Search

This study investigated for the first time brain ischemic involvement in 19 consecutive neurologically asymptomatic PNH patients by non-enhanced cerebral MRI, and by intracranial arterial and venous angio-MRI. Eleven cases (58%, 7 aged 5 mm, and 5 cases a score >4 by the age-related white matter changes (ARWMC) scale. Compared with age and sex-matched controls (1:2 ratio), patients showed an increased frequency of periventricular WM vascular degeneration (32% versus 5.2%, p = 0.04) and of severe lesions (ARWMC scale score >4) (26% versus 2.6%, p = 0.05), and a higher overall ARWMC scale score (3.5 ± 1.07 versus 2.0 ± 0.8, mean ± SD, p

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.

Objectives To assess the endothelial dysfunction (ED) after bare metal stents (BMS) and sirolimus eluting stents (SES) implantation in the same patient, overcoming the confounding role of individual variables. Background SES reduce restenosis rate compared to BMS but causes more ED. ED is a potentially unsafe phenomenon, since it is the first step in the cascade of atherosclerosis. Studies showing more pronounced ED with drug eluting stents than BMS involved different series of patients, making the comparison difficult because endothelial function (EF) is responsive to many risk factors. Methods we designed a prospective comparison of 6 months post-deployment EF of SES versus BMS implanted in the same patient, but in different coronary segments. Forty-eight lesions were randomly assigned on a 1:1 allocation using block sizing of 4 according to a computer-generated sequence (SAS System, Version 9.1) basis to treatment with SES or BMS. The EF was evaluated by measuring vessel diameter variation in the stented segment, before and after selective intracoronary infusion of acetylcholine (iiAch). Results In eligible patients, the relative magnitudes of major vasoconstriction were 2.6, 2.9, 4.6, and 3.1 at 5 mm proximal and 5, 10 and 20 mm distal to the stent edge. Overall, a 3.5-fold major distal vasoconstriction after iiAch of SES vs. BMS was calculated. Conclusions in the same patients, but treating different coronary segments, SES implantation induces a higher rate of vasoconstriction compared to BMS. The increased vasoconstriction after iiAch is an indicator of ED.© 2013 Wiley Periodicals, Inc.

BACKGROUND: Febrile nonhemolytic transfusion reactions occur in 0.12% of transfusions, usually during transfusion or within 4 to 6 hours after transfusion and are not medically dangerous. CASE REPORT: A patient with thalassemia from Togo with asymptomatic malaria in which the infection became clinically manifest only after blood transfusion, mimicking a febrile nonhemolytic transfusion reaction, is presented. Thirty-two hours after transfusion of 2 O D− red blood cell (RBC) units, the patient (phenotype A2 D+) developed fever and multiorgan failure and was admitted to the intensive care unit. Direct and indirect antiglobulin tests were negative on posttransfusion samples. Blood cultures and infectious diseases testing were negative. No malaria parasites were found at thick blood smear microscopic examination on Days 1 and 2 and the malaria rapid diagnostic test gave inconsistent results. Plasmodium total antibodies were detected in the serum at high levels. On Day 5, routine microscopic examination of blood smear revealed the presence of parasites in a very small number of RBCs. This finding was almost simultaneous to the availability of polymerase chain reaction testing results that were positive for P. falciparum. The sequential agglutination with anti-A antiserum allowed patient's and donors' RBCs to be separated and revealed that the parasitized cells were almost exclusively those of donors (14.4% vs. 0.029%). Malaria infection in implicated donors was excluded. CONCLUSION: In this patient with thalassemia with asymptomatic malaria, the infusion of two normal RBC units provided a favorable environment for a rapid parasite replication leading to a dramatic acute malaria attack.

ObjectivesThe purpose of this analysis was to assess 5-year outcomes of transcatheter aortic valve implantation (TAVI) using the current technology of the self-expanding CoreValve prosthesis (Medtronic Inc., Minneapolis, Minnesota).BackgroundThere is a paucity of evidence on long-term durability of currently available transcatheter heart valves.MethodsStarting in June 2007, all consecutive patients with severe aortic stenosis undergoing TAVI with the third-generation 18-F CoreValve device in 8 Italian centers were prospectively included in the ClinicalService Project. For the purposes of this study, we included only consecutive patients with 5-year follow-up data available (n = 353) treated from June 2007 to August 2009. All outcomes were reported according to VARC (Valve Academic Research Consortium)-1 criteria.ResultsAll-cause mortality rates at 1, 2, 3, 4, and 5 years were 21%, 29%, 38%, 48%, and 55.0%, respectively. Cardiovascular mortality rates at 1, 2, 3, 4, and 5 years were 10%, 14%, 19%, 23%, and 28.0%, respectively. The overall neurological event rate at 5 years was 7.5%, of which more than two-thirds occurred early after the procedure. During follow-up, there were 241 rehospitalizations for cardiovascular reasons in 164 (46%) patients. Among all rehospitalizations, acute heart failure was the most frequently reported (42.7%), followed by requirement of permanent pacemaker implantation (17.4%). On echocardiography, mean transaortic gradients decreased from 55.6 ± 16.8 mm Hg (pre-TAVI) to 12.8 ± 10.9 mm Hg (5-year post-TAVI) (p < 0.001). Late prosthesis failure occurred in 5 cases (1.4%); among these, redo TAVI was successfully carried out in 2 patients (0.6%) presenting with symptomatic prosthesis restenosis. The remaining 3 cases of prosthesis failure did not undergo further invasive interventions. Ten patients (2.8%) showed late mild stenosis with a mean transaortic gradient ranging from 20 to 40 mm Hg. No other cases of structural or nonstructural valvular deterioration were observed. Valve thrombosis or late valve embolization were not reported.ConclusionsTAVI with the currently adopted CoreValve generation was associated with sustained clinical outcomes up to 5-year follow-up, with a low rate (1.4%) of significant prosthetic valve degeneration. The procedure appears to be an adequate and lasting resolution of aortic stenosis in selected high-risk patients.

Although transcatheter aortic valve implantation (TAVI) is still currently guided by transesophageal echocardiography (TEE) in a considerable number of hospitals, exclusive angiographic (Angio) guidance seems a reasonable approach in this setting. To date, however, no studies have directly compared the outcomes of TAVI according to the imaging modality used for procedural guidance. We, therefore, used data from a large multicenter data repository to compare the outcomes of TAVI guided exclusively by Angio and ATEE. All consecutive patients with severe aortic stenosis who underwent TAVI with the CoreValve Revalving System (CRS) in 9 Italian centers from September 2007 to March 2014, dichotomized according to the imaging support used to guide the procedure (ATEE and Angio), were included. Thirty-day and 12-month clinical outcomes were evaluated. Propensity matching analysis was performed to adjust for baseline differences. A total of 625 patients were included (256 and 369 patients were included in the ATEE and Angio groups, respectively). Patients from the ATEE more frequently underwent TAVI under general anesthesia compared with Angio group (37.9% vs 22.8%, respectively, p0.001). Importantly, ∼80% of the patients experienced mild or even less aortic regurgitation as assessed by angiography after the procedure, without between-group differences. Postdilation and valve-in-valve rates were equivalent (24.7% vs 25%, p = 0.934 and 5.5% vs 3.4%, respectively, p = 0.217). No differences were revealed in the rates of death, cardiovascular death, and stroke or transient ischemic attack at 12-month follow-up. These results were sustained after propensity matching analysis. In conclusion, as long as a comprehensive procedural planning is performed, TAVI with CRS may be performed exclusively under angiographic guidance without the need for associated TEE.

