ALBERT

Beschreibung: Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare and complex disease characterized by a severe prothrombotic state caused by a complement system mediated hemolysis. The introduction of the anti-C5 antibody, Eculizumab, has been conducted in many Hematology units worldwide to adopt new diagnostic tools to evaluate new and old PNH patients in order to consider the adequacy of the adoption of this new drug in each case. Magnetic Resonance Imaging (MRI) allows a more adequate and profitable approach in PNH that other radiology techniques used for this purpose. In the last four years Hematology and Radiology units in our Hospital have collaborate in the clinical evaluation of PNH patients performing MRI (cranioencephalic, thoracic and/or abdominal) in acute complications of PNH patients (9 patients) or as a programmed protocoled evaluation previous to consider Eculizumab treatment (14 patients). The protocoled evaluation consists in thoracic and abdominal MRI evaluations in all cases, and cranioencephalic MRI (with independence of the presence of neurological symptoms) in 9 cases. The PNH patients were examined with 1.5 Teslas magnet for cranioencephalic,thoracic and abdominal MRI and with 3.0 Teslas magnet for some cranioencephalic MRI (Achieva Magnets; Philips Healthcare, Best, The Netherlands). Different protocols designed for the study of this pathology, using morphological sequences with different empowerment, functional sequences and angiographic studies after administration of intravenous contrast (gadobutrol) have been used. In the abdominal explorations had been performed calculations of T2 * for the quantification of deposit of iron in liver and kidney. The first group of patients (incidental studies in acute/chronic situations) included Classical and with other bone marrow failure syndrome (BMFS) Parker’s types. The second group (protocoled studies previous consideration of Eculizumab therapy) consisted on 11 Classical Parker’s type patients with active hemolysis (LDH increased 3-13 times over normal levels) and elevated PNH clone (73-99% negative GPI granulocytes by FLAER cytometry); and 3 with BMFS Parker’s type patients (LDH increased 2-6 times over normal levels) with lower PNH clone (43-50% negative GPI granulocytes by FLAER cytometry). Thrombosis was found in four cases, one in the inferior cava and and three arterial (two cerebral and one in descendent aorta). In three patients this finding implied to initiate Eculizumab therapy. Minor ischemic brain changes were displayed by three patients. None of the eighteen patients explored with thoracic MRI, displayed pulmonary hypertension signs despite the elevation of pro-BNP in eight of them. Iron overload in the liver and/or kidneys were very frequent. The finding of a reversal of the normal cortical and medullary intensities on T1 and T2 weighted images of both kidneys was evident in the majority of patients with severe PNH types. Interestingly, one patient with a chronic PNH severe form displayed no renal iron cortical after two years on Eculizumab therapy. This finding was also evident in patients with active hemolysis in the past but with very low PNH clones and clinical remission of the disease. Many other incidental discoveries includes cholelithiasis, splenomegaly, kidney arterial vessel constriction, vascular anomalies, kidney and vesical stones, adrenal adenoma, atheromatosis at different levels, Tornwaldt cyst, hamartoma, hemangiomas and abnormal bone marrow signal. MRI is the best imaging technique to diagnose thrombosis in PNH patients and to control evolution. Moreover, in the cerebrovascular setting allows a more fine and precise diagnosis of the minor pathologic thrombotic changes. MRI is the only imaging technique that permits to evaluate the iron overload that in some PNH cases could be underestimated and needs quelation therapy. In our opinion all new patients with classical severe hemolytic PNH must be evaluated prospectively with MRI. The collaboration of the Radiology team with the Hematologist is fundamental to acquire expertise in this rare disease. Disclosures: Pastrana: Alexion Pharmaceuticals: Speakers Bureau. Ojeda:Alexion Pharmaceuticals: Consultancy, Speakers Bureau.

