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Leukopenia, granulocytopenia, and, occasionally, anemia develop in rats fed a purified diet deficient in riboflavin. Folic acid (L. casei factor) corrects the leukopenia and granulocytopenia. Riboflavin will prevent all the dyscrasias but will correct only the anemia. The bone marrow in granulocytopenic rats is hypoplastic and is almost completely depleted of cells of the granulocytic series. Cells of the erythroid series are decreased in number. The myelogram of rats made folic acid deficient by the inclusion of sulfasuxidine in a purified diet resembles this picture and in both cases the response of the marrow and the blood to folic acid therapy is similar. The bone marrow in riboflavin-deficient rats having both granulocytopenia and anemia is depleted of granulocytic cells but shows an erythroid hyperplasia. This myelogram differs from that seen in sulfasuxidine-induced folic acid deficiency anemia and granulocytopenia in which there is erythroid hypoplasia. The two anemias differ further in that the folic acid deficiency anemia responds to folic acid therapy whereas the riboflavin deficiency anemia responds to riboflavin therapy but not to folic acid therapy.

The current study was initiated to define whether Tc-99m leukocyte imaging, compared to In-111 chloride imaging, could provide additional information on the hematopoietic activity of the bone marrow in diffuse hematologic disorders. A total of 11 patients with hematologic disorders were studied both with Tc-99m leukocytes and In-111 chloride. One patient with myelofibrosis showed disparate tracer distribution in the bone marrow between the two; Tc-99m leukocyte uptake in the central marrow was highly decreased, whereas In-111 chloride showed expanded marrow. The disparate results of granuloid hypoplasia and erythroid hyperplasia obtained by bone marrow biopsy and aspiration in this patient were thought to give an explanation for the radionuclide findings. The remaining 10 patients showed similar tracer distribution in the marrow, and the observation of hematopoietic cellularity confirmed by bone marrow biopsy and aspiration correlated well with the radionuclide findings. These results demonstrate that Tc-99m leukocytes may be a potential imaging agent for evaluating hematopoietic activity in the bone marrow, in particular granulopoietic activity.

Material from 63 cases with primary myelodysplastic syndromes (P-MDS) (French-American-British [FAB] types: refractory anemia [RA] = 21; RA with ring sideroblasts [RARS] = 8; RA with excess of blasts (RAEB) = 10; RAEB in transformation (RAEBt) = 6; chronic myelomonocytic leukemia [CMML] = 10 and unclassifiable = 8, ie, bone marrow aspiration was inadequate and stringent FAB criteria were not applicable) was analyzed for bone marrow histologic and immunohistochemical patterns. Standard Giemsa, hematoxylin and eosin (H&E) and reticulin stains were used for morphologic assessment. To identify the cell lineage precisely, chloroacetate esterase staining and an indirect immunoperoxidase technique using mouse monoclonal antibodies CD15, CD68, HLA-DR, and rabbit polyclonal CD3 and UEA-1 (lectin) was developed on formalin- fixed paraffin embedded bone marrow biopsies (BMB). The immunohistochemical assessment permitted accurate identification of dysplastic features such as mononuclear and binuclear megakaryocytes, Pelger-Huet neutrophils, and binuclear erythroblasts. Additional bone marrow histologic and immunohistochemical features observed were heterogeneity of immunohistochemical staining in various cell lineages, megakaryocytic emperipolesis, alteration of bone marrow microarchitecture, intravascular clusters of hematopoietic cells, and the types of benign lymphoid aggregates. The nature of abnormally localized immature precursors (ALIP) was discerned. Three types of clusters of immature cells were found that were difficult to distinguish on Giemsa and H&E morphology, these were erythroid aggregates (n = 18); megakaryocytic aggregates (n = 4), and immature granulocytic and monocytic aggregates (n = 32). The bone marrow histologic and immunohistologic patterns permitted the identification of four groups of clinical relevance: Group 1, cases with predominant erythroid hyperplasia and without ALIP (n = 15); group 2, cases with prominent myeloid hyperplasia and presence of ALIP (n = 32); group 3, cases with hypoplastic MDS (n = 10); and group 4, cases with hyperfibrotic MDS (n = 6). Statistical analysis showed a significant difference in survival and leukemic transformation between groups 1, 2, 3, and 4, with cases in group 2 showing the worst prognosis with early death due to increased propensity to leukemic transformation and cytopenia-related complications (P less than .0001). We conclude that immunohistochemistry is feasible on routinely processed BMB and the information obtained is of diagnostic and prognostic importance in P- MDS. The phenotype of ALIP varies with the morphologic and histologic subtypes of MDS and the term should be reserved for cases in whom the clusters in the intertrabecular region are of myeloid (granulocytic and monocytic) lineage on immunohistochemistry.

Recombinant rat stem cell factor (rrSCF) administered to rats as a single intravenous injection causes a dose-dependent neutrophilia and lymphocytosis as well as the appearance of immature myeloid cells and occasional blast cells in the circulation. Neutrophilia begins at 2 hours, peaks at 4 to 6 hours, and subsides between 12 and 24 hours. Lymphocytosis occurs at 0.5 hours and has subsided by 2 hours. rrSCF- induced neutrophilia and lymphocytosis are abrogated by boiling, demonstrating that endotoxin-contamination of the rrSCF preparation is not responsible for the observed hematologic effects. The bone marrow at 6 hours after injection of rrSCF shows a left-shifted myeloid and erythroid hyperplasia as evidenced by significant increases in the absolute numbers of morphologically recognizable early myeloid and erythroid precursors. A concurrent decrease in the absolute numbers of mature marrow neutrophils is noted, suggesting that the release of marrow neutrophils contributes to the peripheral neutrophilia. After 2 weeks of daily injections of rrSCF, bone marrow smears demonstrate a remarkable mast cell hyperplasia accompanied by a decrease in total marrow cellularity and by a striking erythroid and lymphoid hypoplasia. rrSCF also causes mast cells to appear in the circulation and causes a systemic increase in embryonic connective tissue-type, but not mucosal- type, mast cells. In vitro long-term culture of lineage-depleted mouse bone marrow cells with rrSCF results in an almost pure outgrowth of mast cells.

Background: Imatinib mesylate has shown promising results in chronic myeloid leukemia (CML) in all phases. This drug is an effective treatment for patients with CML in chronic phase as it induces hematological remission in nearly all patients and cytogenetic responses in many. The bone marrow changes produced by this drug are different from the treatment modalities used earlier in CML. Materials & Methods: We studied 80 patients of CML on treatment with Imatinib at doses of 400-800 mg per day. Morphological and cytogenetic evaluation (Ph analysis) of bone marrow aspirates was done at six months of treatment. Result: In our study, 95% (76 out of 80) patients showed complete hematological response and 63.3% showed major cytogenetic response at the end of six months of treatment. The most commonly observed changes in the bone marrow aspirates at the end of six months of therapy were in the form of reduction in the cellularity, reduction in the M: E ratio to a mean of 2:1, presence of relative erythroid hyperplasia, normalization of megakaryocytic morphology and variable increase in the bone marrow lymphocytes. None of these changes had significant correlation with the patient's Ph status. Conclusion: We advise study of trephine biopsies to overcome the often-faced problem of hemodiluted aspirates in these cases and evaluation of sequential bone marrows to check the durability of these morphological changes and their correlation with the cytogenetic response with emphasis on cytogenetic changes other than Ph positivity.

Mild nonregenerative anemia was detected in a 9-year-old neutered male domestic shorthair cat during a routine examination. Bone marrow core biopsy revealed erythroid hyperplasia; however, a specific cause was not identified. Over the next 8 months the anemia progressed, eventually becoming mildly regenerative, and moderate thrombocytopenia developed. On ultrasonographic examination, marked splenomegaly, mild hepatomegaly, and abdominal lymphadenopathy were found. Cytologic evaluation of splenic aspirates revealed increased numbers of mildly to moderately pleomorphic histiocytes that frequently had phagocytosed RBCs, leukocytes, and occasionally platelets. Histopathologic examination of the spleen and liver revealed effacement of splenic architecture by a histiocytic sarcoma (HS), and neoplastic histiocytes in hepatic sinusoids. A second bone marrow aspirate revealed neoplastic infiltration by similar cells. The histiocytes in all tissues were mildly to moderately pleomorphic and markedly erythrophagocytic. The immunophenotype of histiocytes in the spleen was CD1c(-)/CD11b(+)/CD18(+)/MHC-II(+), supporting a macrophage cell lineage. The clinical, pathologic, and immunophenotypic findings in this cat were similar to those in hemophagocytic HSs in dogs. To our knowledge, this is the first report of a HS of purported macrophage phenotype in a cat.

An eight-month-old male West Highland white terrier (WHWT) presented with mild exercise intolerance and splenomegaly caused by a severe and highly regenerative haemolytic anaemia that persisted until the dog died at two years of age. There was marked erythroid hyperplasia in the bone marrow and progressive osteosclerosis. Erythrocyte pyruvate kinase (PK) deficiency was confirmed by demonstrating a block in glycolysis at the PK step, abnormal erythrocyte PK kinetics, absence of R-type PK isoenzyme, and the presence of M2-type PK not normally expressed in erythrocytes. From the study of family members it is concluded that erythrocyte PK deficiency in the WHWT is inherited as an autosomal recessive trait. Heterozygotes have about half-normal erythrocyte PK activity, do not express the M2-type PK isoenzyme and are asymptomatic. This disease closely resembles erythrocyte PK deficiency in the basenji breed and should be considered as a differential diagnosis in young dogs with severe haemolytic anaemia.