The clinical outcome, response to treatment, and occurrence of acute complications were retrospectively investigated in 308 primary autoimmune hemolytic anemia (AIHA) cases and correlated with serological characteristics and severity of anemia at onset. Patients had been followed up for a median of 33 months (range 12-372); 60% were warm AIHA, 27% cold hemagglutinin disease, 8% mixed, and 5% atypical (mostly direct antiglobulin test negative). The latter 2 categories more frequently showed a severe onset (hemoglobin [Hb] levels ≤6 g/dL) along with reticulocytopenia. The majority of warm AIHA patients received first-line steroid therapy only, whereas patients with mixed and atypical forms were more frequently treated with 2 or more therapy lines, including splenectomy, immunosuppressants, and rituximab. The cumulative incidence of relapse was increased in more severe cases (hazard ratio 3.08; 95% confidence interval, 1.44-6.57 for Hb ≤6 g/dL; P < .001). Thrombotic events were associated with Hb levels ≤6 g/dL at onset, intravascular hemolysis, and previous splenectomy. Predictors of a fatal outcome were severe infections, particularly in splenectomized cases, acute renal failure, Evans syndrome, and multitreatment (4 or more lines). The identification of severe and potentially fatal AIHA in a largely heterogeneous disease requires particular experienced attention by clinicians.

We reviewed clinical and molecular data of 23 consecutive unrelated patients affected by paroxysmal nocturnal hemoglobinuria (PNH) (19 with hemolytic PNH, 3 with aplastic anemia/PNH, and 1 with myelodysplasia/PNH syndrome) with a mean follow-up of 11.8 years. Five patients had thrombotic episodes, and 10 needed regular blood transfusions; 2 died for cerebral hemorrhage and kidney failure, and 2 spontaneously recovered from PNH. Twenty different PIG-A gene mutations were detected in 21/23 patients: 15 frameshift, 1 splicing, 2 nonsense, and 2 missense mutations. Two mutations (DelG341 and IVS2 +1g-a) were detected twice. A PIG-A mutated clone was also revealed in the two patients in complete clinical remission. One patient with aplastic anemia/PNH syndrome was treated with two courses of antilymphocyte globulin and cyclosporin with partial sustained response. Six patients were given rHu-EPO 150 U/kg/day s.c. for at least 6 months: one became transfusion-independent for 8 months and then discontinued treatment for clinical complications; one displayed a mean rise of Hb of 1.5 g/dL and is currently maintaining Hb levels higher than 9 g/dL after 54 months of therapy. Mutation specific quantitative-competitive PCR showed that the rise of hemoglobin was related to an increase of PIG-A negative molecules, suggesting that the efficacy of rHu-EPO therapy may be due to the stimulation of the abnormal clone.

The spontaneous development of a factor VIII (FVIII) inhibitor is a rare occurrence that has an approximate incidence of one per million of population per year [1]. It most commonly arises in elder...

ABSTRACT We evaluated the iron status and searched for mutations C282Y and H63D in the hereditary hemochromatosis gene ( HFE ) in 34 pyruvate kinase (PK)-deficient patients from 29 unrelated families. Nine had received multiple transfusions. Thirteen of the 25 nontransfused patients displayed increased serum ferritin concentration, in the absence of conditions known to raise this parameter. HFE genotype was abnormal in 9 of 34 patients. The allele frequency was 1.8% for mutation 845G→ (C282Y) and 16.1% for mutation 187C→:G (H63D). Nontransfused subjects with abnormal genotype had serum ferritin and transferrin saturation values significantly higher than those with wild-type genotype. Of the 12 adult nontransfused patients with increased iron status parameters, 1 was C282Y homozygous, 1 compound heterozygous for C282Y and H63D, 3 H63D heterozygous, and 7 had a normal HFE genotype. Serum ferritin and transferrin saturation were not related to hemoglobin, reticulocytes, and bilirubin concentration. At multivariate analysis serum ferritin was independently associated with age and gender, but not with splenectomy and HFE genotypes. The retrospective evaluation of the iron status profile of 10 patients (3 with abnormal and 7 with wild-type HFE genotype) with at least 10 years follow-up showed that overt iron accumulation requiring iron chelation had occurred only in the 3 patients (2 of whom were splenectomized) with the mutated HFE gene.

We studied the PK-LR gene in 16 unrelated patients with congenital haemolytic anaemia associated with erythrocyte pyruvate kinase deficiency. Fifteen different mutations were detected among the 28 mutated alleles identified: two deletions (del 1010G, del 1042–1044); one four nucleotide duplication (nt 1515–1518, GGTC); one splice site [IVS6(−2)t]; nine missense (991A, 1003A, 1151T, 1160G, 1181T, 1181A, 1456T, 1483A, 1529A); and two nonsense (721T, 1675T) mutations. Eight of them [del 1010G, del 1042–1044, dupl 1515–1518, IVS6(−2)t, 1003A, 1160G, 1181T, 1181A] were novel. The deletion 1042–1044 causes the loss of Lys 348. Deletion 1010G and duplication 1515–1518 determine a frameshift and the creation of a stop codon at nucleotides 1019 and 1554 respectively. Mutation IVS6(−2)t leads to an alteration of the 5′ and 3′ splice site consensus sequence; the cDNA analysis shows a 67-bp deletion in the first part of exon 11 (del 1437–1503). All the four new missense mutations involve highly conserved amino acids. The most frequent mutation in Italy would appear to be 1456T. Correlation was made between mutations, biochemical characteristics of the enzyme and clinical course of the disease.

PNH is a rare disorder characterized by hemolytic anemia, marrow failure and thrombosis, due to a deficiency in GPI-anchored proteins. Thrombotic events in PNH are commonly described in hepatic, portal, mesenteric, splenic, and renal veins, along with anecdotic case reports of cerebral venous sinus thrombosis and arterial ischemic strokes. This study was aimed at investigate brain involvement in 19 asymptomatic PNH patients by non-enhanced cerebral magnetic resonance imaging (MRI), and by intracranial arterial and venous angio-MRI. Neuroradiological findings were completed with a neuro-psychiatric evaluation, and correlated with clinical/hematologic features and therapy. Seventeen out of 19 patients were classical hemolytic (63% transfusion dependent before treatment with eculizumab and 1 patient also after), and 2 PNH in the context of aplastic anemia (all transfusion-dependent until treatment with ATG-CyA). Median age at diagnosis was 44 years (range 17-80); asthenia and dyspnea on exertion were present in all patients, abdominal pain in 8, and a thrombotic event in 4. Median Hb was 9.6 g/dL (range 6.7-12.9), LDH 3.7-fold (range 1.2-16.3) over upper limit of normal (ULN), and 73% of patients displayed a clone size greater than 50% GPI negative cells; 10/19 of patients were on eculizumab at the moment of the study. On MRI, 11 subjects showed pathological findings: 9 cases displayed white matter (WM) abnormalities related to chronic ischemic small vessel disease, of whom 6 periventricular WM vascular degeneration, and 8 deep WM focal chronic ischemic lesions (5 cases have both sites involved). Moreover, 1 subject showed a focal abnormality >5 mm and 5 subjects a score > 4 as evaluated in WM and basal ganglia by the age related white matter changes (ARWMC) scale. No subject displayed active or previous bleeding, nor were focal alterations of the basal nuclei by the ARWMC scale found. Two patients (80 and 81 yrs) showed atrophy of the cerebral hemispheres. Regarding vascular abnormalities, one subject had hypoplastic left transverse sinus with irregularities in the sinus wall, suspected for prior partial venous thrombosis. Three further cases displayed hypoplastic transverse sinus associated with collateral draining cortical veins, indistinguishable from anatomical variants. Intracranial artery stenosis or aneurysm, and Moya-Moya like alterations were not observed. Finally, cerebral MRI was unremarkable in 8/19 subjects. By comparing patients with or without any MRI abnormality (WM and vascular alterations), mean age was significantly higher in the former (60+17 vs 43+8 yrs, mean+SD, p Neurological examination was unremarkable in all patients but one, who complained of progressive rest tremor in his left arm and leg (twelve years after PNH diagnosis), and was diagnosed with possible Parkinson disease. Previous history of neurological disorders was observed in 3 patients: one typical transient global amnesia (at the age of 60), one seizures (at 5 years), and one headache before the diagnosis of PNH. Regarding psychiatric evaluation, 3/18 patients had a psychiatric disorder according to structured clinical interview for axis I DSM-IV-TR disorders (1 generalized anxiety disorder, 1 bipolar disorder type 2, and 1 adjustment disorder as a consequence of PNH diagnosis). No relationship was observed between MRI findings and neuro-psychiatric assessment. In conclusion, brain MRI revealed chronic ischemic and vascular lesions in 58% of asymptomatic PNH patients. Brain MRI is advisable at diagnosis and during the course of the disease, and WM lesion burden may be useful in the decision to start therapy. Disclosures Barcellini: Agios: Consultancy.