Beschreibung: Since 1964, a total of 56 patients with Paroxysmal Nocturnal Hemoglobinuria clone (PNH) were evaluated in our Hematology Unit. The PNH was evaluated with Ham’s and/or sucrose tests until 1993, when the firsts cytometric analysis of PNH were performed in our Laboratory with CD55 and CD59 markers on granulocytes mainly, and since 2011 also with the FLAER technique. According with PNH Parker´s Classification, most of the patients were Classical PNH type (28 patients), and the remaining included in the other subsets such as 21 PNH in the setting of another bone marrow failure syndrome (BMFS) and 7 PNH subclinical. Most of the patients (70%) displayed an Aplastic Anemia (AA) before or concomitantly with the diagnosis of PNH, and received immunosuppressant drugs (Steroids with/out antithymocyte globulin & Cyclosporine). In four patients an Allogeneic Hematopoietic Transplantation was performed due to a Severe Aplastic Anemia (2 patients), a Classical Severe PNH (before Eculizumab era) or a Myelodysplastic Syndrome. Another patient received a Liver Transplantation because of advanced Hepatitis C related liver failure. In our PNH series, an unexpected high incidence of cancer has appeared, with 8 patients (14,5%) displaying different hematological or non-hematological cancers in the lasts years:SexAge Diagnosis PNHParker’s ClassificationYear Diagnosis PNHYear Diagnosis CancerPrevious ImmunosuppressionCancerYear Death♂16Classical19692011YesLymphoma2011AA & Liver Tx♀30Classical19732003NonePancreatic2006♂38Classical19741995NoneGastric20122012Pulmonary♂26Classical19892013Yes, SteroidsCerebralAlive♀25Classical19942005YesLymphoma2006AA & Cord-Blood Tx♂40BMFS19951995Yes, SteroidsLiver1995♂75BMFS20112009NoneSeminoma2012*♂56Subclinical20101999Yes, SteroidsProstaticAlive*Dead because bone marrow failure. In our PNH series, cancer reports as one of the most frequent final cause of death, with thrombosis with similar incidence (11 patients of the 56 are dead, with 5 patients dead because thrombosis), although the high incidence and severity of thrombosis episodes in this cohort of patients (20 patients experienced thrombosis with a total of 40 events). As displayed supra, some of the cancers could be attributed to the therapy applied in particular patients: The two secondary lymphomas after organ transplantation could be explained by the immunosuppression employed in these procedures. Only three patients did not received immunosuppressant drugs before cancer diagnosis. This high mortality cancer rate precludes the indiscriminate use of steroids in PNH patients. This result, never reported before in PNH, merits an investigational survey of cancer incidence in PNH patients in the PNH International Registry. Disclosures: Ojeda: Alexion Pharmaceuticals: Consultancy, Speakers Bureau.

Beschreibung: Thrombosis, mainly venous but also arterial, is the leading threat in Paroxysmal Nocturnal Hemoglobinuria (PNH) patients, caused by the continuous hemolysis. It constitutes the first cause of death in all reported series. Since 1964, a total of 56 patients with PNH clone were evaluated in our Hematology Unit. According with PNH Parker´s Classification, most of the patients were Classical type (28 patients), and the remaining included in the other subsets: 21 in the setting of another bone marrow failure syndrome (BMFS) and 7 subclinical. Since November 2007, Eculizumab (an anti-C5 antibody) is employed in the disease to abrogate the hemolysis in our patients. In the last years, sixteen patients have been treated with this drug in our series. PNH patients previously anticoagulated with warfarin because thrombosis, continued on therapy after the addition of Eculizumab. Also, patients with more than 50 % PNH clone (established by Cytometry with FLAER on granulocytes) and platelets 〉50 x109/L received oral anticoagulation. The incidence and localization of thrombotic events in the patients without Eculizumab was as follows:Parker’s ClassificationClassicalBMFSSubclinical28216Thrombosis cases (%)14 (50)4 (20)2 (33)Thrombotic episodes:2675Deep calf642Multiple cerebral ischemic infarcts212 (1 death)Large Cerebrovascular311Budd-Chiari11 (1 death)Portal2 (1 death)Retinal2Cava1Pulmonary thromboembolism2 (1 death)Myocardial infarction3 (1 death)Arterial ischemia2 (1 amputation)Skin ischemic2 (vasculitis, livedo reticularis) After introduction of Eculizumab, sixteen patients have been treated with this drug and active thrombosis resolved in all cases, as was the case of a patient with a large persistent thrombosis in the inferior cava vein despite the isolated anticoagulation therapy. Only one patient on Eculizumab therapy experienced a thrombotic event and suffered a transient ischemic attack with aphasia after a prolonged catheter ablation procedure for an atrial fibrillation. This patient had previous signs of small vessel disease in MR imaging techniques. The episode occurs despite heparin anticoagulation and anticipated additional Eculizumab dose and resolves thereafter. Eculizumab had a clear favourable impact in preventing thrombosis complications in our series of PNH patients. Careful monitoring of LDH levels and shortening the Eculizumab interval doses it is indicated in any chirurgical or invasive procedures in these patients. Disclosures: Pastrana: Alexion Pharmaceuticals: Speakers Bureau. Ojeda:Alexion Pharmaceuticals: Consultancy, Speakers Bureau.