On cytological bone marrow examination we distinguished between pure sideroblastic anaemia (PSA), which is confined to dyserythropoiesis, and refractory anaemia with ring sideroblasts (RARS), which is characterized by additional dysplastic features of granulopoiesis and/or megakaryopoiesis. In a follow-up study of 94 patients with AISA diagnosed according to FAB criteria for myelodysplastic syndromes we found a striking difference in the risk of leukaemic transformation between PSA and RARS (5 year cumulative rate 1.9% v. 48%). Overall survival was much better in PSA than in RARS (5 year cumulative chance 69% v. 19%). Infections and haemorrhages were frequent causes of death in RARS but not in PSA. Bone marrow culture studies (CFU-GM) were performed on 10 consecutive patients with PSA and RARS, respectively. RARS patients showed grossly impaired colony growth, typical of the myelodysplastic syndromes. Patients with PSA had persisting colony formation, even if moderately decreased in frequency, with numbers of CFU-GM being inversely correlated with the degree of erythroid hyperplasia in the bone marrow. We conclude that on cytomorphological grounds AISA can be divided into pure (dyserythropoietic) sideroblastic anaemia (PSA) and a true myelodysplastic form (RARS), with both types differing considerably in terms of survival, risk of leukaemic transformation and findings on bone marrow culture (CFU-GM).

Multi-colony stimulating factor (Multi-CSF, interleukin-3, IL-3) and granulocyte-CSF (G-CSF) administered concurrently as an intravenous (IV) injection induce a peripheral neutrophilia that is approximately additive in comparison with the neutrophilia induced by IL-3 and G-CSF individually. The bone marrow (BM) at 12 hours is depleted of mature neutrophils and shows a left-shifted myeloid hyperplasia, consistent with the neutrophil-releasing and myeloproliferative activities of both IL-3 and G-CSF individually. The BM at 24 hours shows a replenished reserve of mature neutrophils and a synergistic left-shifted myeloid hyperplasia as compared with IL-3 and G-CSF alone. Daily IV injections of IL-3 plus G-CSF for 1 week also induce an approximately additive daily peripheral neutrophilia. The BM after a week's administration of IL-3 plus G-CSF shows a generalized myeloid hyperplasia with a synergistic increase in mature neutrophils as compared with IL-3 or G- CSF alone. Daily injection of IL-3 plus G-CSF induced a significant decrease in erythroid, lymphoid, and eosinophilic marrow precursors, possibly owing to a myelophthisic effect of the myeloid hyperplasia and despite the fact that IL-3 alone induced a significant erythroid hyperplasia.

Relapsing Polychondritis (RPC) is a rare disorder of unknown etiology which affects mucopolysaccharide-rich tissues such as cartilage. A 64-year-old man developed auricular and nasal chondritis with complaints of arthralgia of the hands and cervical pain. The auricular biopsy established the diagnosis of RPC. The hematological data revealed normocytic, slightly hypochromic anemia, a persistently elevated reticulocyte count, slightly increased bilirubin, and decreased haptoglobin. The presence of hemolytic anemia was confirmed by the shortened half-life of erythrocytes and erythroid hyperplasia of the bone marrow. This case illustrates the coexistence of RPC and hemolytic anemia which has been only rarely reported. The pathogenesis of RPC is also discussed may elucidate the pathogenesis of this disease.

Infection of erythroid progenitor cells by Friend spleen focus-forming virus (SFFV) leads to acute erythroid hyperplasia and eventually to erythroleukemia in susceptible strains of mice. The viral envelope protein, SFFV gp55, forms a complex with the erythropoietin receptor (EpoR) and a short form of the receptor tyrosine kinase Stk (sf-Stk), activating both and inducing Epo-independent proliferation. Recently, we discovered that coexpression of SFFV gp55 and sf-Stk is sufficient to transform NIH 3T3 and primary fibroblasts. In the current study, we demonstrate that sf-Stk and its downstream effectors are critical to this transformation. Unlike SFFV-derived erythroleukemia cells, which depend on PU.1 expression for maintenance of the transformed phenotype, SFFV gp55-sf-Stk-transformed fibroblasts are negative for PU.1. Underscoring the importance of sf-Stk to fibroblast transformation, knockdown of sf-Stk abolished the ability of these cells to form anchorage-independent colonies. Like SFFV-infected erythroid cells, SFFV gp55-sf-Stk-transformed fibroblasts express high levels of phosphorylated MEK, ERK, phosphatidylinositol 3-kinase (PI3K), Gab1/2, Akt, Jun kinase (JNK), and STAT3, but unlike virus-infected erythroid cells they fail to express phosphorylated STATs 1 and 5, which may require involvement of the EpoR. In addition, the p38 mitogen-activated protein kinase (MAPK) stress response is suppressed in the transformed fibroblasts. Inhibition of either JNK or the PI3K pathway decreases both monolayer proliferation and anchorage-independent growth of the transformed fibroblasts as does the putative kinase inhibitor luteolin, but inhibition of p38 MAPK has no effect. Our results indicate that sf-Stk is a molecular endpoint of transformation that could be targeted directly or with agents against its downstream effectors.

Many dogs and cats with immune-mediated haemolytic anaemia (IMHA) lack a bone marrow erythroid regenerative response. To better understand the failure of the bone marrow to respond to the anaemia, bone marrow pathology associated with non-regenerative IMHA and pure red cell aplasia (PRCA) was reviewed. Eighty-two affected dogs and 57 affected cats were identified from a population presenting to a referral hospital over a 10-year period. Fifty-five dogs had non-regenerative IMHA (38 had bone marrow erythroid hyperplasia and 17 had erythroid maturation arrest) and 27 had pure red cell aplasia (PRCA). Twenty-eight cats had non-regenerative IMHA (24 had erythroid hyperplasia and 4 had erythroid maturation arrest) and 29 had PRCA. A variety of pathological changes were observed in bone marrow aspirates and core biopsy specimens taken from these animals. These changes included dysmyelopoiesis, myelonecrosis, myelofibrosis, interstitial oedema, haemorrhage, acute inflammation, haemophagocytic syndrome, lymphocyte aggregation, and lymphocyte or plasma cell hyperplasia. In both dogs and cats, dysmyelopoiesis, myelonecrosis, myelofibrosis, interstitial oedema, haemorrhage, acute inflammation and haemophagocytic syndrome were primarily noted in bone marrow specimens where there was evidence of erythroid hyperplasia. These animals were also more often neutropenic and thrombocytopenic, and had decreased 60 day survival when compared with dogs or cats with non-regenerative anaemia associated with erythroid maturation arrest or PRCA. Therefore, the pathogenesis of the non-regenerative anaemia in non-regenerative IMHA may involve both antibody-mediated destruction of bone marrow precursor cells and pathological events within the bone marrow that result in ineffective erythropoiesis.

Introduction: Based on the 2008 World Health Organization classification (WHO 2008), erythroleukemia is defined by the presence of ≥50% erythroid precursors in bone marrow (BM) and ≥20% myeloblasts in the non-erythroid cell population. Multilineage dysplasia is almost always present with high rates of MDS-like cytogenetic abnormalities, specially complex karyotypes. Therefore an extensive comparison with myelodysplastic syndromes (MDS) with ≥50% erythropoesis seems crucial to elucidate whether erythroleukemia and MDS with erythroid hyperplasia should be considered as different biological entities. Aim: To elucidate this issue, the outcome and cytogenetic alterations of erythroleukemia patients were studied and compared to MDS patients with ≥50% erythropoesis with Methods: We retrospectively analyzed 448 de novo MDS with ≥50% erythropoesis and 59 de novo erythroleukemias from the MDS Spanish registry (RESMD). Diagnosis was done according to WHO 2008 and patients with ≥80% erythropoiesis with less than 20% of myeloblasts in the non-erythroid cell compartment were excluded assuming a diagnosis of pure erythroid leukemia. Results: Median age of presentation was 74 years (25-94 years), median follow-up was 29.4 months, 63% were males. Median overall survival (OS) of MDS patients with ≥50% erythropoiesis and Conclusion: Erythroleukemia and RAEB-1 with ≥50% erythropoiesis share clinico-biological features and outcome. Our findings suggest that erythroleukemia is a continuum of MDS with erythroid hyperplasia and karyotype rather than an arbitrary blast cut-off is the main prognostic marker in this subset of patients. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Valcárcel: Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Díez-Campelo:Celgene: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Janssen: Research Funding. Ramos:GlaxoSmithKline: Honoraria; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; Celgene Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria.

Introduction: WHO classification of MDS is based on cytopenias, dysplasia, percentage of blasts in PB and BM, and cytogenetics. IPSS-R establishes BM blast subgroups (≤2%,2-10%) with independent impact in OS. Erythroid hyperplasia (≥50% of total BM cells) is common in MDS. Concerning MDS with expanded erythropoiesis, there is no consensus whether the proportion of BM blasts should be considered on the basis of all nucleated cells (approach-A) or in non-erythroid cells (approach-B). Aim: To elucidate this issue, we reassess percentage of BM blasts of MDS with erythropoiesis ≥50% from the Spanish registry (RESMD), according to both definitions. Methods: We performed a retrospective analysis of 507 primary MDS diagnosed according to WHO 2008. Proportion of red-cells was calculated in 500 nucleated cells. Erythroid hyperplasia was documented in 10.4% of patients from RESMD. Results: Median age of presentation was 74years (25-94years) and 63% were males. Median follow-up was 29.4 months and median OS was 47.14 months. Table 1 shows distribution of WHO subtypes of the series according to both approaches. Of note, following WHO recommendations, RAEB-2 diagnosis was not possible; formally all of them were diagnosed with erythroleukemia. Distribution of patients according to IPSS-R blast-categories by both methods is shown in Table 2. It is noteworthy that 14/389pts (3.6%) with blasts 10% (10-20% vs. >20%; 20 vs. 14 months, p =0.53). Figure 1. Applying approach-A, significant differences in proportion of patients with high-risk karyotype defined by IPSS were seen between ≥5% blasts vs. 20%. Therefore, high-risk patients (>10% blasts and high-risk cytogenetics) were better defined by the second method. Conclusion: In agreement with WHO committee recommendations, these results suggest that there is no a uniform criteria for assessment of medullary blasts. Considering percentage of blasts on the basis of non-erythroid compartment might improve the classification and prognostication of MDS with ≥5% blast cells. However in low-blast count patients this approach could overestimate their risk. Disclosures Valcárcel: Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Ramos:GlaxoSmithKline: Honoraria; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; Celgene Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria.