Little evidence is available on antithrombotic strategies after transcatheter aortic valve implantation (TAVI). We aimed to evaluate the use and clinical impact of different antithrombotic regimens after TAVI. Patients with severe aortic stenosis undergoing TAVI with CoreValve™ (Medtronic, MN)

SummaryWe studied the effects of fluvastatin and bezafibrate in monotherapy and in combination on plasma fibrinogen, t-plasminogen activator inhibitor (PAI-1) and C reactive protein (CRP) in patients with coronary artery disease (CAD) and mixed hyperlipidaemiaIn this randomised, double blind, multicentre trial 333 patients with stable angina pectoris or previous myocardial infarction or coronary revascularisation and mixed hyperlipidaemia (LDL-cholesterol 135-250 mg/dl and triglycerides (TG) 180-400 mg/dl) were randomised to fluvastatin 40 mg, bezafibrate 400 mg, fluvastatin 20 mg + bezafibrate 400 mg or fluvastatin 40 mg + bezafibrate 400 mg treatments for 24 weeks.Plasma fibrinogen significantly decreased after treatment with the combinations fluvastatin+bezafibrate (−14 and −16%) and with bezafibrate monotherapy (−9%). No significant reduction was observed after fluvastatin monotherapy (−4%). No significant changes were observed in PAI-1 and CRP plasma levels. Combination therapy significantly decreased both LDL-C and TG, and significantly increased HDL-C.The combined effects on fibrinogen and plasma lipids achieved by fluvastatin and bezafibrate combination treatment might be more useful than the simple reduction of cholesterol in preventing ischaemic cardiovascular disease.

Summary. A case of disseminated granulomatous Geotrichum capitatum infection is reported. A young patient with blastic crisis of chronic myelogenous leukaemia developed septicaemia caused by G. capitatum in the post-chemotherapy aplastic phase. Subsequently, disseminated infection of the liver, spleen, pancreas and kidneys was observed. Treatment with high cumulative doses of a lipid formulation of amphotericin B (Amphocil®, 20.2 g in 11 weeks) and maintenance with itraconazole resolved clinical manifestations of G. capitatum granulomatous disseminated disease and controlled reactivation of the infection during the two subsequent courses of cytotoxic chemotherapy. Zusammenfassung. Es wird uber einen Fall von disseminierter, granulomatoser Geotrichum capitatum-Infektion berichtet. Eine junge Patientin mit blastischer Krise bei chronisch-myeloischer Leukamie entwickelte eine Septikamie, verursacht durch G. capitatum in der ablastischen Phase nach zytotoxischer Chemotherapie. Anschliesend wurden Disseminierungen in Leber, Milz, Pankreas und Nieren beobachtet. Die Behandlung erfoigte mit hohen Dosen einer Amphotericin B-Lipid-Formulierung, namlich 20.2 g Amphocil®uber 11 Wochen, sowie einer Erhaltungstherapie mit Itraconazol. Diese Behandlung fuhrte zur Heilung der klinischen Manifestationen der Pilzin-fektion und verhinderte die Reaktivierung des Prozesses wahrend zweier nachfolgender Behand-lungsperioden mit zytotoxischer Chemotherapie.

In this article we report a case of erythroblastic synartesis, a rare disease characterized by ineffective erythropoiesis, clusters of erythroblasts due to membrane invaginations, in which an autoimmune pathogenesis is hypothesized. We investigated the presence of anti-erythroblast autoimmunity in bone marrow cultures using a mitogen-stimulated direct antiglobulin test, a method reported to be able to disclose a latent autoimmunity in various diseases. The test revealed the presence of erythroblast-bound IgG, supporting the hypothesis of the autoimmune pathogenesis of erythroblastic synartesis. Supernatants induced the same specific morphological features, i.e erythroblastic clustering and diserythropoietic signs (multiple nuclei, nuclear inclusions, and intercellular bridges) in normal progenitors.

Febrile nonhemolytic transfusion reactions occur in 0.12% of transfusions, usually during transfusion or within 4 to 6 hours after transfusion and are not medically dangerous.A patient with thalassemia from Togo with asymptomatic malaria in which the infection became clinically manifest only after blood transfusion, mimicking a febrile nonhemolytic transfusion reaction, is presented. Thirty-two hours after transfusion of 2 O D- red blood cell (RBC) units, the patient (phenotype A(2) D+) developed fever and multiorgan failure and was admitted to the intensive care unit. Direct and indirect antiglobulin tests were negative on posttransfusion samples. Blood cultures and infectious diseases testing were negative. No malaria parasites were found at thick blood smear microscopic examination on Days 1 and 2 and the malaria rapid diagnostic test gave inconsistent results. Plasmodium total antibodies were detected in the serum at high levels. On Day 5, routine microscopic examination of blood smear revealed the presence of parasites in a very small number of RBCs. This finding was almost simultaneous to the availability of polymerase chain reaction testing results that were positive for P. falciparum. The sequential agglutination with anti-A antiserum allowed patient's and donors' RBCs to be separated and revealed that the parasitized cells were almost exclusively those of donors (14.4% vs. 0.029%). Malaria infection in implicated donors was excluded.In this patient with thalassemia with asymptomatic malaria, the infusion of two normal RBC units provided a favorable environment for a rapid parasite replication leading to a dramatic acute malaria attack.

Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disease characterized by ineffective erythropoiesis, hemolysis, erythroblast morphological abnormalities, and hypoglycosylation of some red blood cell (RBC) membrane proteins. Recent studies indicated that CDAII is caused by a defect disturbing Golgi processing in erythroblasts. A linkage analysis located a candidate region on chromosome 20, termed the CDAN2 locus, in the majority of CDAII patients but the aberrant gene has not so far been elucidated. We used a proteomic-genomic approach to identify SEC23B as the candidate gene for CDAII by matching the recently published data on the cytoplasmic proteome of human RBCs with the chromosomic localization of CDAN2 locus. Sequencing analysis of SEC23B gene in 13 CDAII patients from 10 families revealed 12 different mutations: six missense (c.40C>T, c.325G>A, c.1043A>C, c.1489C>T, c.1808C>T, and c.2101C>T), two frameshift (c.428_428delAinsCG and c.1821delT), one splicing (c.689+1G>A), and three nonsense (c.568C>T, c.649C>T, and c.1660C>T). Mutations c.40C>T and c.325G>A were detected in unrelated patients. SEC23B is a member of the Sec23/Sec24 family, a component of the COPII coat protein complex involved in protein transport through membrane vesicles. Abnormalities in this gene are likely to disturb endoplasmic reticulum (ER)-to-Golgi trafficking, affecting different glycosylation pathways and ultimately accounting for the cellular phenotype observed in CDAII.

In paroxysmal nocturnal hemoglobinuria (PNH) hemolytic anemia is due mainly to deficiency of the complement regulator CD59 on the surface of red blood cells (RBCs). Eculizumab, an antibody that targets complement fraction 5 (C5), has proven highly effective in abolishing complement-mediated intravascular hemolysis in PNH; however, the hematologic benefit varies considerably among patients. In the aim to understand the basis for this variable response, we have investigated by flow cytometry the binding of complement fraction 3 (C3) on RBCs from PNH patients before and during eculizumab treatment. There was no evidence of C3 on RBCs of untreated PNH patients; by contrast, in all patients on eculizumab (n = 41) a substantial fraction of RBCs had C3 bound on their surface, and this was entirely restricted to RBCs with the PNH phenotype (CD59−). The proportion of C3+ RBCs correlated significantly with the reticulocyte count and with the hematologic response to eculizumab. In 3 patients in whom 51Cr labeling of RBCs was carried out while on eculizumab, we have demonstrated reduced RBC half-life in vivo, with excess 51Cr uptake in spleen and in liver. Binding of C3 by PNH RBCs may constitute an additional disease mechanism in PNH, strongly enhanced by eculizumab treatment and producing a variable degree of extravascular hemolysis.

Autoimmune hemolytic anemia (AIHA) is a greatly heterogeneous condition both in terms of clinical presentation and response to treatment, usually classified as warm (WAIHA), cold (CAD), mixed, and atypical forms. The aim of this study was to identify predictors of outcome and response to therapy considering in particular the serological characteristics and the severity of anemia at onset. We evaluatedretrospectively 307 patients (112 M and 195 F, median age at diagnosis 63, range 1-97), diagnosed between 1978 and 2013 and followed-up for a median of 33 months (range 12-372); 60% of cases were WAIHA, 27% CAD, 8% mixed, and 5% atypical (14 DAT- and 1 DAT+ for IgA only). Hemoglobin values were lower in mixed (median 5.8, range 2-10.7 g/dL) atypical (6.2, 3-9), and in IgG+C3 DAT+ WAIHA (6.9, 2.9-11.5). Twenty-one subjects were diagnosed with Evans’ syndrome, the majority of them WAIHA, with a severe onset. Considering anemia at onset, 27% of cases had Hb levels 10 g/dL; the most severe cases were mainly mixed and atypical forms (P=0.0001). Regarding therapy, 47% of cases were treated with one therapy line only, 26% with two, 13% with three, and 4% with four or more lines. Sixty % of WAIHA received first line steroid therapy only, 20 CAD required no treatment, and patients with IgG+C DAT+ WAIHA, mixed, and atypical forms were more frequently treated with 2 or more therapy lines (P As regards complications, infections occurred in 26 cases (10 grade 3, 11 grade 4, and 5 grade 5), irrespective of serological AIHA type and severity at onset, and of the number of therapy lines; on the contrary, they were observed more frequently in splenectomized cases. Acute renal failure was recorded in 6 cases and was not associated with AIHA clinical or serological characteristics. A thrombotic event was recorded in 11% and was associated with severe onset, higher median LDH levels, and previous splenectomy. At the time of the analysis 63 cases (21%) have died, of whom 11 because of AIHA (3.6%); death was not associated with the severity of anemia at onset, nor with the serological type of AIHA; at variance, it was associated with infections (HR 11.47, 95% CI 3.43-38.4, p=0.0004), acute renal failure (HR 17.99, 95% CI 4.73-68.40, p=0.001), Evans’ syndrome (HR 6.8, 95% CI 1.99-23.63, P=0.0074), previous splenectomy (HR 3.21, 95% CI 0.92-11.25), and multi-treatment (4 or more lines of therapy; HR 9.1, 95% CI 2.41-34.36, p=0.0076). Death was not associated with thrombotic events, nor with the type of treatment, in particular immunosuppressants or rituximab. In conclusion, we showed that AIHA cases with a severe onset, mostly mixed and atypical forms, are frequently refractory to different therapies. Although obtained retrospectively, our results suggest to put forward rituximab among second line options, given its efficacy and safety. In addition, standard rituximab doses should be preferred in CAD, whereas lower doses may be equally effective in WAIHA and mixed forms. Finally, we suggest to defer splenectomy after rituximab, given the increased risk of thromboembolism, infections and fatal outcome in splenectomized patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The 30-day mortality in catheter-based reperfusion therapy in patients with acute myocardial infarction varies widely in the literature and only some factors, such as cardiogenic shock, are clearly associated with the risk. This non-randomized, single center study investigates the potential factors influencing the 30-day mortality in 586 consecutive patients with ST-elevation myocardial infarction, treated with primary coronary angioplasty (PTCA).In the whole series and in two subgroups (with and without cardiogenic shock) the clinical, angiographic and procedural variables were used to develop multivariate statistical models for the prediction of the endpoint.The overall 30-day mortality was 7.3%: 35.8 and 4.5% in patients with and without cardiogenic shock, respectively (p0.001). Independent predictors of the 30-day mortality included: a) in the entire series: shock, PTCA angiographic success, time to treatment, age, and coronary artery disease extension; b) in patients with cardiogenic shock: PTCA angiographic success, time to treatment, coronary artery disease extension, and use of abciximab; c) in patients without cardiogenic shock: time to treatment, age, and coronary artery disease extension.In patients with ST-elevation myocardial infarction submitted to primary PTCA, the 30-day mortality rate is a highly predictable endpoint. The role of abciximab therapy and of other independent predictors varies according to the presence or otherwise of cardiogenic shock.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal disorder due to a PIG-A gene mutation, resulting in deficient expression of GPI-anchored-proteins. Both immune-mediated suppression of hematopoiesis and cytokine alterations have been reported in aplastic anemia, a disease closely related to PNH whereas no data are available on PNH itself. The aim of this study was to investigate the effect of exogenous cytokines on clonogenic activity in PNH.We evaluated burst-forming units-erythroid (BFU-E) and colony-forming units-granulocyte-macrophage (CFU-GM) in bone marrow mononuclear cells (BMMC) from 5 PNH patients and 5 controls, alone or in the presence of transforming-growth-factor (TGF)-beta, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and specific antibodies. Molecular analysis of the PIG-A gene was performed by polymerase chain reaction (PCR) and direct sequencing on every single colony.Patients' cells showed less clonogenic activity than did control cells. In PNH, addition of TGF-beta inhibited both BFU-E and CFU-GM; IFN-gamma and TNF-alpha inhibited BFU-E alone. In patients cytokines modulated normal and mutated clones differently: TGF-b reduced the number of PIG-A- and PIG-A+ colony-forming-cells (CFC), whereas TNF-alpha and IFN-gamma reduced PIG-A+ CFC only. BMMC from patients showed higher TGF-beta production than did BMMC from controls.TGF-beta could contribute to the genesis of the unfavorable bone marrow microenvironment but does not seem to play a role in the in vivo dominance of PIG-A deficient cells. Mutated clones were more resistant to the inhibitory effects of IFN-gamma and TNF-alpha, suggesting that PNH cells may have a growth advantage in an unfavorable microenvironment.

