[Show abstract][Hide abstract] ABSTRACT: The European Hematology Association (EHA) Roadmap for European Hematology Research highlights major achievements in diagnosis and treatment of blood disorders and identifies the greatest unmet clinical and scientific needs in those areas to enable better funded, more focused European hematology research. Initiated by the EHA, around 300 experts contributed to the consensus document, which will help European policy makers, research funders, research organizations, researchers, and patient groups make better informed decisions on hematology research. It also aims to raise public awareness of the burden of blood disorders on European society, which purely in economic terms is estimated at Euro 23 billion per year, a level of cost that is not matched in current European hematology research funding. In recent decades, hematology research has improved our fundamental understanding of the biology of blood disorders, and has improved diagnostics and treatments, sometimes in revolutionary ways. This progress highlights the potential of focused basic research programs such as this EHA Roadmap. The EHA Roadmap identifies nine sections in hematology: normal hematopoiesis, malignant lymphoid and myeloid diseases, anemias and related diseases, platelet disorders, blood coagulation and hemostatic disorders, transfusion medicine, infections in hematology, and hematopoietic stem cell transplantation. These sections span 60 smaller groups of diseases or disorders. The EHA Roadmap identifies priorities and needs across the field of hematology, including those to develop targeted therapies based on genomic profiling and chemical biology, to eradicate minimal residual malignant disease, and to develop cellular immunotherapies, combination treatments, gene therapies, hematopoietic stem cell treatments, and treatments that are better tolerated by elderly patients.
[Show abstract][Hide abstract] ABSTRACT: Autoimmune hemolytic anemia (AIHA) is a relatively uncommon disorder caused by autoantibodies directed against self red blood cells. It can be idiopathic or secondary, and classified as warm, cold (cold hemagglutinin disease (CAD) and paroxysmal cold hemoglobinuria) or mixed, according to the thermal range of the autoantibody. AIHA may develop gradually, or have a fulminant onset with life-threatening anemia. The treatment of AIHA is still not evidence-based. The first-line therapy for warm AIHA are corticosteroids, which are effective in 70-85% of patients and should be slowly tapered over a time period of 6-12 months. For refractory/relapsed cases, the current sequence of second-line therapy is splenectomy (effective approx. in 2 out of 3 cases but with a presumed cure rate of up to 20%), rituximab (effective in approx. 80-90% of cases), and thereafter any of the immunosuppressive drugs (azathioprine, cyclophosphamide, cyclosporin, mycophenolate mofetil). Additional therapies are intravenous immunoglobulins, danazol, plasma-exchange, and alemtuzumab and high-dose cyclophosphamide as last resort option. As the experience with rituximab evolves, it is likely that this drug will be located at an earlier point in therapy of warm AIHA, before more toxic immunosuppressants, and in place of splenectomy in some cases. In CAD, rituximab is now recommended as first-line treatment.
[Show abstract][Hide abstract] ABSTRACT: The clinical outcome, response to treatment, and occurrence of acute complications, were retrospectively investigated in 308 primary autoimmune hemolytic anemia (AIHA) cases and were 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 DAT-negative). The latter two categories showed more frequently a severe onset (Hb levels ≤6 g/dL) along with reticulocytopenia. The majority of warm AIHA 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 (HR 3.08, 95% CI 1.44-6.57 for Hb ≤6 g/dL, p<0.001). Thrombotic events were associated to 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 multi-treatment (4 or more lines). The identification of severe and potentially fatal AIHA in a largely heterogeneous disease requires particular experienced attention by clinicians.
[Show abstract][Hide abstract] ABSTRACT: We studied monosomy and deletions of chromosome 7 in 208 patients with myeloid disorders; we found 39 patients (19%) with monosomy or deletion of chromosome 7: 24 patients with chromosome 7 deletion and 15 with monosomy 7. In the 24 patients with chromosome 7 deletions, studied with copy-number variants, short-tandem repeats, microsatellites, single nucleotide polymorphisms, and deletion polymorphisms, the most common deleted region was 7q31.1 (20 patients). Deletion polymorphism studies performed in these 20 patients showed an interstitial deletion of at least 140 kilobase in 6 patients; the deletion spans between the genes forkhead box P2 and Myo D family inhibitor domain containing. Because both genes do not seem to be involved in leukogenesis, we suggest to look carefully into this deletion for the presence of tumor suppressor genes and microRNAs.
[Show abstract][Hide abstract] ABSTRACT: Although the issue of informed consent for human research biobanks has been analyzed in a number of publications, revolving on speculative as well as empirical perspectives, it remains a critical matter within the ethical community globally. Considering that ethical requirements for information and consent vary widely, depending on the international, regional and national regulatory landscape, the development of a harmonized model of informed consent for research biobanks is today a huge challenge. After the analysis of the informed consent issue in pediatric biobanks, this chapter describes the variability of regulations addressing adult research biobanks on a global, regional and national level by focusing on the several consent standards reported into the literature. Afterward, this chapter proposes a template and procedure for developing unified informed consent forms for the storage and use of human specimens for research purposes, based on the experience of an inter- disciplinary group of professionals collaborating with a multispecialty biobank in the context of a public university hospital. Although local, this experience and the related consent template and procedure could serve as models suitable in similar research environments.
