroxysmal nocturnal hemoglobinuria red cells that have bound C3, and with the kinetics of C3 binding. These results are consistent with the notion that by affecting C3 binding the CR1 genotype influences the response to eculizumab treatment, and this emerges as a novel example of pharmacogenetics.

Perisurgical induction of eculizumab in a patient with paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement (C')-induced lysis of PNH red blood cells (RBCs), which are deficient in the expression of CD55 and CD59. Surgery is one of the major clinical situations that trigger hemolytic attack and thrombosis in PNH. We describe here a case of 64-year-old man with classic PNH complicated by early-stage gastric cancer requiring distal gastrectomy under general anesthesia. We administered humanized monoclonal anti-C5 antibody (eculizumab; Soliris) for a limited period (600 mg, once a week × four times) perisurgically. Eculizumab effectively inhibited the C' system and the patient underwent a curative distal gastrectomy without significant surgery-triggered hemolytic attack. Although discontinuation of eculizumab induced mild hemolysis 2 weeks after the last administration, it was treated conservatively without thrombotic complication. Limited-term induction of eculizumab could be an option for PNH patients with transient and anticipated high risks, with careful preparation for the discontinuation-related risks afterwards.

CH50 Activity Correlates with Residual Intravascular Hemolysis in Pts with Paroxysmal Nocturnal Hemoglobinuria Treated by Eculizumab

AbstractAbstract 3193 Background:Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by intravascular hemolysis, which is effectively controlled with eculizumab. However, in some cases, unexplained upper limit lactate dehydrogenase levels (LDH), as well as low haptoglobin levels are not unusual under treatment, suggesting residual low-level hemolysis. C3-mediated clearance of PNH red blood cells has been recently suggested in patients (pts) under eculizumab (Risitano Blood 2010). We hypothesized residual hemolysis may also be due to incomplete C5 blockage under treatment. CH50 activitiy (residual functional C5 activity) as well as C3 deposition on PNH red blood cells were assessed among eculizumab-treated pts. We also examined the hypothesis that mutations in complement genes are implicated in residual hemolytic process. Patients and Methods:From October 2010 to February 2012, 22 pts (7 male, median age 42 years range 21 to 72) with hemolytic PNH treated with eculizumab (900 mg intravenously every 14±2 days) were prospectively followed systematically every 2 weeks. Before each Eculizumab infusion, clinical data (abdominal pain, thrombosis events and transfusion requirements) as well as complete blood count, LDH, bilirubin levels and reticulocytes were systematically collected. Concomitantly, CH50 using sensibilized sheep red blood cells, C3d deposition on red blood cells using flow cytometry as well as C3 and C4 circulating levels were also studied. Complete C5 blockage was defined by a CH50 activity ≤10% (as for pts with hereditary complete C5 deficiency). All pts included in the study have at least 6 months follow-up under treatment. Only measurements performed after at least 6 months of treatment were considered. Residual intravascular hemolysis was defined by upper limit LDH level before eculizumab injection. CH50 was analyzed with a longitudinal tobit regression model accounting for repeated sampling and the limit of detection. The model was fit in a Bayesian framework, so no p-values are presented. Results:Before starting eculizumab, 21 pts were transfusion-dependent with a mean of 7 red blood cell (RBC) transfusions per year and 9 pts had a significant PNH-related complication (9 thrombosis and 7 with a previous history of aplastic anemia). During the study period, 6 pts received at least one transfusion (mean of 3 RBC transfusions per year) and 1 patient presented a deep vein thrombosis. All pts were analyzed for CH50 activity (412 samples; median 19 per patient; range, 4 to 31). Overall, CH50 measurements showed substantial variability for most pts (Figure 1). Residual CH50 activity (>10%) was significantly associated with higher LDH levels whereas pts who were still transfused (as well as pts with lower hemoglobin level and higher reticulocytes counts) tended to have higher CH50. Type III PNH red blood cell C3d deposition (assessed in all but 1 patient; 277 samples; median 14 per patient; range, 2 to 21) was found in all pts evaluated during the study period (Fig. 1). However, we did not find any correlation between C3 opsonization and clinical or biological signs of hemolysis. The association between CH50 and higher C3 deposition was weak (on average −0.8% CH50 per 10%) more C3 deposition (95%CI −1.7 to 0.2) (Fig. 1). Those results were confirmed in the subset of 15 patients with pure hemolytic PNH (no history of aplastic anemia). C3 and C4 circulating levels were in the normal range during the study period in all but one patient (pts#15, Fig. 1), who carry a complement factor H (CFH) mutation, leading to a quantitative Factor H deficiency. Systematic screening was positive for 2 other pts (CFH, pts#1; C3 mutation, pts#14) but with no phenotypical consequence. Conclusion:Our results confirm the excellent overall clinical and biological response of hemolytic PNH pts to eculizumab. We found that unblocked CH50 activity, reflecting the residual C5 activity, was significantly associated with residual intravascular hemolysis while the selective C3d deposition of PNH red cells (found in all pts under treatment) was not. We also identified mutations in C3 and factor H genes. Whether incomplete C5 blockade is due to low residual level of eculizumab, complement mutations or polymorphisms, or other mechanisms are under investigation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.