We investigated regulatory variants of five cytokine genes [tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, transforming growth factor (TGF)-beta, interleukin (IL)-6 and IL-10] in 40 Italian patients affected by paroxysmal nocturnal haemoglobinuria (PNH) and aplastic anaemia (AA). Genotypes associated with high production of TGF-beta and IFN-gamma were more frequent in patients than in controls. Genetic regulation of the immunological pathways involved in the pathogenesis of bone marrow failure is suggested.

Applying an automatic code to the 3D UZC galaxy catalogue, we have extracted a complete sample of about 400 physical compact groups having cz≤10000 km/s and at least 3 member galaxies within a projected distance radius of 200 h −1 kpc. We find interaction pattern to be more common among triplets than among higher multiplicity compact groups, which nicely matches predictions based on the lower velocity dispersion and higher overdensity displayed by triplets. Whilst morphological disturbance are rather common among CG members, we retrieve a relative low fraction of AGNs. Interestingly only Seyfert 2 are retrieved, thus confirming once more that locally dense environments discriminate against Seyfert 1.

The clinical presentation of autoimmune hemolytic anemia (AIHA) is greatly heterogeneous, from mild/compensated to life-threatening forms. The aim of this study was to correlate the clinical and serological characteristics of the disease, usually classified as warm (WAIHA), cold (CHD), and mixed, based on the thermal and isotype characteristics of the anti-RBC antibody (IgG, IgM or both, respectively). One-hundred fifty seven AIHA patients (61 M and 96 F, median age 57, range 5-95) referred to our institution from 1978 to September 2012 were investigated. They had been followed-up for a median of 26 months (range 12-271), and 50% were still in follow-up. As regards the thermal characteristics 40% of cases were WAIHA, 32% CHD, 19% mixed forms and 9% atypical (12 DAT negative and 1 DAT positive for IgA only). Considering the severity of anemia at onset 33% of cases had Hb levels10 g/dL. The most severe AIHA cases were mainly mixed (18/30, 60% p=0.001) and atypical (6/13, 46%) forms, whereas only a small fraction of CHD was characterized by a severe onset (8/51, 16% p=0.002). Reticulocytopenia ( Disclosures: No relevant conflicts of interest to declare.

Abstract 983 Eculizumab, a monoclonal antibody that binds to the complement component C5, controls intravascular hemolysis in PNH patients and causes significant clinical improvement. However, the extent to which patients respond is variable in terms of hemoglobin levels, and some patients remain red blood cell (RBC) transfusion-dependent in spite of regular eculizumab treatment. We have hypothesized that genetic polymorphisms of complement-related genes might contribute to determining this variability of response to eculizumab. In order to test this hypothesis, we have analyzed polymorphic alleles of the Complement Receptor 1 (CR1) gene and of the Complement Component 3 (C3) gene. We have genotyped by standard methods (1) the HindIII restriction length polymorphism (RFLP) in the CR1 intron 27 (rs11118133 A>T) whose co-dominant alleles, H (common) and L (rare), are associated with high and low levels of CR1 expression on RBC, respectively; (2) the single nucleotide polymorphism rs2230199 C>G of the C3 gene, responsible for the allelic electrophoretic variants, S (slow, common allele) and F (fast, rare allele), that influence the complement alternative pathway activity. We have studied 72 patients with hemolytic PNH treated with eculizumab for at least 6 months. Patients with clear evidences of bone marrow failure (platelets ≤ 50,000/μL, neutrophils ≤1000/μL) have not been included in this study because this condition may affect the clinical response independently of the control of hemolysis. In order for our criterion for response to be stringent and objective we used blood transfusion requirement: patients who received no RBC transfusion have been defined as Responders; patients who received any RBC transfusion have been defined as Poor Responders. In this series of 72 hemolytic PNH patients on eculizumab the allelic frequency of the CR1 rare allele L was 0.31, and that of the C3 rare allele F was 0.19. We found no correlation between response and the C3 genotype (Chi square for trend: p=0.939). On the other hand the proportion of Poor Responders is significantly higher in patients who were heterozygotes (H/L) for the CR1 polymorphism, and even higher in those who were homozygous for the rare allele (L/L) of the CR1 HindIII RFLP polymorphism (Chi square for trend: p=0.016): see figure 1. We do not know yet the precise mechanism whereby the CR1 genotype affects the response to eculizumab. CR1 enhances the decay of C3 and C5 convertase by binding to C3b and C4b and it plays a role in the clearance of immune complex and in phagocytosis. Thus, the variable levels of CR1 expression on RBC membrane associated with the HindIII RFLP polymorphism may affect the regulation of complement alternative pathway. Moreover, it is well established that in almost all PNH patients on eculizumab a significant fraction of GPI-negative RBCs are opsonized by C3 and therefore may undergo extravascular hemolysis. Thus, a possible explanation of our findings is that the lower levels of CR1 expression on RBCs associated with H/L and L/L genotypes are responsible for a more intense extravascular hemolysis in PNH patients on eculizumab. In conclusion, our data show that the CR1 HindIII polymorphism may predict the poor response to eculizumab in PNH patients and provide additional insight in the mechanisms responsible for the variable clinical effectiveness of complement blockade. Disclosures: Risitano: Alexion: Membership on an entity's Board of Directors or advisory committees, Research Funding. Peffault de Latour:Alexion: Consultancy. Iori:Alexion: invited speaker Other. Socie:Alexion: Consultancy. Luzzatto:Alexion: Consultancy. Notaro:Alexion: Consultancy.

Abstract 5272 Pyruvate kinase (PK) deficiency, transmitted as an autosomal recessive trait, is the most common erythroenzymopathy of glycolytic pathway (prevalence of 1:20,000) associated with chronic non spherocytic hemolytic anemia from mild to severe. More than 180 mutations in the PK-LR gene have been so far reported, and genotype-phenotype correlation has been established for some of them. Hereditary Spherocytosis (HS) is the most common congenital hemolytic anemia in Caucasians, with an estimated prevalence ranging from 1:2000 to 1:5000. The main clinical features are hemolytic anemia from compensated to severe, variable jaundice, splenomegaly and cholelythiasis. The molecular defect is highly heterogeneous, caused by proteins involved in the attachment of cytoskeleton to the membrane integral domain (spectrin, ankyrin, band 3 and protein 4.2). We describe a case of PK deficiency associated with HS. The propositus was a 13 years-old Italian male with neonatal jaundice and need of blood transfusion (Hb 5.8 g/dL) during an infectious episode. At the time of the study Hb was 13.9 g/dL, MCV 81.8 fL, reticulocytes 207×109/L, unconjugated bilirubin 2.16 mg/dL, LDH 605 U/L, haptoglobin The presence of spherocytes in peripheral blood smear prompted us to investigate for the coexistence of HS. Erythrocyte osmotic fragility was decreased and SDS–PAGE analysis of red cell membrane proteins revealed a 30% spectrin reduction. Family study demonstrated a heterozygous condition for the 994A mutation in the father, who also displayed comparable enzyme deficiency, whereas promoter variant −148T was detected in the mother and in the brother. No red cell membrane abnormalities were present in the family members, although positive EMA binding test and increased osmotic fragility were found in the father and brother. The co-existence of HS and PK deficiency is very rare event, only few cases are described to date. Clinical, family and molecular studies allowed the determination of the interrelationship between the two RBC abnormalities in the patient and his relatives. The reduced PK activity in the propositus and his father is justified by heterozygous 994A mutation. The more severe clinical picture in the propositus could be caused by the coexistence of HS and by the presence of −148T mutation, that although it seems not to have effects on PK-LR mRNA expression, is often detected in PK deficient subjects with heterozygous PK mutations. Disclosures: No relevant conflicts of interest to declare.

