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All Japanese patients with paroxysmal nocturnal hemoglobinuria (PNH) treated with eculizumab were enrolled in post-marketing surveillance (PMS) between June 2010 and August 2019 to assess the long-term effectiveness and safety of eculizumab. The reduction in intravascular hemolysis, the change in hemoglobin (Hb) level, and the change in renal function were assessed to determine the effectiveness of eculizumab. The types and frequencies of adverse events (AEs) were assessed to determine its safety. A total of 632 patients were enrolled and the median treatment duration was 3.6 years. Treatment with eculizumab significantly reduced lactate dehydrogenase (LDH) levels and significantly increased Hb levels. These changes were maintained for up to 5 years of treatment. An estimated glomerular filtration rate ≥ 60 ml/min/1.73 m

Small populations of glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells accounting for up to 0.01% of total granulocytes can be accurately detected by a high-sensitivity flow cytometry (FCM) assay established by the Clinical and Laboratory Standards Institute (CLSI method) and have a prognostic value in bone marrow failure (BMF); however, the significance of GPI(-) granulocytes accounting for 0.001-0.009% of granulocytes remains unclear. To clarify this issue, we examined the peripheral blood of 21 BMF patients in whom minor (around 0.01%) populations of GPI(-) granulocytes had been previously detected by a different high-resolution FCM method (OPTIMA method, which defines ≥ 0.003% GPI(-) granulocytes as an abnormal increase) using both the CLSI and OPTIMA methods simultaneously. These two methods detected an "abnormal increase" in GPI(-) granulocytes in 10 patients (48%) and 17 patients (81%), respectively. CLSI detected 0.002-0.005% (median, 0.004%) GPI(-) granulocytes in 7 patients who were deemed positive for PNH-type cells according to the OPTIMA method, which detected 0.003-0.012% (median 0.006%) GPI(-) granulocytes. The clone sizes of GPI(-) cells detected by each assay were positively correlated (r = 0.994, p < 0.001). Of the seven patients who were judged positive for PNH-type cells by OPTIMA alone, five received immunosuppressive therapy, and all of them achieved a partial or complete response. GPI(-) granulocytes detected in BMF patients by the CLSI method should thus be considered significant, even at percentages of < 0.01%.

Complement C5 inhibition is the standard of care (SoC) for patients with paroxysmal nocturnal hemoglobinuria (PNH) with significant clinical symptoms. Constant and complete suppression of the terminal complement pathway and the high serum concentration of C5 pose challenges to drug development that result in IV-only treatment options. Crovalimab, a sequential monoclonal antibody recycling technology antibody was engineered for extended self-administered subcutaneous dosing of small volumes in diseases amenable for C5 inhibition. A 3-part open-label adaptive phase 1/2 trial was conducted to assess safety, pharmacokinetics, pharmacodynamics, and exploratory efficacy in healthy volunteers (part 1), as well as in complement blockade–naive (part 2) and C5 inhibitor–treated (part 3) PNH patients. Twenty-nine patients were included in part 2 (n = 10) and part 3 (n = 19). Crovalimab concentrations exceeded the prespecified 100-µg/mL level and resulted in complete and sustained terminal complement pathway inhibition in treatment-naive and C5 inhibitor–pretreated PNH patients. Hemolytic activity and free C5 levels were suppressed below clinically relevant thresholds (liposome assay

Background: Small populations of paroxysmal nocturnal hemoglobinuria (PNH)-type cells ( Methods: Patients diagnosed with PNH, AA, MDS or indistinguishable BMF without malignant diseases were prospectively recruited to the study between April 1 st, 2016 and December 31 st, 2019 in Japan. Participants were excluded from the study if treated with eculizumab or ravulizumab. Peripheral blood samples were obtained with informed consent and sent to the single laboratory every 12months (mos) for 36 mos. A high-resolution flow cytometry assay known as OPTIMA method (Ann Hematol 97(12):2289-2297) was used to precisely detect a small population of GPI(-) cells, which defines ≥0.003% PNH-type granulocytes (Gran) and ≥0.005% erythrocytes as an abnormal increase. The quality of life (QOL) of the pts was assessed using the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue) and European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) instruments. All other lab data and clinical information were obtained at each participating institute or hospital on sample collection, and were accumulated by the Japan PNH Study Group for analysis. Results: Total 1,985 pts were registered and 1,813 pts were eligible for analysis. Median age was 67 with 50.5% male patients. PNH-type cells were positive in 50.4% (235/466) of AA, 19.7% (70/355) of MDS, 22.3% (61/273) of suspected PNH, and 33.9% (232/685) of undiagnosed BMF. PNH-type cells were increased in nearly 30% of the pts with RCUD, RCMD, MDS-U, and 5q-, but not in RARS, RAEB-1, or RAEB-2. Time-course data of the size of PNH-type cells were available in 651 pts at 12mos and in 210 pts at 36mos. Small ( Conclusion: PNH-type cells were detected exclusively in AA and low-risk MDS, supporting the hypothesis that the increase of PNH-type cells in BMF underpin the benign immune-mediated feature of the disease. The presence of PNH-type cells predicts a better response to IST in BMF, which is consistent with previous reports. Detection of subclinical PNH-type cells was associated with an improvement of QOL scores in multiple items at 36mos. Those small populations of PNH-type cells stayed subclinical in most of the cases, but caution should be exercised in monitoring the sizes as some may evolve into clinical PNH. Figure 1 Figure 1. Disclosures Ueda: Chugai Pharmaceutical: Consultancy, Honoraria, Research Funding; Alexion Pharma: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Yonemura: Alexion Pharma: Honoraria, Research Funding; Chugai Pharmaceutical: Research Funding; Novartis Pharma: Honoraria. Obara: Novartis Pharma: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Alexion Pharma: Honoraria, Research Funding. Ando: Novartis: Honoraria; Kyowa Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Celgene: Honoraria; Astellas Pharma: Honoraria; Takeda Pharmaceutical: Research Funding. Kawaguchi: Alexion Pharma: Honoraria. Nishimura: Alexion Pharma: Consultancy; Chugai Pharmaceutical: Consultancy; Novartis Pharma: Consultancy; Roche: Consultancy; apellis pharmaceuticals: Consultancy; Biocryst: Consultancy; Sanofi: Consultancy. Nakao: Kyowa Kirin: Honoraria; Novartis Pharma: Honoraria; Symbio: Consultancy; Alexion Pharma: Research Funding.