[Show abstract][Hide abstract] ABSTRACT: Anti-red blood cell (RBC) autoantibodies are the causative agents of autoimmune hemolytic anemia (AIHA), whose estimated incidence is one to three cases per 100,000 per year. The most frequent are immunoglobulin (Ig)G, which are usually directed against epitopes of the Rh system, react at 37 °C, mainly determine extravascular hemolysis, and are responsible for the ''warm'' forms (WAIHA). IgM are pentameric autoantibodies able to fix complement more efficiently than other isotypes, cause intravascular hemolysis, are directed against the I/i system, have an optimal temperature of reaction at 4 °C, and are responsible for the ''cold'' forms of autoimmune hemolytic anemia (cold hemoagglutinin disease, CHD). AIHAs can be distinguished as primary (idiopathic) and secondary to other diseases (autoimmune, infections, lymphoproliferative, or neoplastic diseases) and show great clinical heterogeneity, from compensated forms without anemia to fulminating disease. The gold standard for the detection of anti-RBC antibodies is the direct antiglobulin test (DAT) or Coombs test. DAT-tube is the traditional agglutination technique usually performed with broad-spectrum Coombs reagents; the use of monospecific anti-IgG, anti-IgM, and anti-C3 antisera is recommended to define the class of the autoantibody, along with its thermal characteristics. It is worth mentioning a simple test, the spontaneous agglutination of RBCs at 20 °C, a characteristic feature indicating the presence of cold IgM autoantibodies. DAT-tube may give false-negative results due to the small number of RBC-bound IgG molecules below the threshold of the test (estimated 400 molecules per RBC). Moreover, the tube technique with polyspecific (IgG+C) may fail to detect IgA autoantibodies; the use of monospecific antisera against IgA can overcome the DAT negativity. Another cause of DAT-negativity may be the presence of low-affinity autoantibodies, which can be detected by low ionic strength solutions (LISS) or cold washings. More sensitive methods include microcolumn, solid-phase, complement-fixation antibody-consumption test, enzyme-linked and radiolabelled tests, flow-cytometry, mitogen-stimulated-DAT, and the dual direct antiglobulin test. Despite all the above-mentioned tests, 5-10% of AIHA may be DAT negative, and the diagnosis is made after exclusion of other causes of hemolysis and on the basis of the clinical response to therapy.
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT Toll-like receptors (TLR) represent major agents of innate immunity and initiators of adaptive immunity. TLR4 and TLR9 gene expression was related with the occurrence of infections, autoimmunity and disease progression in 95 B-CLL patients, grouped according to stage, therapy, and known prognostic markers, and followed prospectively (median 33.6 months, range 25-50). A retrospective analysis (median 6.8 years, range 6-26) was also performed. TLR4 gene expression was decreased and TLR9 increased in patients versus controls, the former being more pronounced in advanced and multi-treated disease, and in patients with unmutated IgVH and unfavorable cytogenetic. Patients with reduced TLR4 had an increased risk of disease progression and development of autoimmune complications. No relationship was found between reduced TLR4 expression and infectious episodes, which were observed in advanced stages and treated patients. These findings suggest that impaired innate immunity identifies B-CLL patients with poor prognosis and reduced ability to silence autoreactive phenomena.
No preview · Article · Oct 2013 · Leukemia & lymphoma
[Show abstract][Hide abstract] ABSTRACT: to evaluate the sustained response to low-dose (LD)-rituximab in autoimmune hemolytic anemia (AIHA), the ex vivo effect on anti-RBC antibody production by mitogen-stimulated direct antiglobulin test (MS-DAT), and the in vitro dose-effect of the drug on the production of anti-RBC antibodies.
32 patients, 18 warm (W)AIHA and 14 cold hemagglutinin disease (CHD), were treated with LD-rituximab (100 mg fixed dose x4 weekly infusions) along with a short course of oral prednisone. Complete clinical examination, blood counts and hemolytic markers were performed at enrollment, and at month 6, 12, 24, and 36.
Hematologic parameters significantly improved at all time points compared to enrollment. The overall response was 90%, 100%, 100% and 89% and the relapse free survival 87%, 79%, 68% and 68% at 6, 12, 24 and 36 months, respectively. Response rates were slightly better in WAIHA than in CHD, and relapse risk was greater in cold than warm forms (HR 2.1, 95% CI 0.6 -7.9). Four patients were retreated (one patient twice), all achieving a response, lasting a median of 18 months (range 9-30). Treatment was well tolerated without adverse events or infections. Anti-RBC antibody production by MS-DAT significantly decreased over time. In vitro studies showed that rituximab effectively inhibited anti-RBC antibody production at 50 μg/mL, 1/6 of the drug concentration after therapy with standard doses.