Congenital dyserythropoietic anemia (CDA) type II is the most frequent type of congenital dyserythropoietic anemia; it is transmitted in an autosomal recessive fashion and is characterized by ineffective erythropoiesis, peripheral hemolysis, bi-multinuclearity in the erythroblasts, and hypoglycosylation of red blood cell (RBC) membrane proteins such as band 3. The disease is generally caused by biallelic mutations in the SEC23B gene. However, there are a small portion of patients with clinical and hematologic features of CDA II that are negative for mutations in SEC23B, suggesting that alternative etiologies for such disturbed erythropoiesis exist. We identified two siblings of Italian origin who had dyserythropoiesis with a chronic macrocytic anemia. Their parents were healthy with normal hematologic parameters. No history of consanguinity for at least three generations was noted. The affected siblings had anisopoikylocytosis on peripheral blood smear with stomatocytes (8-9%), spherocytes (4-5%), rare ovalocytes, and dacryocytes. RBCs osmotic fragility was increased but the red cells had normal eosin-5-maleimide (EMA)-binding. Serum ferritin and transferrin saturation were increased in only one sibling. Bone marrow morphology revealed erythroid hyperplasia (myeloid: erythroid ratio = 0.6) with binuclearity and megaloblastic changes, as well as occasional cytoplasmic bridging between cells at different stage of maturation; electron microscopy of bone marrow erythroblasts showed multiple membranes that ran parallel to the plasma membrane or that were grouped in stacked segments, possibly attributable to residual endoplasmic reticulum (ER) cisternae. SDS-PAGE analysis of RBC ghosts from both siblings demonstrated hypoglycosylation of band 3 and GLUT1, as well as residual residual Protein Disulphide Isomerase (PDI) positive ER remnants, as observed in classical CDA II cases. However, in contrast to CDAII, the Ham's test performed with 15 normal serum samples was negative, and no mutations were detected in the SEC23B gene. To uncover the underlying etiologies, whole-exome sequencing was conducted on all available family members. After filtering for common variants, only a single gene had biallelic mutations in the affected siblings, which were transmitted from the unaffected heterozygous parents. The identified mutations resided in the PARP4 gene, which encodes a poly-ADP ribose polymerase enzyme, and were predicted to be deleterious. We demonstrate that knockdown of PARP4 using shRNA in primary human erythroid progenitors results in impaired erythroid differentiation and increased apoptosis. In addition, morpholino-mediated knockdown of the PARP4 orthologue in the zebrafish resulted in dyserythropoiesis and anemia in developing embryos. Sequencing of PARP4 in additional rare cases of CDA II without an identified molecular basis will help to uncover the frequency and spectrum of PARP4 mutations leading to dyserythropoiesis. The finding of a new gene implicated in a similar type of CDA with features such as redundant ER membranes offers the potential for more mechanistic dissection of the role of both SEC23B and PARP4 in erythroid development and suggests that new insight can be gained into the underlying pathophysiology of both normal and disordered erythropoiesis through the study of such rare cases. Disclosures No relevant conflicts of interest to declare.

Severe iron overload is a reported complication of certain erythroid disorders which are characterized by increased erythropoietic activity. Proposed mechanisms include enhancement of iron absorption secondary to increased erythroid activity and coexistent heterozygosity or homozygosity for haemochromatosis. We performed PCR-based analysis for the haemochromatosis-related HFE C282Y mutation in an extended family with inherited haemolytic anaemia in which several members exhibited iron overload. The results demonstrated iron overload was associated with homozygosity but not heterozygosity for this mutation. Such an association may also exist in other erythroid disorders in which iron overload has been reported.

We report a 6-year-old Iranian boy with silvery-gray hair, eyelashes and the eyebrows who was admitted because of seizures and subsequent stupor. He had previous history of acute hemiparesis at 1 year of age and hepatitis-like syndrome 3 months ago. Microscopic examination of the patient's hair shaft revealed different sized clumps of melanin seen in the center of the shafts. Bone marrow aspiration revealed erythroid hyperplasia and erythrophagocytic cells. Bilateral frontal cortical and subcortical high signal lesions, dirty white matter, high signal areas in the upper pons and in both caudates and lentiform nuclei in T2 WI were the brain MRI findings of the patient. He died in the accelerated phase of Griscelli Syndrome (GS) type 2. To our knowledge we report the first case of GS from Iran.

Thymectomy is an inevitable therapy for thymoma. Therefore, determining hemato-immune changes post-thymectomy is important. Twenty-six normal LEW/Sea rats thymectomized (Tx) at the ages of 38 +/- 5 days were followed without any treatment for 4 months (experiments [Exp] A and B). In addition, 16 LEW/Sea rats Tx at the age of 42 days (Exp C and D) and 10 non-Tx control LEW/Sea rats (Exp E) were immunized with syngeneic male liver cells 2 to 3 times and followed 2.7 months after the first immunization. Flow cytometric (FCM) analysis of mesenteric lymph nodes (MLN) and peripheral blood (PB) showed as follows: among the 26 Tx rats (Exp A and B), MLN lymphocyte population at 4 months postthymectomy was characterized by decreased numbers of CD4+ cells (22%-36%) or alphabeta T-cell receptor (TCR)-positive cells (16%-54%) and increased numbers of interleukin 2 receptors (IL-2R) (>90%). In the 16 Tx-immunized rats (Exp C and D), both alphabeta TCR (MLN) and CD45R (PB) expression on lymphocytes was suppressed with rather high numbers of CD4. Bone marrow (BM) and PB hematological studies of the partially and totally Tx rats indicated the following: 8 BM of the 10 totally Tx males (Exp A) and the 7/10 male spleen showed erythroid hyperplasia with maturation arrest at the stage of basophilic erythroblasts and reticulocytopenia in the PB. One of the 10 males, which had both BM myeloid to erythroid (M/E) ratio of 4.3 and spleen myeopoiesis, was in a more advanced stage, a prestage of pure red cell aplasia (PRCA) with 22% of CD4+ MLN cells. Syngeneic liver cell immunization resulted in the Tx rats as follows: hepatotoxicity based on the immunization was weaker in the 16 Tx rats (Exp C and D) than in the 10 non-Tx rats (Exp E). Polymerase chain reaction (PCR) of PB and MLN showed compensatory activated leptin receptor (LR) and T-bet DNA in 19 Tx rats (Exp A and B) with abnormal FCM findings. The ineffective erythropoiesis at 4 months after thymectomy was explained by the erythroblast LR/erythropoietin receptor (EPOR) dysfunction. The low grade hepatotoxicity in the Tx rats (Exp C and D) was explained by the disturbed Th1 reactions (or the disturbed T-bet gene transcription) at postthymectomy.

A 19-year-old man who developed extensive oesophageal lye (Alkali) stricture and received long-term enteral nutrition (eight months) with a jejunostomy tube developed macrocytic anaemia (Hb: 41 g/L) with leucopenia (white blood cell [WBC]: 3.0 x 109/L). The patient's serum vitamin B12, folate, iron and liver function tests were normal. Bone marrow examination revealed gross erythroid hyperplasia and cytoplasmic vacuolization of erythroid and myeloid elements. Further investigations revealed low serum copper (0.3 μmol/L) and ceruloplasmin concentrations (

A young woman presented with non-specific generalized musculoskeletal pain. Investigations revealed anaemia, raised reticulocyte count, basophilic stippling and erythroid hyperplasia of the bone marrow. Further history showed that she had been taking home-made Chinese medicines for acne for two months and the blood lead level confirmed the diagnosis of lead poisoning. The medications were stopped and her condition gradually improved. Incorporating the use of herbal medicines into the routine medical history is important in early identification of herbal poisoning.

Owing to the risks of heavy metals-induced severe haematopoietic disorders, it is important to investigate these chemicals for their haematotoxicity and the possible ways to ameliorate their toxicity. The effects of melatonin on lead-induced haematotoxicity have, therefore, been examined in rat blood and bone marrow. When adult male rats were injected intramuscularly with lead acetate (10 mg kg(-1)) daily for 7 days, the erthrocytic count, haematocrite value and haemoglobin content were significantly decreased. The counts of platelets, total leucocytes and lymphocytes in the peripheral blood were also significantly lower in lead-treated rats than in control animals. The total granulocyte count was significantly elevated in the peripheral blood of the same lead-treated rats. Significant decreases in polychromatic and pyknotic erythroid series as well as lymphocytes in bone marrow of the lead-intoxicated rats were also demonstrated. Meanwhile, the neutrophiles were increased in the same treated rats. The erythropoietin level was significantly decreased and the lead concentration was increased in the plasma of the lead-treated rats compared with the control rats. Bone marrow examination of the rats treated with lead for 7 days showed erythroid hyperplasia with a sign of dyserythropoiesis and demonstrated ringed sideroblasts in varying proportions. Daily pretreatment with melatonin (30 mg kg(-1)) intragastricaly, concurrently with lead injection for 7 days significantly prevented the changes recorded in the peripheral blood parameters. The changes observed in the bone marrow polychromatic erythroid, lymphocytes and the neutrophiles were significantly ameliorated by coadministration of melatonin and lead compared with lead-treated rats, while the pyknotic erythroid series was still significantly low. The levels of erythropoietin and lead in plasma were not changed in melatonin+lead-treated group compared with lead only treated rats. In addition, melatonin administration ameliorated the decrease in erythroid cell count in bone marrow. Less dyserythropoiesis and megaloblastic changes were observed in bone marrow film when melatonin was concurrently administered with lead. In the same animals, iron staining of the bone marrow cells showed absence of ringed sideroblasts. In conclusion, the present results indicate that melatonin has the ability to protect the haematopoietic cells from the damaging effects of exposure to lead. This protection might be attributed to the antioxidative power of melatonin.

Introduction Thalassemia1,2 is an inherited autosomal recessive disorder. It occurs in two major degrees of severity. Thalassemia minor is the heterozygous type with decreased severity of symptoms. Thalassemia major is the homozygous type characterised by decreased production of normal globin chains resulting in intravascular hemolysis, profound anemia, erythroid hyperplasia, extramedullary hematopoiesis, splenomegaly and severe bone deformities. It usually results in death by 2nd decade of life3.