Background Inhibition of complement C5 is the current standard of care for paroxysmal nocturnal hemoglobinuria (PNH). C5 inhibition reduces transfusion requirements and improves anemia and quality of life by ameliorating intravascular hemolysis. However, current therapies require lifelong intravenous infusions at regular intervals, and breakthrough hemolysis may occur due to inadequate C5 inhibition. Crovalimab is a novel anti-human C5 antibody characterized by pH-dependent target binding, enhanced recycling by the neonatal Fc receptor, and high bioavailability, allowing for small-volume (2 × 2 mL) subcutaneous administration every 4 weeks. Part 3 of the Phase I/II COMPOSER study (Sostelly et al. Blood. 2019; Röth et al. Blood. 2020) demonstrated that in patients switched from eculizumab, enhanced crovalimab clearance occurred during the switching phase due to the formation of drug-target-drug complexes, with a risk of temporary loss of complete complement inhibition. Part 4 of COMPOSER tested a dosing regimen optimized to maintain complete complement inhibition in C5 inhibitor-naive patients and those switched from eculizumab to crovalimab. Objectives To evaluate the safety, pharmacokinetic (PK), and pharmacodynamic (PD) effects of an optimized crovalimab regimen. Study Design and Methods Crovalimab is being evaluated in the ongoing Phase I/II COMPOSER study (NCT03157635). Parts 1, 2, and 3 of COMPOSER assessed the PK and safety of crovalimab in healthy volunteers, C5 inhibitor-naive patients, and patients switched from eculizumab, respectively. In Part 4, patients received an intravenous loading series of crovalimab 1000 mg on day 1 followed by 340 mg subcutaneous on days 2, 8, 15, and 22. Maintenance dosing of 680 mg subcutaneous every 4 weeks was started on day 29 (week 5). Plasma concentrations of crovalimab, lactate dehydrogenase (LDH), and free and total C5, as well as complement activity were determined at follow-up visits. The primary endpoints were PK and PD effects of the dosing strategy. Patients were followed up for safety and efficacy, including transfusion avoidance, breakthrough hemolysis events, and hemoglobin stabilization. Results Data for 15 patients (8 naive, 7 switched) treated with the optimized crovalimab dosing strategy in Part 4 are presented here. The data cutoff was January 29, 2020. The PK profiles showed that crovalimab exposure was maintained above the target concentration (100 µg/mL) for complete complement inhibition in all patients, naive and switched. Complete complement inhibition, as measured by liposome immunoassay < 10 U/mL, was achieved immediately following the initial dose and maintained throughout the study treatment period. Consistent with these results, crovalimab-free C5 levels declined rapidly following the initial dose and remained low throughout the follow-up period. As expected based on the target-disposal properties of crovalimab, limited total C5 accumulation was observed in naive patients and a decline was seen in switched patients. Median LDH rapidly declined to ≤ 1.5-fold the upper limit of normal (ULN) in naive patients, remained ≤ 1.5-fold the ULN in switched patients, and remained below this level throughout the observation period in both patient groups (Figure). Crovalimab was well tolerated, and no serious treatment-related adverse events were observed. Conclusions These data showed that the optimized dosing strategy for crovalimab in PNH did not raise any safety concerns and suggest that the strategy results in sustained complete complement inhibition in patients naive to complement inhibitor therapy and those switched from eculizumab. These data support the continued development of crovalimab. Disclosures Peffault De Latour: Alexion Pharmaceuticals Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Apellis: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Sica:F. Hoffmann-La Roche Ltd: Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland., Research Funding. Ramos:F. Hoffmann-La Roche Ltd: Other: All authors received editorial support for this abstract, provided by Scott Battle, PhD, of Health Interactions and funded by F. Hoffmann-La Roche.; Genentech, Inc: Current Employment, Other: Fellowship support. Hernández-Sánchez:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Kim:Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. Kiialainen:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received editorial support for this abstract, provided by Scott Battle, PhD, of Health Interactions and funded by F. Hoffmann-La Roche.. Yoon:F. Hoffmann-La Roche: Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland., Research Funding; Janssen: Consultancy; Novartis: Consultancy, Honoraria; YuhanPharma: Research Funding; Amgen: Consultancy, Honoraria; Kyowahako Kirin: Research Funding. Sreckovic:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Soubret:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Ninomiya:F. Hoffmann-La Roche Ltd: Other: All authors received editorial support for this abstract, provided by Scott Battle, PhD, of Health Interactions and funded by F. Hoffmann-La Roche., Research Funding; Alexion: Honoraria; Chugai: Membership on an entity's Board of Directors or advisory committees. Sostelly:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Panse:F. Hoffmann-La Roche Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Grunenthal: Consultancy, Membership on an entity's Board of Directors or advisory committees; Apellis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees; Alexion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Blueprint Medicines: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Chugai: Speakers Bureau; Pfizer: Speakers Bureau; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees. Buatois:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Schrezenmeier:Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. Paz-Priel:Genentech, Inc: Current Employment; F. Hoffmann-La Roche Ltd: Current equity holder in publicly-traded company, Other: All authors received editorial support for this abstract, provided by Scott Battle, PhD, of Health Interactions and funded by F. Hoffmann-La Roche.. Nishimura:F. Hoffmann-La Roche Ltd: Consultancy, Other: All authors received editorial support for this abstract, provided by Scott Battle, PhD, of Health Interactions and funded by F. Hoffmann-La Roche.; Alexion: Honoraria, Research Funding; Chugai: Consultancy. Röth:Alexion Pharmaceuticals Inc.: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Biocryst: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Apellis: Consultancy, Honoraria. OffLabel Disclosure: Yes. Crovalimab is an anti-C5 monoclonal antibody being evaluated as a therapy for paroxysmal nocturnal hemoglobinuria

Data characterizing the safety and effectiveness of eculizumab in patients with paroxysmal nocturnal hemoglobinuria (PNH) are limited. We describe the safety and effectiveness of eculizumab in PNH patients enrolled in a post-marketing surveillance study. Types and frequencies of observed adverse events were similar to those reported in previous clinical trials and no meningococcal infection was reported. Effectiveness outcomes included the reduction of intravascular hemolysis, the change in hemoglobin (Hb) level, the withdrawal of transfusion and corticosteroids, the change of renal function, and overall survival. The effect of eculizumab on intravascular hemolysis was demonstrated by a reduction in lactate dehydrogenase levels at all measurements after baseline. Significant increases in Hb levels from baseline were also observed after 1 month’s treatment with eculizumab (p < 0.01). Of those who were transfusion-dependent at baseline, the median number of transfusions decreased significantly from 18 to 0 unit/year after 1 year of treatment with eculizumab (p < 0.001). An increase in Hb and a high rate of transfusion independence were observed, especially in patients with platelet count ≥150 × 109/L. Approximately 97 % of patients showed maintenance or improvement of renal function. Overall survival rate was about 90 % (median follow-up 1.9 years). These results suggest an acceptable safety profile and favorable prognosis after eculizumab intervention.