The Progetto Lombardo Atero-Trombosi (PLAT) Study was a prospective, multicenter, multidisciplinary study of the association among hemostatic variables, conventional risk factors, and atherothrombotic events in four groups of patients with preexisting vascular ischemic disease (335 myocardial infarction survivors, 123 patients with stable angina pectoris, 160 with transient ischemic attacks, and 335 with peripheral vascular disease). In the myocardial infarction group, univariate analysis showed that atherothrombotic events were associated with high fibrinogen (p = 0.001), factor VIII:C (p less than 0.001), and von Willebrand factor antigen (vWF:Ag) (p = 0.004) levels and with low high density lipoprotein cholesterol (p = 0.043), factor VII (p = 0.019), and protein C (p = 0.044) levels; multivariate analysis produced associations with high fibrinogen and factor VIII:C levels and low protein C levels. By both univariate and multivariate analysis, events in the angina pectoris group were associated with high vWF:Ag (p = 0.026) and leukocyte (p = 0.033) levels and the presence of carotid arterial stenosis (p = 0.063); associations with high leukocyte (p = 0.037) and factor VIII:C (p = 0.186) levels, family history (p = 0.031), and diabetes (p = 0.061) were also found in the group with transient ischemic attacks. In those with peripheral vascular disease, events were associated with Fontaine stage greater than or equal to IIB (p = 0.024), high factor VIII:C levels (p = 0.073), and low protein C (p = 0.028), fibrinogen (p = 0.030), antithrombin III (p = 0.054), and factor VII (p = 0.057) levels by univariate analysis and with Fontaine stage and low fibrinogen levels by multivariate analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disorder characterized by an acquired PIG-A gene mutation. The clinical symptoms are intravascular hemolysis and hemoglobinuria. A variety of mutations in PIG-A gene have been so far reported. The aim of the study was to investigate the molecular defect in 8 patients with hemolytic PNH and in a 51 years old female who spontaneously recovered from severe hemolytic PNH (SL). The study of the entire codifying region and flanking intronic sequences of PIG-A gene was done by SSCP analysis and sequencing. 8 new and one known mutations were detected. Results are reported in the table. Mutation C55T has been already described in 5 PNH patients, but its nature was controversial. C55T was the only mutation found in patient SL. It was also detected in patient DTI, in association with T728C. To exclude the somatic origin of this variant, the mucous membrane cells were analyzed and found to be positive. C55T was also present in 1/100 normal alleles investigated, thus confirming its polymorphic nature. Mutations so far identified could be useful as molecular markers for monitoring the course of the disease during therapy.

Abstract 4028 Poster Board III-964 CDAII, the most frequent type of congenital dyserythropoietic anemia family, is an autosomal recessive disease characterized by ineffective erythropoiesis, peripheral hemolysis, erythroblasts' morphological abnormalities and hypoglycosylation of some RBC membrane proteins. Recent studies indicated that CDAII is not a distinct glycosylation disorder but caused by a defect disturbing Golgi processing in erythroblasts. Linkage analysis located the CDAII gene in a 5 cM region on chromosome 20 and several candidate genes have been excluded. We recently investigated the cytoplasmic proteome of human red blood cells (RBCs) using a combinatorial peptide ligand library as a capturing agent to amplify the signal of low- and very-low abundance proteins: 1578 proteins, most of them unexpected, were identified allowing a deep exploration of the RBC pathways (Roux-Dalvai, Mol Cell Proteomics, 2008). In this study we used a proteomic-genomic approach to identify the candidate gene for CDAII by matching the data on the cytoplasmic proteome of human RBC with the chromosomic localization of CDAN2 locus. The analysis of RBCs cytoplasmic proteome allowed us to identify 17 proteins codified by genes located in the chromosomic region between 20p11.23 and 20q11.23: SNX5, SEC23B, DTD1, NAT5, GINS1, BCL2L1, MAPRE1, CHMP4B, EIF2S2, AHCY, ACSS2, GSS, EIF6, CPNE1, EPB41L1, RPRD1B(C20orf77), TGM2. Most of them were excluded because found to be associated to other diseases, already excluded in CDAII by previous works, or because not related by function to CDAII. Among the remaining proteins we focus on SEC23B for its possible role in the endoplasmic reticulum-to Golgi trafficking and its localisation on 20p11, the region with the highest LOD-score in CDAII after the recent mapping of the markers on the current contigs (Denecke et al, Biochim Biophys Acta, 2009). The 20 exons and intronic flanking regions of SEC23B gene were analysed by direct sequencing in 16 CDA II patients from 13 families; 13 different mutations were detected among the 28 mutated alleles identified: 6 of them were missense, 2 frameshift, 1 splicing and 4 stop codon. All the missense mutations affected highly conserved aminoacids, and were not found in 100 normal alleles examined. Two of them (c.40C>T and c.325G>A) were detected in various unrelated patients. Despite all exons and flanking intronic regions were sequenced, one patient failed to show mutations and two cases displayed only one mutation, suggesting the possibility that a second gene could be involved in CDAII. Patients' data are summarised on the table (*,° = members of the same family). SEC23B is a member of the SEC23/SEC24 family, a component of COPII coat protein complex which is involved in protein trafficking through membrane vesicles. Very recently it has been shown that knockdown of Zebrafish SEC23B leads to aberrant erythrocyte development (Schwarz et al, Nat Genet, 2009). Even if the exact function of human SEC23B is not completely clarified, abnormalities in this gene are likely to disturb ER-to Golgi trafficking affecting different glycosylation pathways, and ultimately accounting for the cellular phenotype observed in CDA II. Case Sex Origin Hb g/dL Retics 10e9/L Band3 hypoglyc Mutations Exons Effects 1 F N Italy 9.7 160 Yes c.40 C>T/c.428 A>CG 2/5 Arg 14 Trp/Frameshift 2 F C Italy 11.4 44 Yes c. 1821 delT/? 16/? Frameshift/? 3 F Bolivia 9.9 61 Yes c.568 C>T/c.1808 C>T 5/16 Arg 190 STOP/Ser 603 Leu 4 M Rumania 9.8 102 Yes c.40 C>T/c.1660 C>T 2/14 Arg 14 Trp/Arg 554 STOP 5 M S Italy 10.4 nd Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 6 F C Italy 8.3 103 Yes c.40 C>T/Ivs6 +1g>a 2/Ivs6 Arg 14 Trp/Splicing 7 M C Italy 9.7 100 Yes c.1489C>T/c. 2101 C>T 13/18 Arg 497 Cys/Arg 701 Cys 8° F N Italy 9.2 121 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 9° M N Italy 11.3 115 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 10° F N Italy 11.7 63 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 11 F N Italy 11.6 104 Yes c.40 C>T/c.1043 A>C 2/9 Arg 14 Trp/Asp 348 Ala 12* M S Italy 11.9 90 Yes c.40 C>T/c.649 C>T 2/6 Arg 14 Trp/Arg 217 STOP 13* F S Italy 7.8 61 Yes c.40 C>T/ c.649 C>T 2/6 Arg 14 Trp/Arg 217 STOP 14 F N Italy 9.5 105 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 15 M N Italy 10.4 236 Yes c.367 C>T/? 5/? Arg 123 STOP/? 16 F S Italy 10.7 87 Yes ?/? ?/? ?/? Disclosures: No relevant conflicts of interest to declare.