Case 1 A 12-month-old boy, born to a nonconsanguineous Hindu couple was brought for evaluation of recurrent severe pallor, skin bleeds and hearing impairment. There had been short lasting response to prior treatment with Packed Red Blood Cells (PRBC), oral iron, folic acid and vit. B12. Examination revealed severe pallor, and multiple petechiae over the body. There was no response to sounds. Investigations revealed fasting sugar of 160 mg/dL, hemoglobin of 4.3 g/dL, platelets of 20,000/cu.mm, normal total and differential leucocyte counts (TLC and DLC), and dimorphic anemia on peripheral smear. Serum ferritin, folic acid and B12 levels were normal. Bone marrow examination revealed erythroid hyperplasia with megaloblastic changes. Brainstem audiometry revealed no identifiable waves bilaterally. On oral thiamine (150 mg per day), his hemoglobin increased to 7 g/dL and platelets to 700,000/cu.mm in 5 days. Insulin therapy of 1 IU/kg/day was stopped on day 16. Following discontinuation of thiamine 1 month later, random blood sugar levels increased to 230 mg/dl within 1 week. After restarting thiamine, euglycemia was restored. The child has been periodically followed up on daily thiamine for 5 years now. Both the hemoglobin and glycosylated hemoglobin have remained in the normal range. His deafness has persisted.

Introduction The congenital dyserythropoietic anaemias (CDA) are a rare group of inherited haemolytic anaemias characterized by ineffective erythropoiesis and dyserythropoiesis. Due to the rarity of this disorder (and in Africa where haemoglobinopathies are the commonest cause of congenital haemolytic anaemias), the diagnosis can be missed. We report a six year old girl with recurrent anaemia, jaundice, hepatosplenomegaly and history of multiple transfusions who was diagnosed with CDA. Case Report A six year old girl presented to the paediatric clinic with a two year history of passing coke-coloured urine, recurrent anaemia requiring regular three-monthly transfusions and persistent jaundice. She was not a known sickle cell anaemia or thalassaemia patient. History of prenatal period, birth and delivery were normal (birth weight 3.5kg/ 7.7lbs). There was no history of neonatal jaundice or family history of similar illness. At presentation, she was acutely ill-looking, conscious but weak. She was afebrile, severely pale, icteric, tachypneic and in respiratory distress (respiratory rate of 52 cycles/minute). She was small for age; weight 15kg / 33.1lbs (below the 3rd percentile), height 104cm (below the 3rd percentile) with frontal bossing and gnathopathy. The liver was 6cm below the right costal margin and tender, with splenomegaly- 5cm below the costal margin. There was no lymphadenopathy. Her chest was clear, heart rate 140 beats/minute, and blood pressure of 100/50mmHg with presence of a haemic murmur. A preliminary clinical diagnosis of recurrent severe haemolytic anaemia in heart failure secondary to haemoglobinopathy was made. Full blood count showed severe anaemia with haemoglobin (Hb) concentration of 5.1g/dL, normal red cell indices, white cell count- 13.6 X 109/L, platelets- 191 X 109/L and reticulocyte count- 1.3%. The Hb genotype was AA, direct and indirect antiglobulin test were negative. Liver functions tests showed normal enzyme and protein values; but high total bilirubin (104mmol/L) and unconjugated bilirubin- 17.4umol/L. Serum uric acid was normal. HIV, hepatitis B and C were negative. Urinalysis was positive for blood, urobilinogen, bilirubin, with a pH of 8.0. Urine microscopy showed granular, epithelial and red blood cell casts. Glucose-6-phosphate dehydrogenase assay- 12.5 μ/Hb. She was admitted, placed on oxygen, transfused and discharged on the third day in stable condition. However she was brought back to the hospital about 3 months later for similar symptoms. Urgent Hb concentration was 6.0g/dL. Peripheral blood film revealed anisopoikilocytosis, some macrocytes, tear drop cells, polychromasia, basophilic stippling, fragmented red cells and presence of nucleated red cells (12/100 white cells)- a few of which were multinucleated (Fig 1). HPLC showed low HbA (74.1%), severely increased HbF (23.8%) and HbA2 (2.1%). Bone marrow (BM) aspiration showed a hypercellular marrow, erythroid hyperplasia (myeloid/erythroid ratio 1:2), dyserythropoiesis with erythroid multinuclearity in >10% of late erythroid precursors and significant karyorrhexis. Myelopoiesis and megakaryopoiesis were essentially normal (Fig 2). Serum ferritin was elevated (2,658ng/ml). In the absence of availability of electron microscopy or molecular studies, a diagnosis of CDA type II was made based on clinical, laboratory and characteristic bone marrow findings. She was transfused, placed on iron chelation therapy, her parents counseled on treatment options and she is being followed up. Discussion The CDAs are classically grouped into 3 types based on bone marrow morphology. Type I has erythroblasts joined by an internuclear bridge. Type II erythroblasts have multinuclearity of late erythroblasts while type III has gigantoblasts (erythroblasts with ≥8 nuclei). Inheritance is autosomal recessive and diagnosis is usually in childhood or early adult life. Common clinical findings are anaemia, jaundice and splenomegaly; however these are seen in other more common inherited haemolytic anaemias. BM examination is the gold standard for diagnosis. Patients with CDA usually have high serum ferritin that may require iron chelation therapy. CDA although rare must be considered in a child who has recurrent anaemia in whom other causes have been excluded. BM examination remains a key diagnostic tool in identification of the CDAs. Fig 1: Blood Film Fig 1:. Blood Film Fig 2: Bone Marrow Fig 2:. Bone Marrow Disclosures No relevant conflicts of interest to declare.

Congenital dyserythropoietic anemia type I (CDA I) is an autosomal recessive disorder marked by specific morphological abnormalities of erythroblasts in the bone marrow, resulting in ineffective erythropoiesis and anemia. CDA I principally affects the erythroid lineage, while other non-erythroid hematopoietic lineages are unaffected. Light microscopy of bone marrow shows erythroid hyperplasia and binucleated erythroblasts connected by internuclear bridges. Electron microscopy of bone marrow demonstrates erythroblasts with spongy heterochromatin and invagination of the nuclear membrane. The gene responsible for causing CDA I, CDAN1, encodes for a ubiquitous protein, codanin-1, with unknown function. The goal of our study is to understand how codanin-1 regulates erythropoiesis and how codanin-1 defects result in CDA I. To determine codanin-1 expression pattern during erythroid differentiation, we analyzed codanin-1 RNA and protein levels in erythroid primary cells and cell lines. RNA-seq conducted in erythroid-induced human primary CD34+ cells revealed codanin-1 RNA expression is maintained in erythroid lineage. Similarly, immunoblotting shows codanin-1 protein is expressed at high levels in human erythroleukemia cell line stimulated down the erythroid lineage. Codanin-1 has been identified as a chromatin-binding protein. Thus, we conducted ChIP-seq analysis to determine the location of DNA binding sites for codanin-1 in K562 cells. The ChIP-seq results identified 2534 binding sites for codanin-1. Interestingly, many of these sites belong to key erythroid genes. Furthermore, 28% of these DNA binding sites overlap with GATA-1 bound sites. Because putative loss of function mutations in CDAN1 result in CDA I, we studied the effects of codanin-1 gene silencing on erythroid differentiation. shRNA-mediated knockdown reduced codanin-1 mRNA by 50% and protein expression levels by 90%. To assess the effects of codanin-1 perturbation on erythroid differentiation, hemoglobin synthesis was quantified using Drabkin’s reagent in which the absorbance measured at 540 nm is proportional to hemoglobin content in the sample. Erythroid-induced knockdown cells displayed decreased hemoglobin content when compared to non-targeting control shRNA (0.20 versus 0.48, measured in 3 separate experiments; p To establish a robust CDA I cell system, the first well-characterized CDAN1 mutation (R1042W) was studied because codanin-1 deletion mutants are embryonic lethal in mice and do not occur in humans. Endogenous codanin-1 knockdown cells were transduced with mutant or wild type codanin-1 and induced down the erythroid lineage. Because CDA I is characterized by abnormal heterochromatin appearance, we conducted electron microscopy studies to examine the ultrastructural features. Studies show that knockdown of endogenous codanin-1 coupled with ectopic expression of mutant codanin-1 results in recapitulation of the CDA I phenotype in the K562 cell line model. Because CDA I is almost exclusively restricted to the erythroid lineage, understanding its pathophysiology has the potential to identify critical pathways in erythropoiesis. Here we show that the full phenotype of CDA I is recapitulated upon not only the loss of function of the codanin-1 protein but also on the expression of the mutant form of codanin-1. Our analysis demonstrates that codanin-1 binds to important erythroid loci, suggesting that codanin-1 affects the expression of erythroid genes. Further work will focus on studying codanin-1 function in human primary erythroid cells and in animal models. Disclosures No relevant conflicts of interest to declare.

Although the physiological role of tissue-specific translational control of gene expression in mammals has long been suspected on the basis of biochemical studies, direct evidence has been lacking. Here, we report on the targeted disruption of the gene encoding the heme-regulated eIF2alpha kinase (HRI) in mice. We establish that HRI, which is expressed predominantly in erythroid cells, regulates the synthesis of both alpha- and beta-globins in red blood cell (RBC) precursors by inhibiting the general translation initiation factor eIF2. This inhibition occurs when the intracellular concentration of heme declines, thereby preventing the synthesis of globin peptides in excess of heme. In iron-deficient HRI(-/-) mice, globins devoid of heme aggregated within the RBC and its precursors, resulting in a hyperchromic, normocytic anemia with decreased RBC counts, compensatory erythroid hyperplasia and accelerated apoptosis in bone marrow and spleen. Thus, HRI is a physiological regulator of gene expression and cell survival in the erythroid lineage.