Background Appropriate hemostasis is essential for clear visualization of the neural structures and cleavage planes. It is also essential for avoiding heat-induced injury, minimizing blood loss, and reducing operative time. Objective To determine the role of cerebrospinal fluid (CSF) in platelet-dependent hemostasis during neurosurgery. Methods The amplification of aggregation, activation of integrin αIIbβ3, intrinsic and extrinsic coagulation pathways, and activation of signaling cascades in platelets were evaluated. For comparison, various concentrations of a commercially available artificial CSF solution (aCSF), an artificial CSF solution prepared by the authors, and normal saline (NS) were used. Differences between aCSF and NS in obtaining in vivo hemostasis were assessed by measuring the tail vein bleeding time in C57BL/6N mice. Results Platelet aggregation was directly amplified by the addition of aCSF through increased activation of integrin αIIbβ3, phosphatidylserine exposure, and P-selectin expression. However, the prothrombin time and activated partial thromboplastin time were not primarily related to coagulation activity with the addition of aCSF. Activation of Src kinase was related to platelet activation by aCSF. The elimination of sodium bicarbonate from aCSF and the addition of the selective inhibitor of the HCO3/Cl exchanger, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt, significantly inhibited platelet aggregation. The bleeding time in aCSF-treated mice was significantly shorter than that in NS-treated mice. Conclusion Sodium bicarbonate facilitates hemostasis through the amplification of platelet aggregation function. The existence of CSF and irrigation with aCSF provide better conditions for physiological hemostasis and they have the potential of improving hemostasis by bipolar coagulation or with irrigation during neuroendoscopic procedures.

In the original publication of this article, Tables 2, 3 and 4 were published incorrectly. The corrected tables 2, 3 and 4 are given in the following pages.

Minor populations of glycosylphosphatidylinositol-anchored protein-deficient (GPI[−]) cells in the peripheral blood may have a prognostic value in bone marrow failure (BMF). Our objective is to establish the optimal flow cytometry (FCM) assay that can discriminate GPI(−) populations specific to BMF from those of healthy individuals. To identify a cut-off that discriminates GPI(−) rare cells from GPI(+) cells, we determined a position of the borderline that separates the GPI(−) from GPI(+) cells on a scattergram by testing more than 30 healthy individuals, such that no GPI(−) dot fell into the upper left quadrant where fluorescein-labeled aerolysin (FLAER)−CD11b+ granulocytes and CD55−CD59− glycophorin A+ erythrocytes were positioned. This method allowed us to define ≥ 0.003% CD11b+FLAER− granulocytes and ≥ 0.005% glycophorin A+CD55−CD59− erythrocytes to be specific to BMF patients. Longitudinal cross-validation studies showed minimal (

In paroxysmal nocturnal hemoglobinuria (PNH), various symptoms due to intravascular hemolysis exert a negative impact on patients’ quality of life (QOL). To determine clinical factors related with improvements in QOL in PNH patients treated, we analyzed changes in QOL scales in PNH patients treated with eculizumab based on data collected from post-marketing surveillance in Japan. Summary statistics were obtained using figures from QOL scoring systems and laboratory values, and evaluated by t test. One-year administration of eculizumab improved the most QOL items in comparison with the baseline. In particular, significant improvement of EORTC QLQ-C30 was observed in fatigue, dyspnea, physical function, and global health status. Canonical correlation analysis revealed a high correlation between QOL and laboratory values. Changes in serum lactate dehydrogenase (LDH) and hemoglobin showed strong correlations with QOL improvement. Quality of life improvement was independent of patients’ baseline characteristics of co-occurrence of bone marrow failure (BMF), or the degree of LDH. In this analysis, we found that the degree of QOL improvement was independent of the baseline LDH before eculizumab treatment and of co-occurrence of BMF. Paroxysmal nocturnal hemoglobinuria patients who have not received eculizumab treatment due to mild hemolysis may benefit from eculizumab treatment.

Thromboembolic event is the most important complication of paroxysmal nocturnal hemoglobinuria (PNH) affecting its mortality. The pathophysiological mechanisms underlying thrombosis in PNH have not been fully clarified; multiple factors are likely to be involved. They are likely to be related to the complement activation on/around red blood cells (RBC), white blood cells, and particularly platelets. Intravascular hemolysis also contributes to the mechanisms through the release of free hemoglobin, depletion of nitric oxide (NO), and generation of RBC-derived microparticles (MP). Elevated MP in the plasma which derive from platelets, endothelial cells, and RBC, in total, enhance the prothrombotic states in the circulation. Recent researches have revealed close interactions between coagulation and complement systems, enhancing each other. Continuous activation of complement makes a vicious loop enhancing the coagulation/complement systems interacting with blood cells and endothelial cells in PNH.

Summary. Background: Eltrombopag is an oral, non-peptide thrombopoietin receptor agonist that has shown efficacy and safety in chronic immune thrombocytopenia (ITP). However, ethnic differences in eltrombopag exposure have been reported: area under the curve exposure to eltrombopag was 87% greater among ITP patients of East Asian descent than among ITP patients of non-East Asian ITP descent. Objectives: To evaluate the efficacy and safety of eltrombopag by using, in Japanese ITP patients, lower starting (12.5 mg) and maximum (50 mg) doses of eltrombopag than the standard starting (50 mg) and maximum (75 mg) doses approved in the USA and Europe. Patients: We examined 23 Japanese patients with previously treated chronic ITP with a platelet count of

c-Maf is one of the large Maf (musculoaponeurotic fibrosarcoma) transcription factors that belong to the activated protein-1 super family of basic leucine zipper proteins. Despite its overexpression in hematologic malignancies, the physiologic roles c-Maf plays in normal hematopoiesis have been largely unexplored. On a C57BL/6J background, c-Maf−/− embryos succumbed from severe erythropenia between embryonic day (E) 15 and E18. Flow cytometric analysis of fetal liver cells showed that the mature erythroid compartments were significantly reduced in c-Maf−/− embryos compared with c-Maf+/+ littermates. Interestingly, the CFU assay indicated there was no significant difference between c-Maf+/+ and c-Maf−/− fetal liver cells in erythroid colony counts. This result indicated that impaired definitive erythropoiesis in c-Maf−/− embryos is because of a non–cell-autonomous effect, suggesting a defective erythropoietic microenvironment in the fetal liver. As expected, the number of erythroblasts surrounding the macrophages in erythroblastic islands was significantly reduced in c-Maf−/− embryos. Moreover, decreased expression of VCAM-1 was observed in c-Maf−/− fetal liver macrophages. In conclusion, these results strongly suggest that c-Maf is crucial for definitive erythropoiesis in fetal liver, playing an important role in macrophages that constitute erythroblastic islands.

Thrombosis in paroxysmal nocturnal haemoglobinuria (PNH) has been suggested to be due to several pathophysiological states: a suppressed fibrinolytic system, increased leucocyte-derived tissue factor, complement (C')-mediated damage to platelets and endothelia, or increased platelet- and endothelium-derived microparticles (MPs). Because haemolytic attack is often accompanied by thrombosis in PNH, we studied the role of C'-induced release of MPs in the thrombogenesis of PNH. C' activation induced procoagulant alteration in PNH red blood cells (RBC), when assessed by thrombin generation in the presence of C'-activated PNH RBC, which was abolished by their subsequent treatment with annexin V. Significant amounts of procoagulant MPs, measured by phosphatidylserine-binding prothrombinase activity, were released from PNH RBC in association with the formation of C5b-9, but not significantly before C5b-8. Generation of procoagulant, annexin V-binding, MPs from C'-activated RBC was studied also by flow cytometry. While phorbol 12-myristate 13-acetate, an activator of protein kinase C (PKC), induced the release of MPs from normal RBC as well as PNH RBC, C'-induced release of MPs from PNH RBC was Ca(2+) -independent and not associated with the activation of PKC, calpain or caspase. Procoagulant properties of MPs released from PNH RBC could contribute to the thrombogenesis of PNH.