Beschreibung: Introduction.Transfusion-associated Chagas’ disease has been transmitted by labile blood components in endemic countries. In addition, at least 6 cases have occurred in the USA and Canada, due to platelets. In many countries, no test to prevent transfusion-transmission of T. cruzi is in routine use. This study evaluated the efficacy of photochemical treatment (PCT) to inactivate T. cruzi in contaminated platelet components. Methods. Paired platelet components were prepared as follows. 3 pooled ABO matched buffy-coat platelet components (BC-PLT) were combined, and then re-divided into 3 BC-PLT units of approximately 300 mL. Each BC-PLT unit was inoculated with a high dose (5x103/mL) of viable T. cruzi metacyclic trypomastigotes obtained from mice infected with either the G, Tulahuen (T) or Y strains. Five pools of 3 BC-PLT units each were assayed for infectivity before and after PCT with the INTERCEPT system (150 μM amotosalen + 3 J/cm2). Infectivity was determined with 3 different methods by assaying 10 mL samples for parasite viability as follows: 1) in vitro culture to detect viable epimastigotes; 2) 3H-Thymidine incorporation in culture; and 3) in vivo inoculation into Interferon-γ Receptor (IFN-γR) deficient mice, highly susceptible to T. cruzi infection. For all BC-PLT units, 10 mL aliquots of each platelet unit were taken before inoculation as negative controls (NC), after parasite inoculation before PCT as positive controls (PC), and after PCT. In vitro cultures were seeded, observed weekly and parasitemia calculated at 30 days post-inoculation(d.p.i) as parasites/mL (p/mL) by limiting dilution analysis. At 30 d.p.i, cultures were pulsed with 3H-Thymidine for 16 hours, cultures were harvested and counts per minute (c.p.m.) measured. In the in vivo study, mice were inoculated intraperitoneally with BC-PLT (10 mL concentrated to 1 mL) and parasitemia was monitored at 7, 14, 21 and 28 d.p.i. Survival of mice was followed up for more than 2 months post inoculation. Results. After 30 days follow up, no viable parasites were detected in PCT BC-PLT by any of the 3 viability assays, and with any of the 3 T. cruzi strains tested. Only the untreated PC samples showed the presence of the replicative form of the parasite by in vitro cultures derived from the 3 different T. cruzi strains. PC from BC-PLT contaminated with the different strains contained 7812, 550, and 100 parasites/mL for the Y, T and G strains, respectively, by in vitro cultures. PC, but not PCT BC-PLT samples, showed significant incorporation of 3H-Thymidine. In addition, in vivo studies showed that only mice inoculated with PC died as a result of the infection. Moreover, 100% of mice died at 14, 28 and 38 days after inoculation with PC containing the Y, T and G strains, respectively. All mice inoculated with NC and PCT BC-PLT aliquots were alive after more than 2 months post-exposure. Conclusions.The pathogen reduction system using Amotosalen HCl and UVA demonstrated robust efficacy for inactivation of high doses of 3 different strains of T. cruzi . This PCT system offers the potential to make the platelet supply safer in countries with T. cruzi endemicity and in countries with growing immigrant blood donor populations from Latin America and other regions in which T. cruzi is endemic.