A biópsia de medula óssea propicia a avaliação da celularidade global, dos precursores imaturos de localização anormal e de fibrose nas sídromes mielodisplásicas. O método tem sido considerado importante também para o diagnóstico e prognóstico da síndrome. O objetivo deste estudo foi o de observar a influência de parâmetros histológicos como a celularidade, a relação eritróide-mielóide, a presença de precursores imaturos de localização anormal e fibrose medular na sobrevida de pacientes com a síndrome. De abril de 1985 a junho de 1998, 46 pacientes diagnosticados segundo os critérios do grupo Franco, Americano, Britânico foram estudados. A casuística era composta de 20 pacientes do sexo masculino e de 26 do sexo feminino com idade média de 61 anos. Foram revisados 46 esfregaços de aspirado de medula óssea e 36 cortes histológicos de biópsia de medula óssea. A sobrevida média dos casos de hipocelularidade foi de 64,8 meses e dos casos que eram hiper ou normocelulares foi de 31.8 meses. Pacientes com a predominância de hiperplasia tiveram sobrevida média de 50,8 meses, que foi superior aos que apresentavam hiperplasia mielóide (20,3 meses). Não houve diferença estatística na sobrevida dos pacientes que apresentaram ou não fibrose medular. A biópsia de medula óssea deve ser considerada útil na identificação de parâmetros que influenciam no prognóstico da síndrome mielodisplásica. A hipocelularidade e a hiperplasia eritrocitária está relacionada com a sobrevida maior, enquanto a hiperplasia mielóide com a sobrevida mais curta. Bone marrow biopsy allows evaluation of cellularity, abnormal localization of immature precursors and fibrosis in myelodysplastic syndrome. It has been considered important to make diagnosis and prognosis of this disorder. The object of this study evaluated the influence of histopathological parameters, such as cellularity, erythroid/myeloid ratio, abnormal localization of immature precursors and marrow fibrosis, on survival of myelodysplastic syndrome patients. Forty-six patients, admitted from April 1985 to June 1998, and diagnosed as being myelodysplastic syndrome according to French-American-British criteria, were selected. There were 20 males and 26 females, with median age of 61 years. Forty-six bone marrow smears and 36 trephine biopsies were reviewed. Mean survival of hypocellular cases was 64.8 months and of hyper and normocellular cases was 31.8 months. Patients with predominance of erythroid hyperplasia had mean survival of 50.8 months, greater than those with predominance of myeloid hyperplasia (20.3 months). There was no statistical difference in survival of patients with or without abnormal localization of immature precursors and with or without marrow fibrosis. Bone marrow biopsy is a useful tool for the identification of parameters that influence prognosis in myelodysplastic syndrome. Hypocellularity and erythroid hyperplasia were correlated with longer survival while myeloid hyperplasia with poorer survival. Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Hematologia e Hemoterapia Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de Patologia Aplicada UNIFESP, EPM, Hematologia e Hemoterapia UNIFESP, EPM, Depto. de Patologia Aplicada SciELO

A 57-year-old woman presented because of swelling of the abdomen and legs for several weeks. There was a history of “elevated blood count.” Several years earlier, a bone marrow biopsy was performed and was reported as “mildly hypocellular” with erythroid hyperplasia and decreased iron stores. For diagnosis and discussion, see the following page. DIAGNOSIS: Budd-Chiari syndrome (BCS) secondary to paroxysmal nocturnal hemoglobulinuria.

The effects of both intravenous administration of recombinant human erythropoietin (rhEPO) and blood withdrawal, either individually or in combination, on the frequency of micronucleated immature erythrocytes (MIE) in the bone marrow were investigated in 10-week old male Han Wistar rats. Clear increases in MIE frequency and evidence of erythroid hyperplasia were seen in the bone marrow after 1350 IU kg−1 rhEPO but only marginal increases in MIE were seen at the lower dose of 135 IU kg−1. At the lower dose, blood levels of rhEPO were found to be approximately 120 pg mL−1 within one hour of dosing. Withdrawal of 5 blood samples of 0.45 mL on each occasion during 24 hours, the maximum allowed by the Home Office licence, did not increase MIE frequency but did result in levels of endogenous EPO of 15–19 pg mL−1 24 to 72 hours after the start of treatment, with some evidence of increased erythropoiesis. Although the increased plasma amounts of EPO, resulting from blood withdrawal, were lower than the blood levels achieved by the administration of exogenous rhEPO that were required to give increases in micronucleus frequency, the possibility that blood withdrawal might influence the MIE response to genotoxins cannot be totally excluded without further investigation.

Congenital dyserythropoietic anaemia type III (CDA III) is a rare disease inherited in an autosomal dominant way and characterized by mild to moderate haemolytic anaemia. Most patients are adapted to their disease and have no or few complaints. Bone marrow examination shows a characteristic picture with erythroid hyperplasia and multinucleate erythroblasts. 20% of patients in a Swedish family affected with the CDA III condition have monoclonal gammopathy or multiple myeloma. By linkage and recombination analysis in the same family, the gene linked to the CDA III condition (CDAN3) has been located to chromosome 15q22. In this paper we report the observation of visual disturbances with macular degeneration and angioid streaks in six patients with CDA III and discuss the apparent association between CDA III, angioid streaks and monoclonal gammopathy. We suggest that this triad forms a previously unreported syndrome.

The prognostic value of clinicopathological parameters, recorded at diagnosis, in idiopathic myelofibrosis (IMF) was retrospectively analyzed in a consecutive series of 32 patients followed for a minimum of 5 and maximum of 134 months in the period 1983-1994. Of the 32 patients, 18 were males and 14 were females. The mean age was 59.8 years (S.D., 10.4; range, 44-78 years). At the time of closure of the study (May 1995), 10 patients were dead, 18 were alive, and four were lost to follow up. The mean survival was 41 months (range 2-130 months). Univariate analysis showed that the following features were associated with a significantly shorter survival: (1) anemia (hematocrit30%), (2) platelet count100,000/mm3. Age, sex, size of spleen and liver, WBC count, increased reticulin and collagen fibrosis were not of prognostic significance. Clinicopathological correlation was found between the sinusal hemopoiesis and the number of WBC precursors in peripheral blood, and between WBC precursors and spleen size. Medullary fibrosis was found to be associated with striking predominance of large and very mature megakaryocytes. Erythroid hyperplasia in the bone marrow correlated negatively with WBC precursors and spleen and liver size, and positively with reticulocyte count.

Investigations showed that the patient had severe haemolytic anaemia on presentation, the haemoglobin was 6.2 g/dl, with a positive haemolytic screen of reticulocytosis, indirect hyperbilirubinaemia and erythroid hyperplasia in the bone marrow. According to the blood picture it is a spherocytic anaemia and the positive DAT indicates that it is immune mediated. Since it is positive for both C3d and IgG, it is considered as warm autoimmune hemolytic anaemia. However, subsequently her urine was strongly positive for haemosiderin which showed evidence of intravascular haemolysis. References

The survival, self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPC) are tightly regulated by extrinsic signals, and intrinsically by transcription factors and their regulatory networks. The molecular and cellular mechanisms, which regulate the complex process of hematopoiesis, depend upon the correct expression of transcription factors and their regulators. One such family of regulators is the inhibitor of DNA binding/differentiation (Id), which is helix-loop-helix proteins that function by acting as dominant negative regulators of transcription factors such as E proteins, ETS, Pax, and retinoblastoma proteins. Expression of Id2, one of the Id family proteins, is regulated by growth factor independence-1 (Gfi-1) encoding a transcriptional repressor. Gfi-1 is required for the development of multiple cell lineages including HSPC and ultimately differentiated blood cells. Although genes have been identified to mediate hematopoietic defects observed in Gfi-1 knockout (Gfi-1 KO) mice including the maturational and developmental defects in granulocyte (CSF-1, RasGRP1, and PU.1) and B cell (PU.1 or Id2), and myeloid hyperplasia (Id2 or HoxA9), Gfi-1-target genes that mediate the defects in radioprotection, maintenance of HSC, and erythroid hyperplasia in Gfi-1 KO mice are unknown. Since Id2 expression is elevated in HSPC of Gfi-1 KO mice and Id2 promotes cell proliferation, we hypothesized that lowering Id2 expression could rescue the HSPC defects in the Gfi-1 KO mice. By transplanting Gfi-1 KO mouse bone marrow cells (BMC) into lethally-irradiated recipient mice, we observed that short-term reconstituting cell (STRC) activity in Gfi-1 KO BMC is rescued by transplanting Gfi-1 KO; Id2 Het (heterozygosity at the Id2 locus) BMC, while the long-term reconstitution defect of HSC was not. Interestingly, lineage- Sca-1- c-Kithi HPC, which enriched for megakaryocyte-erythroid progenitor (MEP) as one of the STRC, were fully restored in mice transplanted with Gfi-1 KO; Id2 Het BMC, in contrast to lack of the HPC in Gfi-1 KO BM-transplanted mice. The restoration of donor c-Kithi HPC was directly correlated with increased red blood cell (RBC) levels in recipient mice, which was produced after donor BM engraftment. Furthermore, we identified that reduced Id2 levels restore erythroid cell development by rescuing short-term hematopoietic stem cell, common myeloid progenitor and MEP in the Gfi-1 KO mice. In addition, burst forming unit-erythroid (BFU-E) colony assay showed that hemoglobinized BFU-E development was restored in Gfi-1 KO BM and spleen by lowering Id2 levels. Unlike Id2 reduction, reducing other Id family (Id1 or Id3) levels in Gfi-1 KO mice does not rescue the impaired development of erythroid and other hematopoietic lineages including myeloid, T and B cells. Abnormal expansion of CD71+ Ter119-/low erythroid progenitor cells was rescued in Gfi-1 KO; Id2 Het BMC compared to those in Gfi-1 KO mice. Thus, we hypothesized that erythroid development was blocked at the early stage of erythropoiesis due to the ectopic expression of Id2 in Gfi-1 KO mice. Using Id2 promoter-driven YFP reporter mice, we found that Id2 is highly expressed in the CD71+ Ter119-/low erythroid progenitors, and decreases as the cells mature to pro-erythroblasts and erythroblasts, suggesting that repression of Id2 expression is required for proper erythroid differentiation in the later stages. The dramatic changes of Id2 expression during erythroid development support our findings that the overexpression of Id2 in the absence of Gfi-1-mediated transcriptional repression causes impaired erythropoiesis at the early stage. To identify the molecular mechanisms that could account for how reduced Id2 levels rescue erythropoiesis in Gfi-1 KO mice, we compared the expression of genes and proteins in Gfi-1 KO; Id2 Het and Gfi-1 KO BMC. Using microarray, qRT-PCR and western blot, we discovered that reduction of Id2 expression in Gfi-1 KO BMC results in increased expression of Gata1, EKlf, and EpoR genes, which are required for erythropoiesis. However, the expression levels of cell cycle regulators were not altered by lowering Id2 expression in Gfi-1 KO mice. These data suggest a novel molecular mechanism in which Gfi-1 modulates erythropoiesis by repressing the expression of Id2 that reduce the levels of Id2 protein, binding to E2A and inhibiting the formation of E2A/Scl transcription enhancer complex. Disclosures: No relevant conflicts of interest to declare.