Summary. Background: The pro-apoptotic BH3-only protein Bim is recognized as a pivotal regulator of apoptosis induced by the depletion of cytokines. In the present study, we examined the role of Bim in megakaryopoiesis. Methods: Megakaryocyte (MK) progenitors obtained from bim knockout (KO) mice were analyzed in vitro for liability to apoptosis after the depletion of cytokines, ability to differentiate into MKs and proliferation/cell cycle progression in response to thrombopoietin (TPO). The production of platelets in vitro was evaluated by assaying the formation of proplatelets in MKs. Megakaryopoiesis in vivo was observed in a mouse model of thrombocytopenia induced by injecting fluorouracil (5-FU). Results: Bim-deficient CD34-/c-kit+/Sca-1+/Lineage- stem cells and MKs were highly resistant to apoptosis induced by cytokine depletion, suggesting that Bim is involved in the apoptotic process in both stem cells and MKs. As bim KO mice exhibited splenomegaly and thrombocytopenia, splenectomized mice were used for experiments in vivo. Platelet recovery after 5-FU-induced thrombocytopenia was significantly delayed in bim KO mice. Corresponding with this, numbers of MKs in the recovery phase bone marrow were significantly reduced in bim KO mice. Culture of c-kit+/Lineage- progenitors with TPO revealed that Bim-deficient cells poorly proliferate and differentiate into CD41+ cells in comparison with wild-type (WT) cells. However, once differentiated into MKs, these cells matured normally. Furthermore, cell cycle analyses demonstrated that transition from the G1 to the S phase was delayed in Bim-deficient stem cells. Conclusions: In the present study, we demonstrated that Bim plays a pivotal role in the regulation of cell cycle progression in hepatopoietic progenitors during megakaryopiesis.

2 patients with acute megakaryoblastic leukaemia (AMKBL) were successfully treated with a combination of aclarubicin hydrochloride (an anthracyline), enocitabine (a derivative of cytosine arabinoside) and 6-mercaptopurine (6-MP) or 6-thioguanine (6-TG). They achieved a complete remission following 1 or 2 courses. They remained well and in complete remission throughout 3 courses of consolidation therapy, a total of 9 weeks. The results of remission induction therapy of AMKBL have been reviewed in the literature. 4 of 7 adult patients, including our cases, treated with 3 drugs, anthracycline, cytosine arabinoside or its derivative and 6-TG or 6-MP, achieved a complete remission. AMKBL may not have so poor a prognosis as previously believed.

To determine whether anti-platelet autoantibodies react with megakaryocytes, as well as with platelets, in immune thrombocytopenia (ITP), 38 ITP patients were studied. They were classified into four groups; anti-platelet glycoprotein Ib-positive (group A, n = 5), anti-platelet glycoprotein II/b/IIIa-positive (group B, n = 2), positive to both antibodies (group C, n = 3), and negative to both antibodies (group D, n = 28). The number and size of megakaryocytes in each group were compared. The number of megakaryocytes in groups A, B, C, and D was 12.8 +/- 8.9, 75.2, 29.1, and 17.0 +/- 21.7/mm2, respectively. The mean cytoplasmic area of megakaryocytes in groups A, B, C, and D was 1001 +/- 26.3, 1621, 1109, and 1311 +/- 235.6/micrograms2, respectively. This finding indicated that, in the presence of anti-platelet glycoprotein Ib, megakaryocytes were not increased in number and were small in size, whereas, in the presence of anti-platelet glycoprotein IIb/IIIa, megakaryocytes were increased in number and in cytoplasmic area. Our study suggested that anti-platelet glycoprotein Ib may impair platelet production by megakaryocytes in ITP.

To elucidate whether caspase activation is involved in megakaryopoiesis, we characterized megakaryocytes (MKs) in vav-bcl-2 transgenic (Tg) mice, in which Bcl-2 is overexpressed in hematopoietic cells. To exclude the effect of splenomegaly in Tg mice on megakaryopoiesis, splenectomy was performed. After splenectomy, basal platelet counts in peripheral blood were not significantly different between Tg and wild-type (WT) mice. However, when experimental thrombocytopenia was induced by injecting 5-fluorouracil into splenectomized mice, overshoot of platelet counts during the recovery phase was hardly observed in Tg mice. Analyses of MK ploidy during the recovery phase showed that MKs less than 16 N ploidy were significantly decreased in Tg mice, suggesting that MK supply from progenitors is impaired. Supporting this, differentiation of CD34-/c-kit+/Sca-1+/Lineage- stem cells into MKs was significantly hampered in Tg mice, whereas megakaryocyte-erythroid progenitors (MEPs) normally differentiated into MKs. It suggests that differentiation into MKs is impaired in Tg mice before the stage of MEP. Furthermore, MK colony formation in WT cells was dose-dependently inhibited in the presence of a caspase inhibitor. Contrary, Bcl-2-overexpressing MKs showed normal ability for in vitro platelet production. We thus believe that caspase activation is involved in the differentiation of progenitors into megakaryocytic lineage but not in platelet production.

Summary Paroxysmal nocturnal haemoglobinuria (PNH) is a haemolytic disease characterized by complement-sensitive red blood cells (RBC). PNH-affected RBC (PNH-RBC) should have a shortened mean lifespan (MLS); however, direct measurement is difficult. We have recently developed a sensitive flow cytometric assay to analyse PNH-affected reticulocytes that may closely correspond to the PNH clone-derived erythropoiesis. Naturally, the CD59-negative populations in reticulocytes were larger than those in whole RBC in PNH. We estimated the MLS of PNH-RBC in six PNH patients from the differences in the ratios of CD59-negative populations between reticulocytes and whole RBC. The MLS of PNH-RBC was calculated using the following formula: W/100 = R × M/[(100 − R) × 120 + R × M], where W, percentage CD59-negative whole RBC; R, percentage CD59-negative reticulocytes; M, MLS (days) of CD59-negative RBC. The MLS of PNH-RBC, estimated as 16–45 days in the PNH patients, showed a weak positive and a weak negative relation with RBCs and percentage reticulocytes, respectively, among the patients. The MLS, in individual patients, altered irrespective of RBC and percentage reticulocytes. The MLS calculated from our methods may be a parameter that evaluates the haemolytic conditions in PNH.