These data confirm that LD-rituximab treatment is effective and induces sustained responses in AIHA, and that a lower dose of the drug is enough to down-regulate autoantibody production. This article is protected by copyright. All rights reserved.
No preview · Article · Sep 2013 · European Journal Of Haematology
[Show abstract][Hide abstract] ABSTRACT: Hereditary spherocytosis (HS) and pyruvate kinase (PK) deficiency are the most common causes of congenital hemolytic anemia. We describe a case of HS with defective PK activity initially misdiagnosed as PK deficiency.
Hematologic investigation, SDS-PAGE analysis of red cell membrane proteins and sequencing of the PKLR gene were performed.
The molecular characterization of the PKLR gene showed a heterozygous mutation 994G > A (Gly332Ser) associated with the promoter substitution -148C > T, whose role in the pathophysiology of PK deficiency is debated. Further investigations revealed spectrin deficiency; the family study demonstrated that the hemolysis was exclusively attributable to HS.
The present case pinpoints to the need for extensive family investigations to correctly diagnose chronic hemolytic anemia, in particular when molecular characterization does not fully explain the clinical phenotype.
No preview · Article · Jun 2013 · Clinical laboratory
[Show abstract][Hide abstract] ABSTRACT: This overview describes two groups of nonimmune hereditary hemolytic anemias caused by defects in membrane proteins located in distinct layers of the red cell membrane. Hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary pyropoikilocytosis (HPP) represent disorders of the red cell cytoskeleton. Hereditary stomatocytoses represents disorders of cation permeability in the red cell membrane. The current laboratory screening tests for HS are the osmotic fragility test, acid glycerol lysis time test (AGLT), cryohemolysis test, and eosin-5'-maleimide (EMA)-binding test. For atypical HS, SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins is carried out to confirm the diagnosis. The diagnosis of HE/HPP is based on abnormal red cell morphology and the detection of protein 4.1R deficiency or spectrin variants using gel electrophoresis. None of screening tests can detect all HS cases. Some testing centers (a survey of 25 laboratories) use a combination of tests (e.g., AGLT and EMA). No specific screening test for hereditary stomatocytoses is available. The preliminary diagnosis is based on presenting a compensated hemolytic anemia, macrocytosis, and a temperature or time dependent pseudohyperkalemia in some patients. Both the EMA-binding test and the osmotic fragility test may help in differential diagnosis of HS and hereditary stomatocytosis.
Preview · Article · Mar 2013 · International journal of laboratory hematology
[Show abstract][Hide abstract] ABSTRACT: Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible phosphotransfer reaction from 1,3-bisphosphoglycerate to MgADP, to form 3-phosphoglycerate and MgATP. Two isozymes encoded by distinct genes are present in humans: PGK-1, located on Xq-13.3, encodes a ubiquitous protein of 417 amino acids, whereas PGK-2 is testis-specific. PGK1 deficiency is characterized by mild to severe hemolytic anemia, neurological dysfunctions and myopathy; patients rarely exhibit all three clinical features. Nearly 40 cases have been reported, 27 of them characterized at DNA or protein level, and 20 different mutations were described. Here we report the first Italian case of PGK deficiency characterized at a molecular and biochemical level. The patient presented during infancy with hemolytic anemia, increased CPK values, and respiratory distress; the study of red blood cell enzymes showed a drastic reduction in PGK activity. In adulthood he displayed mild hemolytic anemia, mental retardation and severe myopathy. PGK-1 gene sequencing revealed the new missense mutation c.1112T>A (p.Ile371Lys). The mutation was not found among 100 normal alleles, and even if located in the third to the last nucleotide of exon 9, it did not alter mRNA splicing. The p.Ile371Lys mutation falls in a conserved region of the enzyme, near the nucleotide binding site. The mutant enzyme shows reduced catalytic rates toward both substrates (apparent k(cat) values, 12-fold lower than wild-type) and a decreased affinity toward MgATP (apparent K(m), 6-fold higher than wild-type). Moreover, it lost half of activity after nearly 9-min incubation at 45°C, a temperature that did not affect the wild-type enzyme (t(1/2)>1 h). The possible compensatory expression of PGK2 isoenzyme was investigated in the proband and in the heterozygote healthy sisters, and found to be absent. Therefore, the highly perturbed catalytic properties of the new variant p.Ile371Lys, combined with protein instability, account for the PGK deficiency found in the patient and correlate with the clinical expression of the disease.
Full-text · Article · May 2012 · Molecular Genetics and Metabolism