A 60 year old woman with congenital dyserythropoietic anaemia (CDA) type III developed a malignant T cell lymphoma with cutaneous and widespread nodal involvement. Bone marrow aspirates showed erythroid hyperplasia and dyserythropoiesis with multinucleate erythroblasts and gigantoblasts, in keeping with CDA type III. Electron microscopy showed multinucleate erythroblasts with notably irregular nuclear outlines and intranuclear clefts. The development of malignant lymphoma in this patient, together with a documented high prevalence of monoclonal gammopathy and multiple myeloma and a single case of Hodgkin's disease, may indicate an increased incidence of lymphoproliferative disease in CDA type III.

Polycythemia vera (PV) was diagnosed in a four-year-old domestic shorthair evaluated for hind-limb ataxia, extension of all claws, and difficulty in jumping to elevated surfaces. Mild cardiac hypertrophy also was diagnosed. Initial laboratory evaluation revealed polycythemia (packed cell volume [PCV], 75%) and normal serum total protein (7.5 g/dl). Definitive diagnosis of PV was reached by excluding causes of relative and secondary absolute polycythemia using radiography, ultrasonography, and blood gases, and by measuring serum erythropoietin concentration by radioimmunoassay (13 mU/ml) and an enzyme-linked immunosorbent assay (ELISA) method (8.0 mU/ml). Bone-marrow biopsy revealed relative erythroid hyperplasia characteristic of myeloproliferative disease. Clinical signs were controlled with hydroxyurea (12.2 mg/kg body weight) and occasional phlebotomy. Polycythemia vera is an uncommon feline disease, and clinical reports on the use of hydroxyurea to manage the condition in the cat are lacking.

Abstract 248 β-thalassemia, the most common congenital anemia, is caused by mutations in β-globin gene resulting in partial or complete absence of β-globin protein chains. In the absence of properly paired α- and β-globin chains, the α-globin protein accumulates causing proteotoxicity and apoptosis of erythroid cells. Hemolysis and ineffective erythropoiesis together cause severe anemia in thalassemia syndromes. Increased proliferation with arrest of terminal erythroid differentiation and accelerated apoptosis is the hallmark of ineffective erythropoiesis in β-thalassemia. In chronic patients, blood transfusions are required for survival, but result in severe iron overloading. Non-transfusion dependent thalassemia (NTDT) patients however, are also affected by ineffective erythropoiesis, anemia and iron overload. Recombinant EPO therapy is ineffective and rarely used for β-thalassemia patients, as it does not affect the later stages of erythroid differentiation. Therefore, a pharmacological approach is necessary that can increase hemoglobin levels, prevent splenomegaly, bone abnormalities and iron overloading in β-thalassemia patients. Several members of the TGFβ-superfamily are involved in erythropoiesis. ACE-536 is a modified activin type IIb (ActRIIb) receptor fusion protein that acts as a ligand trap. Unlike wild type ActRIIb, ACE-536 does not inhibit activin A induced signaling but inhibits signaling induced by other members of the TGF-β superfamily such as GDF11. While EPO increases proliferation of erythroid progenitors, ACE-536 promotes maturation of terminally differentiating erythroblasts. We hypothesized that ACE-536 treatment will promote terminal erythroid differentiation, as well as reduce anemia, ineffective erythropoiesis and associated co-morbidities in β-thalassemia. We investigated the efficacy of RAP-536 (murine ortholog of ACE-536) in a mouse model of β-thalassemia intermedia (Hbbth1/th1). β-thalassemic mice were severely anemic and had significantly decreased RBC (−31.6% p Following two months of treatment, RAP-536 treated β-thalassemic mice had significantly increased RBC (+32.9%, p In summary, these data demonstrate that RAP-536 attenuates ineffective erythropoiesis, ameliorates anemia and improved associated co-morbidities in a murine model of β-thalassemia. ACE-536 represents a novel potential therapy for patients with β-thalassemia and these preclinical data provide a rationale for clinical studies of ACE-536 in β-thalassemia patients. Disclosures: Suragani: Acceleron Pharma Inc: Employment, Equity Ownership. Li:Acceleron Pharma Inc: Employment, Equity Ownership. Cawley:Acceleron Pharma Inc: Employment. Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding. Pearsall:Acceleron Pharma Inc: Employment, Equity Ownership. Kumar:Acceleron Pharma Inc: Employment, Equity Ownership.

Abstract 2407 Background and Methods: Cyclin E-Cdk2 is a cell cycle regulator that promotes the G1-S transition by phosphorylating proteins involved in cell division and genome duplication. Cyclin E activity is frequently increased in many cancers, usually as a result of deregulation of mechanisms that control cyclin E protein levels and activity. Cyclin E T74A/T393A mice, in which cyclin E has been rendered resistant to degradation by the Fbw7 ubiquitin ligase by ablating two phosphorylation sites, exhibit enhanced epithelial proliferation and erythroid hyperplasia with inefficient erythropoiesis and macrocytic anemia. However, despite this widespread hyperproliferation, cyclin E T74A/T393A mice do not develop spontaneous tumors. To accelerate tumorigenesis and identify cooperating oncogenes in cyclin E T74A/T393A mice that complement the block in erythroid differentiation, we performed an insertional mutagenesis screen using the Sleeping Beauty (SB) transposon system. Results: Expression of SB by the inducible Mx-Cre caused rapid leukemiagenesis within 9–15 weeks post poly (I/C) injection in both control and cyclin E T74AT393A mice. The majority of tumors were T-cell lymphomas/leukemias and erythroid leukemias. Surprisingly, both wild type and cyclin E T74AT393A mice developed erythroid leukemias at similar frequencies. The erythroleukemia were composed of homogeneous populations of immature proerythroblasts akin to pure erythroleukemia in humans. Immunophenotypic profiling showed dim CD45 with variable CD117 (c-kit) and CD71 (transferrin receptor) expression. Preliminary sequencing of 29 erythroid leukemias identified 27 common insertion sites (CIS), including several genes associated with AML (Flt3, Erg and Ets1) as well as erythropoiesis (Fli-1 and Epo), highlighting the specificity of the SB-induced mutations for myeloid and erythroid leukemias. Preliminary analyses have also revealed preferential gCISs in erythroid leukemias isolated from cyclin E T74AT393A mice. Conclusion: We conclude that the Mx-Cre SB mutagenesis system provides a unique and rapid murine model of pure erythroid leukemia. We have identified a set of genes that promote erythroleukemia in mice may represent potential therapeutic targets for human erythroid and myeloid leukemias. Disclosures: No relevant conflicts of interest to declare.

Abstract 3796 Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell disorders characterized by peripheral blood cytopenias such as anemia, neutropenia or thrombocytopenia. Ineffective erythropoiesis due to increased proliferation and abortive maturation of precursors leads to severe anemia, the most common cytopenia observed in MDS syndromes. Despite elevated erythropoietin (EPO) and erythroid hyperplasia, MDS patients are often given recombinant EPO therapy to stimulate erythropoiesis. However, only a small proportion of patients respond to EPO therapy. Frequent blood transfusions as supportive care result in iron overloading and recently iron overloading is also linked to enhanced progression to AML. Therefore, alternative therapies are necessary to treat anemia in MDS patients. Signaling by members of the TGFβ superfamily are known regulators of erythropoiesis. We developed ACE-536, a ligand trap consisting of a modified activin receptor Type IIB extracellular domain linked to a human Fc domain. In vitro assays revealed that ACE-536 inhibits smad 2/3 ligands of the signaling pathway but not smad 1/5/8 ligands. Dose dependent studies using ACE-536 in mice, rats and monkeys revealed that ACE-536 treatment resulted in increased red blood parameters but did not affect other cell types. These data suggests that ACE-536 inhibits smad 2/3 phosphorylation modulating the expression of downstream genes involved in erythroid development pathway. BFU-E and CFU-E colony formation assays from bone marrow and spleen in mice following ACE-536 treatment revealed that ACE-536 did not affect the proliferation stages of erythropoiesis. In mice, terminal erythroid differentiation analysis by flow cytometry at 72hrs following RAP-536 (10mg/kg) treatment demonstrated decreased basophilic and increased ortho- and poly-chromatophilic erythroblasts and reticulocytes compared to VEH treatment. Cell cycle analysis of bone marrow and splenic erythroblasts counterstained with BrdU and 7-AAD after RAP-536 (10mg/kg, for 24 hours) or VEH treatment to EPO pre-treated (1500 units/kg, for 40 hours) mice (N=5/group) revealed that EPO+RAP-536 treatment resulted in significant decrease in S-phase and increase in G1/G2-phases of cell cycle compared to EPO+VEH treatment. In addition, EPO+RAP-536 treatment resulted in a greater increase in RBC parameters than either of the treatments alone. Together, these results demonstrate that ACE-536 increases red blood cell formation by promoting maturation of late stage erythroblasts. We then investigated the effect of ACE-536 on anemia in NUP98-HOXD13 (NHD13) transgenic murine model of MDS. NHD13 mice develop anemia, neutropenia and lymphopenia, with normal or hyper cellular bone marrow. A Majority of the mice die by 14 months due to severe pancytopenia or progression to acute myeloid leukemia. In this study, mice were divided into three groups based on age. Early (∼4 months old), mid (∼8 months old) and late stage (∼10 months) groups were randomized and dosed with either RAP-536 at 10 mg/kg or VEH twice per week for 6–8 weeks. NHD13 mice in each group had severe anemia characterized by reduced RBC, Hemoglobin and HCT and compared to wild-type littermates prior to treatment. Treatment of RAP-536 for 6–8 weeks significantly increased RBC parameters and reversed anemia at all stages. Peripheral blood smear analysis revealed no indication of increased leukemic progression due to RAP-536 treatment. Cell differential and flow cytometric evaluation of erythroid precursors from bone marrow demonstrated decreased erythroid precursors and hyperplasia after RAP-536 treatment compared to vehicle treated control. Our data demonstrate that RAP-536 can increase hematology parameters by enhancing maturation of terminally differentiated red blood cells. We have shown RAP-536 corrects ineffective erythropoiesis, decreases erythroid hyperplasia and normalizes myeloid: erythroid ratios without enhanced progression to AML in a murine MDS model. Therefore ACE-536 may represent a novel treatment for anemia associated with MDS, particularly in patients that are refractory to EPO therapy. ACE-536 has completed Phase I clinical trials in healthy human volunteers and Phase II study in MDS patients is planned. Disclosures: Suragani: Acceleron Pharma Inc: Employment, Equity Ownership. Li:Acceleron Pharma Inc: Employment, Equity Ownership. Sako:Acceleron Pharma Inc: Employment, Equity Ownership. Grinberg:Acceleron Pharma Inc: Employment, Equity Ownership. Pearsall:Acceleron Pharma Inc: Employment, Equity Ownership. Kumar:Acceleron Pharma Inc: Employment, Equity Ownership.