Pregnancy with paroxysmal nocturnal hemoglobinuria (PNH) is associated with significant risk of complications, such as life-threatening thrombosis. Recently, eculizumab has come into clinical use and revolutionized the treatment of PNH. However, clinical information regarding eculizumab use for PNH during pregnancy is limited. The present report describes pregnancies with PNH treated with eculizumab that were registered with the Japan PNH study group and reviews the literature. In case 1, the patient received eculizumab throughout pregnancy and delivered a healthy neonate at term, although breakthrough hemolysis occurred at 20 weeks of gestation. In case 2, the patient discontinued eculizumab before pregnancy and developed preeclampsia at 27 weeks of gestation. She received eculizumab and delivered a preterm, but healthy, neonate by cesarean section. In case 3, the patient received eculizumab from 18 weeks of gestation and delivered a healthy neonate at term without any complications. Reports of 11 pregnant women treated with eculizumab were identified in the literature. Of 14 pregnancies, including our own cases, breakthrough hemolysis and preeclampsia occurred in five and two cases, respectively. There were no thrombotic complications, maternal or neonatal deaths, or fetal structural abnormalities. Thus, eculizumab appears to be safe and effective for managing PNH during pregnancy.

Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder characterized by the deficiency of glycosyl phosphatidylinositol (GPI)-anchored proteins in the affected blood cell membranes. Analysis of blood cells by flow cytometry is useful to identify the affected blood cells with PNH-specific phenotypes. Because PNH-affected red blood cells (RBC) have shortened life-spans in the circulation, ratios of PNH-affected populations analyzed by flow cytometry in whole RBC are lower than those in PNH-affected erythropoiesis. We developed a reticulocyte-gated 2-color flow cytometry of RBC and revealed that the percentages of PNH-affected, CD59--populations in reticulocytes were higher than in whole RBC in patients with PNH. A serial analysis of a patient with PNH who underwent peripheral blood stem cell transplantation confirmed the usefulness of this method to evaluate PNH-affected RBC populations with high sensitivity; ie, the presence of CD59- reticulocytes in the circulation could be a sensitive marker for PNH-affected erythropoiesis.

A 66-year-old Japanese man developed severe anemia and erythroid hypoplasia in bone marrow without any significant underlying disease. The results of an antiglobulin test were strongly positive, and serum erythropoietin (Epo) was high. The patient was diagnosed as having acquired pure red cell aplasia (PRCA) and was treated with steroids. Anemia was subsided by reticulocyte production in parallel with a decrease in the titer of antiglobulin test and the level of Epo. We studied the immunological mechanism directed against erythroid cells in vitro by using the patient's serum. In vitro analysis indicated the presence of an inhibitor of erythroid precursors at onset, and its disappearance at remission, suggesting the presence of inhibitor against erythroid precursors.

CD69 is an activation-related cell surface molecule on human eosinophils. It has been reported that interleukin (IL)-5, but not platelet-activating factor (PAF), can induce CD69 on human eosinophils in vitro. In this study, PAF induced CD69 intensely on eosinophils from patients with hypereosinophilic syndrome (HES), while only weakly on those from normal donors. Because HES eosinophils contain abundant cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase (5-LO), we examined the roles of several enzymes involved in the metabolism of arachidonic acid in the PAF- or IL-5-induced CD69 expression on eosinophils. The CD69 expression induced by PAF and IL-5 on HES eosinophils and that by IL-5 on normal eosinophils were both inhibited by AA861 and MK-886, inhibitors of 5-LO activity. In addition, AACOCF3, a selective cPLA2 inhibitor, inhibited IL-5-induced CD69 expression on normal eosinophils, although it hardly affected either IL-5- or PAF-induced CD69 expression on HES eosinophils. Moreover, PAF alone induced CD69 only weakly on normal eosinophils, but exogenous arachidonic acid remarkably enhanced PAF-induced CD69 expression on them. These findings suggest that IL-5 activates both cPLA2 and 5-LO but PAF activates only 5-LO. It is suggested that 5-LO plays a critical role in the induction of CD69 on eosinophils.

Complement (C')-mediated haemolysis in paroxysmal nocturnal haemoglobinuria (PNH) is mainly due to the deficiency of glycosyl phosphatidylinositol-anchored membrane proteins with C'-regulatory activities CD55 and CD59 in PNH-affected red blood cells (RBCs). Hydrophobic insertion of C5b-7 to RBC membranes, initiating the formation of a membrane attack complex, readily results in lysis of PNH RBCs due to the deficiency of CD59. We studied the significance of the electrostatic interactions between C5b-6 and RBC membranes preceding the insertion of C5b-7. In vitro, C'-mediated lysis of PNH RBCs (assessed by sucrose haemolytic assay) was inhibited by heparin, low-molecular weight heparin (LMWH) or protamine, indicating the significance of the electrostatic interactions between C' components and RBC membranes in the process of C'-mediated haemolysis. Neuraminidase-treated PNH RBCs became resistant to C' activation, suggesting that the sialic acid moieties on RBC membranes are involved in the interactions of RBC with C' components. By using biotin-labelled C7, we demonstrated that LMWH as well as heparin inhibited the insertion of C5b-7 to RBCs, although they did not inhibit the incorporation of C7 into membrane-associated C5b-6. Neither heparin nor LMWH could inhibit the procoagulant alteration of PNH RBC membranes induced by C' activation even at concentrations which inhibited the haemolysis completely. Because LMWH inhibited the C'-mediated lysis of PNH RBCs in vitro at the range which induced a limited prolongation of activated partial thromboplastin time of normal plasma, we consider that LMWH may be useful for both the inhibition of haemolysis and the prevention of thrombosis, which often follow a haemolytic attack in PNH.

The neural cell adhesion molecule, CD56, is expressed on acute myelogenous leukemia (AML) cells in 17-20% of the patients. However, the clinical and biological significance of its expression in AML has not been well analyzed from the standpoint of CD56 expression and its association with differentiation to a natural killer (NK) cell lineage. Here we present a 78-year-old patient with chronic myelomonocytic leukemia (CMML) whose leukemic cells had features of both monocytes and NK cells. We demonstrated that the leukemic cells were positive for CD4, CD56 and interleukin-2 (IL-2) receptor beta chain (CD112) in addition to myelomonocytic markers such as CD33, CD11b and CD11c. These leukemic cells proliferated well in vitro in response to 10-100 U/ml of IL-2, and functionally showed significant cytotoxicity against K562 target cells in a 4-hour (51) Cr release assay. All the above data indicate that these cells possessed at least some of the biological features of NK cells. Accordingly, we speculate that the leukemic cells in this patient may have been derived from a possible common progenitor of monocytes and NK cells.

A 52-year-old male with acute myeloid leukemia developed pseudoaneurysm of the subclavian artery. Pneumonia due to Xanthomonas maltophilia, which was multi-drug resistant, progressed to a lung abscess even under administration of antibiotics. This lung infection contiguous to the left carotid and subclavian arteries was suggested to have caused the pseudoaneurysm of the subclavian artery. The rupture of the aneurysm by penetration to the trachea amounted to about 1,000 ml of bleeding; fortunately the bleeding ceased spontaneously. Nonetheless, an emergency transcatheter coil embolization prevented re-bleeding. Endovascular treatment should be considered especially for aneurysms which develop in patients with underlying diseases.

巨赤芽球はビタミンB12あるいは葉酸の欠乏により造血細胞のDNA合成が障害され発症する.ビタミンB12欠乏性巨赤芽球性貧血には悪性貧血と胃切除(おもに全摘)後に発症するものが多い.診断にはビタミンB12や葉酸の欠乏を明らかにするとともに,欠乏の成因を明らかにすることが重要である.終生の維持療法が必要となることも多く,合併症に注意した臨床観察が求められる.