Abstract 4186 We report the case of a thalassemic patient with asymptomatic malaria in which the infection became clinically manifest only after blood transfusion, mimicking a febrile non haemolytic transfusion reaction. The patient was a 19 yr old thalassemic girl from Togo regularly and uneventfully transfused every 60-90 days since the age of 5 months and splenectomised at 13 yrs. Two malaria episodes in 1995 and 1997 were treated with quinine and halofantrin respectively. In February 2008 the transfusion interval shortened (15 days) and blood transfusions were constantly followed after 30-40 hours by high fever (39-4028C) and hypotension. The patient was referred to our Hospital to clarify and overcome the transfusion problems. Malaria attacks were excluded by the referring clinic. At admission, Hb was 5.9 g/dL, Hct 18.3%, MCV 71.8 fL, MCH 23.1 pg, MCHC 32.2%, reticulocytes 0.82%. WBC 13.1 × 109/L platelet count 953 × 109/L erythroblasts 12.7% of nucleated cells. Molecular analysis of β and α genes revealed double heterozygosity for β IVS1-1 G>A and β IVS2-849 A>G, associated with deletion 3.7 (-α3.7) at the heterozygous state. G6PD 6.29 IU/g Hb (ref.values 5.97±1), Pyruvate kinase 25.1 IU/gHb (ref. v. 11.9-16.1). Red cell osmotic fragility was decreased. The cytofluorimetric analysis of red cells labelled with eosin 5 maleimide gave normal results. Direct antiglobulin test was negative and red cell alloantibodies were not detected. The patient's blood group assessed by molecular biology testing was: genotype O1A2, phenotype A2; ccDee; K-k+; Jk(a+b-); Fy(a-b+); M+N+S+s-. The patient was given 2 crossmatch negative, filtered O neg units: the post-transfusion Hb values were 9.9 g/dL. Thirty-two hours later the patient developed fever (40°C) and hypotension followed by disseminated intravascular coagulation, metabolic acidosis and anuria. Direct and indirect antiglobulin tests were negative on a post-transfusion sample. Because of the rapid worsening of clinical conditions she was admitted to the Intensive Care Unit of our Hospital. Septic shock was excluded on the basis of negative blood cultures and infectious diseases testing. Microscopic examination of thick blood smear was negative for malaria parasites on 2 consecutive days and the malaria rapid diagnostic test (RDT) (Core Malaria Pan-PV-PF®, Core Diagnostics, U.K.-Effegiemme, Nerviano, Milano, Italy) gave inconsistent results. Anti Plasmodium total antibodies (Malaria EIA New Market Laboratories U.K.- Bio-Rad Italia) were detected in the serum at high levels (odd sample/cut off 22). Vital functions were supported by mechanical ventilation, amine administration and continuous venous-venous hemofiltration (CVVH). Broad spectrum antibiotic therapy was started. Fresh frozen plasma and two units of PRC were transfused on day 1 and 2 without increment of Hb level. Three days later, the examination of the peripheral blood smear for counting schistocytes revealed the presence of parasites in a very small number of red cells. This finding was almost simultaneous to the availability of PCR testing results that were positive for P. Falciparum. The differential agglutination with anti A antibody performed on a blood sample collected at admission to ICU allowed to separate patient's and donors' red cell. Counting the number of parasitized red cells in total blood and in the donor fraction blood revealed that the parasitized cells were almost exclusively those of donors (14.4 % vs 0.029%). Treatment with quinine chlorhydrate 500 mg i.v. every 8 hr for 3 days followed by oral quinine sulphate 500 mg every 8 hr for 3 days was successfully undertaken without significant side effects leading to dramatic improvement of clinical conditions and to eradication of P. falciparum. Two RBC units were effectively transfused without any reaction, and in the following two weeks the patient maintained stable haemoglobin values. After discharge, the transfusion interval returned to the previous values (60-90 days) without any post-transfusion reaction. We therefore think that the transfusion of normal, non malaria-resistant red cells fuelled the P. falciparum infection causing fever, DIC and acidosis giving rise to a very severe, atypical febrile non haemolytic transfusion related reaction. Disclosures: No relevant conflicts of interest to declare.

Abstract 1981 Poster Board I-1003 Congenital dyserythropoietic anemias (CDAs) are a heterogeneous group of disorders characterized by ineffective erythropoiesis with prominent dysplastic features of the erythroid precursors in the bone marrow. Three main subtypes (I, II and III) have been identified, but several variants or atypical forms have been reported over the years. CDA are rarely associated with hydrops foetalis. In the past we described two cases presenting with hydrops foetalis and very severe anemia that were classified as atypical CDAs since they presented CDAII-like erythroblastic morphological features lacking other diagnostic CDAII markers. Very recently we and others (Bianchi et al, Human Mutat 2009, in press; Schwarz et al, Nat Genet 2009, in press) demonstrated that mutations in SEC23B gene, coding for a protein involved in the coat protein complex responsible for vesicle budding from the endoplasmic reticulum, cause CDA II. The aim of this work was to ascertain whether atypical CDAII-like forms presenting as hydrops foetalis could be caused by mutations in SEC23B gene and reclassified as CDAII. Two patients (Cantù-Rajnoldi et al, Br J Haematol 1997, 96: 530-3; Bianchi et al, Blood 1999, 94, suppl 1, 8b) from unrelated families with a history of intrauterine death of hydropic foetuses in previous pregnancies were referred at 20th week gestation following ultrasonic diagnosis of foetal hydrops and severe anemia (2.0 and 1.3 g/dL Hb respectively). In both cases intrauterine transfusions enabled the delivery of the babies. At birth both of them underwent extensive laboratory evaluation (including red cell metabolism and membrane proteins analysis) that was not informative on the causes of anemia. Ham test was repeatedly normal as for Western blotting analysis for GRP78 and glycoslylation pattern of red cell band 3. The bone marrow examination revealed the presence of 30 and 48% of bi- or multinucleated erythroblasts, some of whom presenting typical double outer membranes at transmission electron microscopy. Both babies became transfusion dependent. The 20 exons and intronic flanking regions of SEC23B gene were analyzed by direct sequencing. Both patients displayed mutations in SEC23B gene, in particular mutations c.325 G>A/ c.2101 C>T in patient 1 and c.325 G>A/ c.197G>A in patient 2 (Glu109Lys/ Arg701Cys and Glu109Lys/ Cys66Tyr respectively). c.325 G>A is the most frequent mutation so far described in CDAII and at homozygous level is usually associated with mild anemia. When found in compound heterozygosity with a second missense mutation as in these cases it may result in a very severe clinical pattern, although we cannot exclude that factors other than SEC23B mutations may contribute to worsening the clinical picture. In conclusion, SEC23B gene analysis allowed the correct classification of two very severe CDA cases associated with hydrops foetalis. This finding indicates that CDAII may result in intrauterine death and its frequency may therefore be underestimated. These cases may present as “atypical” for the lack of band3 hypoglycosylation likely due to the early sequestration of the more severely affected cells. Disclosures: No relevant conflicts of interest to declare.