Abstract 2154 Congenital dyserythropoietic anemias (CDA) are a rare, inherited form of blood disorders characterized by dyserythropoiesis in the bone marrow, anemia, jaundice and splenomegaly. There are three major types of CDAs, although rarer variants have been identified. We describe a patient with an unusual type of CDA, of which only four other cases have been reported. Our patient had severe hemolytic anemia, increased fetal hemoglobin and abnormal bone marrow pathology inconsistent with previously described forms of CDA. On further study he was also found to have a mutation in KLF1, the gene encoding Erythroid-Kruppel like growth factor (EKLF). Here we describe the full clinical characteristics of our patient and define the diagnostic clinical features of this new variant of CDA by comparing with one of the previously reported patients designated as ME in the table. EKLF is an erythroid specific transcription factor that is essential for b-globin expression, the switch from fetal to adult globin and definitive erythropoiesis (Siatecka, M and Bieker, J. Blood prepublished May 2011). Various mutations in KLF1 have been identified, some causing the benign In(Lu) type of Lu blood group phenotype. Recently, a missense, dominant-negative KLF1 mutation was reported, c.973G>A, which resulted in a previously unidentified type of CDA (Singleton et al. ASH Abstract 162, 2009; Arnaud et al. Am J Hum Genet 2010,87:721-727). The G-to-A transition in exon 3 of KLF1 results in the substitution of glutamate 325 by a lysine (E325K) in the second zinc finger. The mutated area of the zinc finger was found to be essential for binding of EKLF to DNA motifs causing a profound dysregulation of globin gene expression. The mutation was found to have a dominant-negative effect on the transcriptional activity of EKLF, thus making the heterozygous patients symptomatic. Our patient, JL, is an 8 year old male Taiwanese immigrant found to have hyperbilirubinemia and anemia at birth. He is a developmentally normal child with height 10th centile, weight in the 25th centile and spleen palpable to his suprapubic area. He has chronic hemolytic anemia, with baseline hemoglobin 7–9 g/dL, MCV 83 fL and RDW 22% and reticulocyte count 16%. Peripheral blood smear shows marked anisopoikilocytosis, schistocytes, mild polychromasia and nucleated RBCs, many with double nuclei. Bone marrow aspirate revealed a hypercellular marrow with erythroid hyperplasia and dyserythropoiesis. Electron microscopy analysis of the bone marrow showed rare immature erythroid cells with marked heterochromatin. Several cells showed a peripheral double membrane of the cytoplasm and there was rare invagination of nuclear membrane with intranuclear precipitated material. JL received blood transfusions every 2 months for the first 3 years of his life while living in Taiwan. Since moving to the U.S. in 2003, he has only received 2 transfusions secondary to aplastic crisis. Osmotic fragility testing showed mildly increased increased fragility. Hemoglobin electrophoresis revealed an elevated fetal hemoglobin level of 42%. Gene analysis for alpha or beta globin mutations was negative. RBC enzyme testing revealed an ADA of 6.1 and decreased FFK. Due to the unique combination of anemia, elevated fetal hemoglobin, and bone marrow morphology suggestive of, but not fully diagnostic for CDA I, II or III, we tested his EKLF gene and identified the heterozygous E325K mutation. JL's two siblings, mother and father have normal hemoglobin levels and peripheral blood smears. They also have normal In(Lu) blood group phenotype. Since the E325K mutation is dominant-negative, his phenotypically normal family members were not tested for the mutation. Four other patients have also been identified as having an E325K mutation in their EKLF gene. The patients had severe hemolytic anemia, elevated fetal hemoglobin, and bone marrow morphology showing dyserythropoiesis. Patients' erythrocytes also had low CD44 and water channel AQP1 expression, which is known to be regulated by EKLF. One patient was described with multiple congenital anomalies: hepatomegaly, micropenis, hypospadias, enlarged fontanel and hypertelorism. Taken together, the key clinical characteristics of this rare CDA are: severe normocytic anemia, highly elevated Hb F, presence of nucleated RBCs in the peripheral blood, erythroid hyperplasia with limited dyserythropoiesis in the bone marrow, splenomegaly, and growth delay. Disclosures: No relevant conflicts of interest to declare.

Abstract 1672 Introduction: According to the WHO 2008, acute erythroid leukemia (AEL) is defined by erythroid hyperplasia ≥50% and ≥20% of myeloblasts of non-erythroid cells but Patients/Methods: The cohort consisted of 225 pts (f/78; m/147; median age, 68.8 yrs; 18.5–88.4 yrs) with BM erythroid hyperplasia (≥50%) and different myeloid subtypes strictly defined according to WHO 2008: MDS: n=107; AML-MRC/NOS: n=32; AEL: n=79, pure AEL: n=7 (the WHO cohort “AML with recurrent genetic abnormalities” was excluded). All pts were investigated by MGG staining of BM and chromosome banding/FISH. In addition, we performed analysis for the NPM1 mutations (n=126 investigated), FLT3-ITD (n=135), MLL-PTD (n=136), and NRAS mutations (n=90). Results: MDS subtypes were as follows: RA: n=18; RARS: n=18; RCMD: n=21; RCMD-RS (WHO 2001): n=26; RAEB-1: n=22; RAEB-2: n=2. Most AML pts were categorized as “AML with myelodysplasia related changes; AML-MRC” (27/30 cases; 90%); 3 pts were classified as “AML, not otherwise specified; AML-NOS”, 2 pts were not evaluable for this aspect. We first compared the MDS cohort (n=107) with the AML cohort (all 118 pts with AML-MRC/NOS, AEL, and pure AEL): Overall survival (OS) was better in MDS than in the AML cohort (median: not reached vs. 13.9 months; p Conclusions: MDS with erythroid hyperplasia (≥50%) was clearly separated from the AML cohort (consisting of AML-MRC/NOS, AEL, and pure AEL, all with ≥50% of erythropoiesis) by less adverse cytogenetics and by improved survival. In contrast, no significant differences were observed across the different acute leukemia subentities regarding prognosis and cyto-/molecular genetic features. These data support the separation of MDS and AML with ≥50% of erythroid precursors according to the WHO classification. However, with respect to different AML subgroups, the separation to AEL, pure AEL, and AML-MRC/-NOS having ≥50% erythropoiesis seems arbitrary: these AML subtypes show no significant differences regarding prognosis or genetic risk profiles. This argues in favor of a combined group of AML with erythroid hyperplasia aiming to facilitate the definition for clinical studies and the development of therapeutic strategies. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Different fusion oncogenes in acute myeloid leukemia (AML) have distinct clinical and laboratory features suggesting different modes of malignant transformation. Here we compare the in vitro effects of representatives of 4 major groups of AML fusion oncogenes on primary human CD34+ cells. As expected from their clinical similarities, MLL-AF9 and NUP98-HOXA9 had very similar effects in vitro. They both caused erythroid hyperplasia and a clear block in erythroid and myeloid maturation. On the other hand, AML1-ETO and PML-RARA had only modest effects on myeloid and erythroid differentiation. All oncogenes except PML-RARA caused a dramatic increase in long-term proliferation and self-renewal. Gene expression profiling revealed two distinct temporal patterns of gene deregulation. Gene deregulation by MLL-AF9 and NUP98-HOXA9 peaked 3 days after transduction. In contrast, the vast majority of gene deregulation by AML1-ETO and PML-RARA occurred within 6 hours, followed by a dramatic drop in the numbers of deregulated genes. Interestingly, the p53 inhibitor MDM2 was upregulated by AML1-ETO at 6 hours. Nutlin-3, an inhibitor of the interaction between MDM2 and p53, specifically inhibited the proliferation and self-renewal of primary human CD34+ cells transduced with AML1-ETO, suggesting that MDM2 upregulation plays a role in cell transformation by AML1-ETO. These data show that differences among AML fusion oncogenes can be recapitulated in vitro using primary human CD34+ cells and that early gene expression profiling in these cells can reveal potential drug targets in AML.