We examined withdrawal effects of recombinant mouse Tpo (rm-Tpo) on the apoptosis of mature and immature megakaryocytes in in vitro experiments. Apoptotic megakaryocytes were detected by double staining for acetylcholinesterase and by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. When the purified mature megakaryocytes were cultured with or without rm-Tpo, the numbers of viable megakaryocytes, apoptotic megakaryocytes, and megakaryocytes with cytoplasmic processes were not significantly different between the two groups. In contrast, purified immature megakaryocytes underwent apoptosis when rm-Tpo was absent from the culture system. Murine bone marrow cells were cultured with rm-Tpo (50 U/mL) on days 1-7 to generate immature megakaryocytes and subsequently were cultured with different concentrations of rm-Tpo (0-50 U/mL) on days 8-14. The number of viable megakaryocytes was decreased and that of apoptotic megakaryocytes was increased by rm-Tpo in a dose-dependent manner. These results indicated a clear relation between the rm-Tpo level and the apoptosis of immature megakaryocytes.

A 21-year-old man who had anaplastic large cell lymphoma (ALCL) of the null-cell type with multiple bone involvement is reported. On admission, he had symptoms of incomplete paraplegia and urinary and rectal incontinence. Workup studies for staging revealed para-aortic lymph node swellings and multiple bone involvement including skull, ribs, left iliac bone, and thoracic/lumbar spine. Because paraplegia was rapidly progressive, a decompression operation was performed. The biopsy specimen obtained from the lumbar spine revealed sheetlike proliferation of anaplastic large cells. These cells were positive for CD30 (Ki-1), EMA, vimentin, and p80NPM/ALK, and negative for CD3, CD20 (L26), and CD45 (LCA). Epstein-Barr virus-encoded small RNAs were not detectable in these cells. Thus, the patient was diagnosed as having ALCL of the null-cell type. He was treated with several courses of combination chemotherapy, and finally with total body irradiation plus high-dose chemotherapy supported by peripheral blood stem cell transplantation. However, soon after the treatment, the lymphoma cells massively infiltrated his bone marrow. He died of lymphoma 8 months after admission.

We treated three cases of interstitial pneumonitis (IP) in 26 elderly (> or = 65 years old) patients with non-Hodgkin's lymphoma (NHL) who received the same chemotherapeutic protocol including granulocyte colony-stimulating factor (G-CSF) administration. Fortunately, all three patients recovered from IP spontaneously by discontinuation of G-CSF alone or with administration of corticosteroid. Because the duration and extent of neutrophilia induced by G-CSF administration was not different between the cases complicated by IP and those without IP, underlying pulmonary damage is suggested to be more involved than neutrophil count in the development of IP.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, progressive hematopoietic stem cell disorder characterized by chronic complement-mediated hemolysis leading to life-threatening complications and early mortality. Eculizumab, a humanized anti-C5 monoclonal antibody, inhibits terminal complement activation, reduces hemolysis, decreases the risk of thrombosis, and improves renal function and quality of life in PNH patients. The long-term efficacy and safety of eculizumab in Japanese patients were assessed in a 2-year extension to a 12-week, open-label study (AEGIS). Eculizumab treatment led to an immediate and sustained reduction in intravascular hemolysis (P < 0.001) and red blood cell transfusions (P = 0.0016) compared with baseline levels. There were no reports of thromboembolism during eculizumab treatment. The majority of patients had stable (56 %) or improved (41 %) renal function and an improved quality of life (P = 0.015), with sustained reductions in fatigue and dyspnea. Eculizumab was well tolerated; no deaths or serious hemolytic events were reported, and the rate of infections declined over time. There were no significant differences in the response to eculizumab in patients with or without bone marrow dysfunction. These results demonstrate that eculizumab is an effective, well-tolerated long-term treatment for Japanese PNH patients and leads to continued amelioration of some hemolytic complications.

A 30-year-old man with chronic neutrophilic leukemia (CNL) in association with monoclonal gammopathy is presented. Physical examination on admission revealed moderate hepatosplenomegaly. Initial blood count showed neutrophilic leukocytosis (42.2 x 10(9)/1 with 90% mature neutrophils). Leukocyte alkaline phosphatase (LAP) score was elevated. Bone marrow aspiration showed myeloid hyperplasia without dysplastic features. Karyotypic and molecular analyses of bone marrow cells showed the absence of Philadelphia (Ph1) chromosome and bcr gene rearrangement. Because there was no underlying infection or neoplasm, he was diagnosed as having CNL associated with IgG kappa-type monoclonal gammopathy (IgG, 1,269 mg/dl). In addition to its association with monoclonal gammopathy of undetermined significance (MGUS), the present case was also characterized by spontaneous remission of CNL during the 12-year follow-up, accompanied by a gradual increase in serum IgG levels up to 3,000 mg/dl. As far as we know, there have been 19 cases of CNL associated with monoclonal gammopathy in the literature. The median survival of these cases was 5 years. Although there have been only 6 cases of CNL associated with MGUS, survival of these cases was particularly favorable. Taken together with the observation that leukocytosis and hepatosplenomegaly in the present case subsided without specific treatment, we speculate that myeloid proliferation in the present case may have been a leukemoid reaction to underlying monoclonal gammopathy.