PNH is a hematological disorder characterized by complement-mediated intravascular hemolysis due to lack on RBCs of the complement regulators CD55 and CD59 and subsequent activation of the membrane attack complex (MAC). Eculizumab (EC) is an anti-complement fraction 5 antibody which abolishes intravascular hemolysis in PNH patients, leading to reduction of transfusion requirement and of anemia. Although the effector complement pathway is completely blocked by eculizumab in all patients, response to this agent in terms of transfusion requirement and hemoglobin level varies considerably. We have investigated the notion that patients with suboptimal hematological response may suffer from residual hemolysis mediated by mechanisms other than intravascular hemolysis via MAC. After the initial observation of a positive C3d Coombs test in some PNH patients on EC, we extensively studies C3 coating by flow cytometry in 56 PNH patients (41 of them while receiving EC). We found that in all cases on EC treatment a significant proportion of RBCs were coated with complement fraction 3 (C3); by converse, in 28 untreated PNH patients we found no evidence of C3 on red cells. C3 coating was strictly limited to CD59-neg RBCs, as CD59+/C3+ RBCs (as those seen in cold agglutinine disease patients, positive control) were never found. C3 coating was quite different among EC-treated patients, and correlated with the PNH RBC population. The percentage of C3+ cells within the PNH RBC population (the only subjected to C3 coating) was quite different in individual patients (0.5–61.3%, median 22.6%) and substantially preserved over time. We compared the level of C3 coating with the hematological response: all the 41 EC-treated patients showed marked LDH reduction with a substantial improvement of anemia, leading to transfusion independence in 34/41 patients (83%) and stable resolution of anemia in 14 (34%, defined optimal responders). The optimal responders showed a lower percentage of C3+ cells in comparison to suboptimal responders (20.9±19.0 vs 32.2±17.8; p=0.04). Indeed, patients with lower C3 coating (below the median value of 22.6%) showed a significantly higher rate of optimal response (51% in comparison to 15% of those with coating above the median, p=0.01). C3 coating also correlated with the absolute reticulocyte count (p=0.03), clearly suggesting that C3 coating was associated with ongoing residual extravascular hemolysis in vivo, and with pre-treatment LDH level (p=0.001). To confirm the presence of extravascular hemolysis, in 3 index patients with suboptimal response reduced RBC half-life was demonstrated in vivo by 51Cr RBC survival study, which showed reduced RBC half-life and excess uptake in liver and spleen. One of these patients underwent video-laparoscopic splenectomy, which led to transfusion independence and significant increase in Hb level. These data demonstrate that C3 coating of PNH RBCs is a common phenomenon in PNH patients on EC; in addition, we provide evidence that this leads to C3-mediated extravascular hemolysis through the reticulo-endothelial system. This novel mechanism of disease may account for residual hemolysis and suboptimal clinical benefit in some EC treated PNH, paving the way for additional therapeutic strategies to optimize the hematological response to this agent.

Hereditary Spherocytosis (HS) is caused by defects of red cell membrane proteins (spectrin, ankyrin, band 3 and band 4.2). The aim of this study is to analyse a large database of 300 HS patients grouped according to SDS-PAGE, 1) to ascertain whether the clinical/haematological features and response to splenectomy are related to the type of molecular defect, and 2) to compare the sensitivity of the most common laboratory screening tests for HS in the various subsets of patients. Three hundred consecutive patients were investigated; 41 patients had been splenectomised before the time of the study, and 21 thereafter. In not splenectomised subjects, anaemia was severe in 7% of cases, mild to moderate in 52% and compensated in the remaining cases. The median number of spherocytes at the peripheral blood smear examination was 7%. The most frequent protein abnormalities revealed by SDS-PAGE were band 3 (54%) and spectrin and/or ankyrin (34%) deficiency. The membrane protein defect was undetectable in 3% of splenectomised versus 11% of not splenectomised patients. In patients evaluated by SDS-PAGE before and after splenectomy surgery allowed the identification of the defect (one band 3 and seven spectrin/ankyrin deficiency) in all the 8 previously unclassified cases. No significant differences were observed among clinical and haematological parameters of patients grouped according to the type of biochemical defect, although the degree of anaemia, haemolysis markers and median spherocyte number were higher in spectrin deficient than in the other groups of patients. Splenectomy was clinically effective in correcting both anaemia and haemolysis, but splenectomised spectrin/ankyrin deficient patients showed after splenectomy a slightly lower median rise in haemoglobin, and a higher reticulocyte number and unconjugated bilirubin level than band 3 deficient patients. The red cell osmotic fragility tests’ sensitivity varies greatly and ranged from 48 to 95%. The sensitivity was similar in patients with band 3 and spectrin/ankyrin deficiency and also in patients without detectable membrane defect. Furthermore, the sensitivity of all the methods investigated increased in splenectomised cases. AGLT displayed the highest sensitivity, and the association of AGLT and NaCl test on incubated blood reached a sensitivity of 99%, enabling the diagnosis of the atypical HS cases, such as those with rare or no spherocytes in blood smears, normal MCHC and reticulocyte counts.

We describe the clinical, immunological and molecular characteristics of a 39 yrs-old male with 8 yrs story of paroxysmal nocturnal hemoglobinuria (PNH) who underwent a reduced intensity conditioning bone marrow transplantation (RIC-BMT) from identical sibling donor. The patient was pancytopenic, transfusion-dependent and had suffered from Budd-Chiari syndrome. We already reported that that RIC-BMT restored clonogenic erythroid activity and cytokine production (IL-2, IFN-gamma and TNF-alpha) in BM. Likewise, IL-2, TNF-alpha and TGF-beta formerly increased in both BM and peripheral blood (PB), became normal after BMT. Finally, the PNH clone was no more detectable in both BM and PB cells by molecular analysis 1 year after RIC-BMT. At 2 years after the transplant the patient is well, with Hb levels of 15.2 g/dL, but with low WBC (2.7x109/L) and platelets (71x109/L). The number of CFU-GM and BFU-E in BM further increased and none of the CFU-GM and BFU-E showed the original PIG-A gene mutation. Likewise, none of the PB granulocytes showed the original PIG-A gene mutation also 2 years after BMT. These immunological, molecular and cytokine changes observed 2 years after RIC-BMT support the curative action of a donor immune-mediated “graft versus PNH” effect. | | Patient before BMT | 1mo after BMT | 6mo after BMT | 12mo after BMT | 24mo after BMT | Controls (mean±SE) | |:------ | ------------------ | ------------- | ------------- | -------------- | -------------- | ------------------ | | CFU-GM | 7 | 10 | 7 | 10 | 13 | 21±9 | | BFU-E | 12 | 23 | 46 | 108 | 115 | 62±18