A 26-year-old Chinese-Malaysian female patient with beta-thalassemia is presented. The main hematological values found in this patient were as follows: 1) normocytic hypochromic anemia (RBC 444 x 10(4)/microliters, Hb 11.8 g/dl) with marked anisopoikilocytosis, 2) erythroid hyperplasia, and 3) increased HbF (HbA 41.4%, HbA2 2.9%, HbF 48.9%). DNA obtained from peripheral leukocytes was analyzed using dot blot hybridization of the polymerase chain reaction (PCR)-amplified DNA with allele-specific oligonucleotide probes. A C----T substitution at position 654 of the second intervening sequence (IVS-2) was detected in her beta-globin clone.

This report describes a case of congenital dyserythropoietic anemia (CDA) type I diagnosed in a 22-year-old woman with a two year history of macrocytic anemia. Light microscopy study of bone marrow disclosed only minor and nonspecific findings, without erythroid hyperplasia. Electron microscopy of bone marrow erythroblasts surprisingly showed most of the classical features of CDA type I. Electron microscopy may be an important tool for the diagnosis of macrocytic anemia of obscure origin, especially in young patients.

Intact transferrin receptor molecules complexed with transferrin were found in human plasma. The concentration of receptors was determined by an enzyme-linked immunosorbent assay that uses polyclonal antibodies. The mean concentration of 8,279 micrograms/L in 56 normal adults appears to be unrelated to age or sex. Additional receptor measurements were performed on plasmas from 260 subjects with erythropoietic disorders. Decreased concentration of plasma receptors was found in patients with erythroid hypoplasia and increased numbers in those with erythroid hyperplasia. Ferrokinetic measurements of erythropoiesis were compared with numbers of receptors in 148 subjects, and a close correlation was found (r = .86). Both sets of values, measured in different conditions and expressed in relation to normal, were consistent with expected values. Receptor values were unproportionally increased only in conditions of iron deficiency. It is concluded that plasma receptors have a constant relationship to tissue receptors, and their number in most instances reflects the rate of erythropoiesis.

Background: V617F gain-of-function mutation in exon 14 of Janus Kinase 2 gene (JAK2) has been recognized as a major pathogenetic event of many cases of chronic myeloproliferative disorders, in particular in Polycythemia Vera (PV). The molecular basis of myeloproliferative disorders in patients without the V617F is still unclear but, recently, four new heterozygous allelic variants affecting JAK2 exon 12 have been described in V617F negative patients receiving a diagnosis of PV or Idiopathic Erythrocytosis (IE) (Scott et al, NEJM 2007). Revised WHO criteria for PV have been proposed considering these mutations as indicative for PV diagnosis (Tefferi et al, 2007). Aim: To determine the JAK2 exon 12 mutational status in patients with IE in a cohort of 40 consecutive IE V617F negative patients; 2 PV patients, V617F negative, were also included. 36 males and 6 females with a median age of 63 years (range: 25–85), median follow-up 109 months (range: 7–205) were studied. IE was diagnosed in presence of increased haemoglobin (>186 gr/L) and hematocrit levels (>51%) and normal white blood cells and platelet counts. In addition, no splenomegaly, and a normal or low serum erythropoietin (Epo) levels were present along with a bone marrow biopsy showing erythroid hyperplasia with normal megakaryopoiesis and granulopoiesis. They were treated only with phlebotomy therapy. PV was diagnosed according to the WHO criteria. Methods: Granulocytes were isolated from the lower interface of a density gradient separation of peripheral blood samples; the granulocytes were then submitted to erythrocytes lysis (mean purity of the granulocytes 97%) and DNA was extracted. Exon 12 was amplified using the couple of JAK2exon12F (5′-ctcctctttggagcaattca-3′) and JAK2exon12R (5′-caatgtcacatgaatgtaaatcaa-3′) primers. The amplicons were submitted to denaturing high performance liquid chromatography (dHPLC) analysis and runs were performed at 53° C. The amplicons showing a heteroduplex profile were sequenced directly in both strands. Results: 4 of the 40 (10%) IE patients were heterozygous positive for N542-E543del JAK2 exon 12 mutation; both PV patients were negative for JAK2 exon 12 mutations (figure 1). Conclusion: N542-E543del JAK2 was found only in 10% of patients with a diagnosis of IE, a frequency lower than that previously reported (Scott et al, NEJM 2007). This result could be due to the low levels of mutation prevalence in peripheral blood granulocytes. The sensitivity could be increase using erythroid colonies grown on colture media. In addition, different patients selection criteria could explain our results. Accordingly with the proposed revised WHO criteria for Polycythemia, a diagnosis of PV instead of IE should be made in these four patients. figure 1: Clinical features of patients with JAK2 exon 12 mutations and Idiopathic Erythrocytosis. figure 1:. Clinical features of patients with JAK2 exon 12 mutations and Idiopathic Erythrocytosis.

Gene therapy for beta-thalassemia is based on the transplantation of genetically-modified autologous hematopoietic stem cells (HSC) into patients affected by the severe form of disease. The genetic treatment of the hemoglobinopathies poses the general challenge of efficient level of gene transfer into HSC and high and persistent transgene expression, in the differentiated progeny of a genetically modified stem cell. The validation of a gene therapy approach to thalassemia requires to obtain results of gene correction in a broad number of patients’ cells, since different molecular defects in the beta-globin gene lead to the clinical phenotype. The heterogeneity in the molecular defects and in the proportion of alpha and non-alpha (beta, gamma and delta) chains will represent a key element to set a threshold in the amount of vector-derived beta-chain required to correct a thalassemic phenotype. Additionally, the impact of some biological parameters, such as the degree of BM erythroid hyperplasia, the BM subpopulations proportion and the apoptotic index, on the successful correction of thalassemic phenotype needs to be studied in the perspective of clinical translation. In order to address these issues, we collected samples from BM aspirates and isolated CD34+ cells from 25 beta+ and beta0 thalassemic patients, characterized by different genotypes and biochemical profiles of globin chains synthesis. A novel, erythroid specific LV expressing human beta-globin from a minimal promoter enhanced by only 2 LCR elements (HS2 and HS3) was used to transduce BM derived CD34+ cells at high efficiency (>80%). The efficacy of the beta-globin LV in correcting the human thalassemic phenotype was tested in an in vitro model of erythropoiesis and in the human-mouse hematological chimera. Upon transduction, normal level of HbA expression was achieved in erythroblastic cultures and BFU-E, associated with a progression towards erythroid maturation, which was impaired in mock-transduced thalassemic cells. Molecular analysis showed proviral integrity, with no detectable rearrangements and an average proviral copy number of 2.4. Analysis of specific globin chains proportion and contribution to phenotype correction in the context of different genotypes is under evaluation.

Several studies showed that the response rate to standard dose of recombinant Human Erythropoietin (rHuEPO) in MDS patients is generally low, with only few cases presenting a significant increase of haemoglobin (Hb) levels. So, currently, the interest has focused on the use of high dose rHuEPO. The rationale for using high dose rHuEPO was elucidated: residual normal stem cells and/or abnormal clone of MDS stem cells, unresponsive to low levels of endogenous EPO, might respond to high doses of rHuEPO. The aim of this study was to assess the efficacy and safety of high dose rHuEPO treatment. EPO alfa 40.000 IU was given subcutaneously twice weekly for 4 weeks. Twenty-five patients with low-risk MDS (17 RA and 8 RARS) and Hb levels ≤ 10 g/dL were included in this study; sixteen patients were female and 9 male; mean age of enrolled patients was 74 years (range 66 – 85). Twenty-two of 25 patients completed the scheduled treatment and were evaluated for response. At 4 weeks eighteen of 22 patients (81%) showed a Hb mayor response (Hb increase ≥ 2 g/dL); Hb mean value at baseline was 8,15 g/dL (range 7 – 10), at 4 weeks was 13,15 g/dL (range 10 – 14,6). In 4 of 22 patients (19%) the high dose rHuEPO did not induce an increase of Hb levels after 4 weeks of treatment; in addition, these patients needed of RBC transfusions to maintain Hb levels ≥ 8 g/dL. The failure of treatment with rHuEPO occurred in patients with diagnosis of RARS. In our study there were no statistically significant differences between the group of patients with erythroid hyperplasia and the group of patients with normal percent of bone marrow erythroid cells (P = 0,4); no significant difference was noted in response rates between patients with RBC pre-treatment transfusion need and those with stable anaemia without prior transfusion (P = 0,09). In our study, Hb mayor response occurred also in one patient with marked marrow fibrosis. In this study all patients presented defective endogenous EPO production related to their degree of anaemia, with serum EPO levels ≤ 100 mU/ml (mean value 43,5; range 6 – 98). The responder patients need continuous maintenance treatment to maintain their response; EPO alfa 40.000 IU was given subcutaneously once a week; at 12 weeks overall response rate was 77%: 13/18 patients maintained their mayor response, 4/18 patients showed decreased Hb levels in comparison to initial response (Hb decrease > 1 < 2 g/dL), 1 patient progressed on RAEB. Hb mean value at 12 weeks was 11,8 g/dL (range 9,2 – 13,5). The median duration of maintenance of the erythroid response was 7,5 months (range 2 – 24 months). Treatment with high dose of rHuEPO is well tolerated; only one adverse event of arterial hypertension of moderate severity was reported as possible episode related to treatment. In conclusion, our study shows that, in low-risk MDS patients with defective endogenous EPO production, EPO alfa 40.000 IU, given subcutaneous twice weekly for 4 weeks, induces rapid, significant and persistent increase of Hb, without important adverse events; continuous maintenance treatment with 40.000 IU/w is necessary for the majority of the responding patients to maintain their response.

A 45-year-old woman with a 17-year history of vitiligo (panel A) was evaluated because of persistent fatigue. She was found to have pancytopenia (white-cell count, 3200 per cubic millimeter; hemoglobin level, 8.1 g per deciliter; and platelet count, 147,000 per cubic millimeter) with macrocytosis (mean corpuscular volume, 125 μm3) and hypersegmented polymorphonuclear leukocytes (Panel B; hematoxylin and eosin, ×100). The serum vitamin B12 level was 84 pmol per liter (normal range, 220 to 660). The serum folate level was normal. Erythroid hyperplasia with left-shifted megaloblastic erythropoiesis and giant metamyelocytes (Panel C, arrow; hematoxylin and eosin, ×100) was seen . . .