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem cell disorder with the expansion of PIGA mutant clone(s), which is deficient in GPI-anchored proteins including CD55 and CD59. The lack of CD55 and CD59 renders PNH-type red blood cells (RBC) susceptible to complement attacks, resulting in intravascular hemolysis. Classic PNH manifests 3 major symptoms: anemia, bone marrow failure, and thrombosis. Small populations of PNH-type cells ( Methods: Patients diagnosed with PNH, AA, MDS or indistinguishable BMF were prospectively recruited to the study since 2011 in Japan. A high-resolution FCM assay was established to precisely detect a small population of PNH-type granulocytes (with FLAER) and RBCs (with anti-CD55 and CD59 antibodies) ≤ 0.01% of the total granulocyte or RBC population based on the Kanazawa method (Blood 2006 107:1308-1314). Six university laboratories across Japan were designated as regional analyzing centers and measured the percentages of PNH-type cells in the study population, as well as collected clinical and laboratory data. Periodic blind cross validation tests using a positive control sample containing 0.01% PNH-type cells and a negative control sample were performed to minimize inter-laboratory variations. Results: As of July 2016, 2,849 patients were enrolled in the study and 2,734 patients were analyzed. Nine hundred twelve patients (33.4%) were positive for PNH-type cells (≥ 0.005% PNH-type erythrocytes and/or ≥ 0.003% PNH-type granulocytes) and 238 (8.7%) patients had more than 1% of PNH-type cells. PNH-type cells were positive in 90/90 PNH (100%), 512/982 AA (52.1%), 132/822 MDS (16.1%), and 141/512 indistinguishable BMF (27.5%) patients. Among the MDS patients, PNH-type cells were positive in approximately 20% of patients with RCUD, RCMD, MDS-U, or 5q- syndrome, but not in any patients with RARS, RAEB-1, and RAEB-2 (Fig. 1). The serum LDH level increased in proportion to the PNH clone size of RBCs, and 63.3% of the patients possessing ≥1.0% RBCs showed LDH levels more than 1.5 times the upper limit of normal. Of 171 patients who completed submission of 3-year follow-up data, BMF patients with PNH-type cells showed a better response rate [CR+PR, 99/107 (92.5%)] to IST compared to those without PNH-type cells [44/64 (68.8%)] (P Conclusions: These interim analyses of the OPTIMA study demonstrate that our high-resolution FCM is reliable in detecting small populations of PNH-type cells and produces consistent results among different laboratories. The presence of PNH-type cells exclusively in patients with AA and low-risk MDS suggests a link between benign pathophysiology of BMF and an increase in the number of PNH-type cells. The better response of PNH-type cell-positive BMF to IST compared to BMF without PNH-type cells was consistent with previous reports. Our data, for the first time, prospectively confirms the significance of small populations of PNH cells in BMF patients in Japan and warrants further worldwide, prospective studies on non-Japanese patients with BMF. Disclosures Ueda: Alexion Pharmaceuticals: Honoraria, Research Funding. Nishimura:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Hosokawa:Aplastic Anemia and MDS International Foundation: Research Funding. Yonemura:Alexion Pharmaceuticals: Research Funding. Obara:Alexion Pharmaceuticals: Honoraria, Research Funding. Shichishima:Alexion Pharmaceuticals, Inc. Japan: Honoraria. Ninomiya:Alexion Pharmaceuticals: Honoraria. Kawaguchi:Alexion Pharmaceuticals: Honoraria. Kanakura:Fujimotoseiyaku: Research Funding; Toyama Chemical: Research Funding; Bristol - Myers: Research Funding; Nippon Shinyaku: Research Funding; Astellas: Research Funding; Eisai: Research Funding; Pfizer: Research Funding; Chugai Pharmaceutical: Research Funding; Shionogi: Research Funding; Kyowa Hakko Kirin: Research Funding; Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding.

The concentrations of interleukin-5 (IL-5), granulocyte-macrophage colony-stimulating factor GM-CSF, and interleukin-3 (IL-3) in serum and in IL-2-stimulated lymphocyte culture medium (L-IL2-CM) prepared from patients with reactive eosinophilia were measured by enzyme-linked immunosorbent assay (ELISA). Serum IL-5 levels were increased in 16 out of 42 cases. GM-CSF and IL-3 were below the detectable levels in all sera examined. The concentrations of IL-5 and GM-CSF in L-IL2-CM were increased in 10 out of 29 patients. IL-3 was below the detectable levels in all L-IL2-CM.

Summary Megakaryopoiesis was examined in 10 patients (eight females and two males) with cyclic thrombocytopenia (CT) to investigate the underlying pathogenesis. Numbers of CFU-Meg and megakaryocytes and the mean cytoplasmic area (mean area) of megakaryocytes at the peak, nadir, ascent mid phase, and descent mid phase of the platelet cycle were determined. The patients were classified as female cases group I (cases 1–4; previously diagnosed as ITP and CT occurred during remission), female cases group II (cases 5–8; persistent CT from initial diagnosis), and male CT (cases 9 and 10). In three of the four female cases in group I, numbers of CFU-Meg and megakaryocytes were normal or increased persistently during the platelet cycle, whereas the mean area fluctuated in synchrony with the platelet cycle, suggesting failure of cyclic production rather than platelet destruction. In the female cases in group II and one female case in group I, numbers of CFU-Meg and megakaryocytes were also normal or increased at four phases of the cycle, but the mean area did not fluctuate, remaining large during the cycle, suggesting cyclic destruction or platelet clearance. In contrast, in the male patients values for numbers of CFU-Meg, megakaryocytes and mean cytoplasmic area fluctuated during the platelet cycle, indicating that cyclic changes in megakaryopoiesis generated the platelet cycle. These findings indicate that the measurement of cytoplasmic area is useful for distinguishing cyclic platelet production from cyclic destruction or clearance in CT.

Eltrombopag is an oral, nonpeptide, thrombopoietin receptor agonist approved for treatment of chronic immune thrombocytopenia (ITP). The safety, tolerability, and efficacy of eltrombopag for up to 3 years were evaluated in 19 Japanese patients with chronic ITP who had completed a prior 6-month study. Patients received eltrombopag once daily at the last dosage received in the prior study (12.5, 25, or 50 mg). Dose adjustments and treatment interruptions were permitted to maintain platelet counts of 50,000–200,000/μL. Primary evaluations were safety and tolerability of long-term eltrombopag treatment. The median duration of exposure was 27.5 months (range, 9.9–32.3). Adverse events were similar to those reported with short-term use of eltrombopag, and none led to treatment discontinuation. Nine serious adverse events were reported. Median platelet counts began to increase after 1 week of treatment and remained above 50,000/μL for most assessments. Bleeding episodes decreased from 63 % at baseline to 21 % after 2 weeks of treatment and remained below baseline for all assessments. Of 15 patients receiving concomitant baseline ITP medications, 10 permanently discontinued or achieved a sustained reduction of at least one treatment without requiring rescue treatment. Long-term treatment with eltrombopag was safe, well tolerated, and effective in Japanese patients with chronic ITP.

Long-term use of the thrombopoietin mimetic romiplostim was examined in Japanese patients with chronic immune thrombocytopenic purpura (ITP) in this open-label extension. The starting dose of romiplostim was the previous trial dose or 3 μg/kg/week, which was titrated up to 10 μg/kg/week to maintain platelet counts between 50 and 200 × 10(9)/L. As of April 2010, 44 patients had enrolled; 71 % women, median age 55.5 years, with five patients discontinuing romiplostim due to patient request (2), administrative decision (2), or not achieving study-defined platelet response (1). Median treatment duration was 100 weeks; median average weekly dose was 3.8 μg/kg. Twenty-eight patients (64 %) self-injected romiplostim. The most frequent adverse events were nasopharyngitis and headache. Nine patients (20 %) had a total of 14 serious adverse events (0.31/100 patient-weeks); of these, only oral hemorrhage was considered treatment related. Fifty hemorrhagic adverse events were reported in 20 patients (46 %) (1.12/100 patient-weeks). Ninety-six percent of patients had a platelet response (doubling of baseline platelet count and platelet count ≥ 50 × 10(9)/L). Of the 25 patients receiving concurrent ITP therapy at baseline, all reduced or discontinued the therapy. Eight patients (18 %) received rescue medications. Administration of up to 3.5 years of romiplostim increased platelet counts and was well tolerated in Japanese patients with chronic ITP.

Hemosiderinuria caused by intravascular hemolysis is a characteristic clinical feature of an acquired hemolytic disorder, paroxysmal nocturnal hemoglobinuria (PNH). We examined the deposition of hemosiderin (iron) in the kidneys of 6 patients with PNH using magnetic resonance imaging (MRI). Three patients with autoimmune hemolytic anemia (AIHA), a hemolytic disorder showing extravascular hemolysis, served as controls. In five of the six patients with PNH, a characteristic T2-weighted MRI of the kidneys, suggesting the deposition of iron (hemosiderin) predominantly in the renal cortex, was obtained. Hemosiderin-deposition was not revealed in the kidneys of any of the patients with AIHA. We conclude that MRI is a sensitive means of detecting hemosiderin deposited in the renal cortex of patients with PNH and that this feature is considerably specific for diseases showing intravascular hemolysis, as represented by PNH.

Paroxysmal nocturnal hemoglobinuria (PNH) is a progressive and life-threatening disease characterized by complement-mediated chronic hemolysis, resulting in serious life-threatening complications and early mortality. Eculizumab, a humanized anti-C5 monoclonal antibody that inhibits terminal complement activation, has been shown to reduce hemolysis in PNH patients. The pivotal open-label, 12-week phase II registration study (AEGIS) was designed to evaluate the efficacy and safety of eculizumab in Japanese patients with PNH. This trial achieved its primary endpoint of reducing intravascular hemolysis with high statistical significance. Twenty-seven of the 29 patients responded to eculizumab treatment, resulting in an 87% reduction in hemolysis (P 0.0001) and subsequent improvement in anemia (P = 0.0003) despite reduction in transfusion requirements (P = 0.006). Fatigue and dyspnea significantly improved within 1-2 weeks of eculizumab treatment and the improvement was independent of changes in hemoglobin. Chronic kidney disease (CKD) was common (66%) and eculizumab treatment improved CKD in 41% of patients at 12 weeks (P 0.001). Elevated thrombotic risk was evident in Japanese PNH patients and eculizumab treatment normalized D: -dimer levels in 45% of patients with elevated D: -dimers at baseline (P 0.001). The AEGIS results demonstrate that eculizumab is effective, safe and well tolerated in Japanese patients with PNH.

Erythroid cells in the marrow express CD71, transferrin receptor, and reticulocytes released from the marrow lose their expression during maturation. The immature reticulocyte fraction, the proportion of reticulocytes with the highest content of RNA, has been determined by hematology analysis. In the present study, we examined CD71 expression on immature reticulocytes by flow cytometry (FCM) in paroxysmal nocturnal hemoglobinuria (PNH) patients with reticulocytosis. We modified 'reticulocyte-gated FCM' to multi-color FCM, i.e. RNA/CD71, RNA-CD59 or CD59/CD71/CD45. In PNH, in addition to the increased number of immature reticulocytes (%CD71-positive), a more immature phenotype in regard to both CD71 intensity and RNA content levels was demonstrated. In seven PNH patients studied, %CD71-positive reticulocytes were significantly increased at 32.2 +/- 11.9% (n = 10, normal 10.4 +/- 3.5%, P = 0.002). RNA content levels (assessed by mean fluorescence index, MFI) in CD71-positive reticulocytes were significantly increased at 812.0 +/- 215.2 MFI in PNH (n = 10, normal 508 +/- 86.1 MFI, P = 0.002). These data indicate that stimulated erythropoietic conditions induce the release of more immature reticulocytes to the peripheral blood than ordinary erythropoietic conditions. CD71 intensity on immature reticulocytes was well correlated with their RNA content levels, indicating the usefulness of CD71 as an immature reticulocyte marker.

We previously reported that long-lasting in vitro hematopoiesis could be achieved using the cells differentiated from primate embryonic stem (ES) cells. Thus, we speculated that hematopoietic stem cells differentiated from ES cells could sustain long-lasting in vitro hematopoiesis. To test this hypothesis, we investigated whether human hematopoietic stem cells could similarly sustain long-lasting in vitro hematopoiesis in the same culture system. Although the results varied between experiments, presumably due to differences in the quality of each hematopoietic stem cell sample, long-lasting in vitro hematopoiesis was observed to last up to nine months. Furthermore, an in vivo analysis in which cultured cells were transplanted into immunodeficient mice indicated that even after several months of culture, hematopoietic stem cells were still present in the cultured cells. To the best of our knowledge, this is the first report to show that human hematopoietic stem cells can survive in vitro for several months.

Acute promyelocytic leukemia (APL) is characterized by chromosomal rearrangements of 17q21, leading to fusion of the gene-encoding retinoic acid receptor alpha (RARA) with a number of alternative partner genes. Signal transducer and activator of transcription 5 beta (STAT5B) is one of the alternative partners. We report a rare case of APL with STAT5B-RARA fusion transcript and the normal chromosome 17 on G-banding. Administration of all trans-retinoic acid improved disseminated intravascular coagulation without decrease of the leukemia cells in his peripheral blood and bone marrow. The molecular mechanism of fusion between STAT5B and RARA by chromosomal rearrangement is discussed based on the data from genome database. Clinical characteristics of APL with STAT5B-RARA are also discussed.

We investigated the complement-susceptibility of paroxysmal nocturnal haemoglobinuria (PNH) lymphocytes in relation to their dysfunction. When assessed by complement-mediated lysis induced by monoclonal antibodies (CD5 or CD20) and rabbit complement, the complement-susceptibility of lymphocytes from patients with PNH, both CD5+ (T cells) and CD20+ (B cells), was greater than that from controls (P less than 0.001). This susceptibility was further enhanced, both in normal controls (P less than 0.01) and in patients with PNH (P less than 0.001), when the activity of decay-accelerating factor (DAF) on the lymphocytes was blocked with anti-DAF monoclonal antibody. DAF amounts in mononuclear cells (MNC) from patients with PNH, measured by an enzyme-linked immunosorbent assay (ELISA), were lower than those of the controls (P less than 0.001). The expressions of DAF on T cells and on B cells from patients with PNH were significantly decreased (P less than 0.05 in T cells: P less than 0.01 in B cells). MNC from patients with PNH responded less to phytohaemagglutinin (PHA) and concanavalin A (Con A) than MNC from the controls (P less than 0.001). In contrast, the responses of PNH MNC to poke weed mitogen (PWM) or lipopolysaccharide (LPS) were not impaired. MNC from a normal donor preincubated with anti-DAF became less responsive to PHA or Con A. We conclude that PNH lymphocytes show enhanced complement-susceptibility and that they are involved in their own expression of DAF as well as in other types of peripheral blood cells. The results of the responses to various lectins suggest the dysfunction of T cells in PNH.

A 41-year-old female patient with mental retardation and generalized epileptic seizure had a nonmosaic idic (X) (pter-q21.32::q21.32-pter) chromosome in peripheral lymphocytes and bone marrow cells. Primary amenorrhea, myelodysplastic syndrome, pigmented nevi and characteristic facial appearance were also observed. A few cases with the nonmosaic idic (X) (q::q) with various breakpoints reported previously commonly showed ovarian failure with dysfunction of relevant hormone. CNS abnormalities of the present case were demonstrated by CT, MRI and SPECT using 123I-IMP. CNS abnormalities were considered to be possibly due to karyotype with a nonmosaic idic (X) (q21.32).