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Pure erythroid leukemia (PEL) is a rare, aggressive subtype of acute myeloid leukemia with a poor prognosis. The diagnosis of PEL is often medically urgent, quite challenging, and is typically a diagnosis of exclusion requiring meticulous distinction from non-neoplastic erythroid proliferations, particularly florid erythroid hyperplasia/regeneration. Given the frequency of TP53 mutations in the molecular signature of PEL, we hypothesize that differential p53 expression by immunohistochemistry (IHC) may be useful in distinguishing PEL versus non-neoplastic erythroid conditions. We performed p53 IHC on 5 normal bone marrow, 46 reactive erythroid proliferations, and 27 PEL cases. We assessed the positivity and intensity of nuclear staining in pronormoblasts and basophilic normoblasts using a 0–3+ scale with 0 being absent (with internal positive controls) and 3 being strong nuclear positivity. A total of 26/27 PEL cases showed strong, uniform, diffuse intense staining by the neoplastic pronormoblasts versus 0/5 and 0/46 normal and reactive controls, respectively. The control cases show various staining patterns ranging from 0 to 3+ in scattered erythroid precursor cells. Uniform, strong p53 positivity is unique to PEL and discriminates this entity from a benign erythroid mimic. Thus, p53 IHC may be a useful marker in urgent medical cases to assist in the confirmation of a malignant PEL diagnosis while awaiting the results of additional ancillary studies such as cytogenetics.

Introduction: Pancytopenia/bicytopenia is not a disease per se rather a spectrum of changes pertaining to reduction in cell numbers. Its presentation as well as causes are diverse in adult and pediatric population. It is imperative to evaluate the causes of pancytopenia/bicytopenia in adults and pediatric subjects as well as to understand the significant differences in their etiology and manifestations. Aims and objectives: Comparative evaluation of pancytopenia/bicytopenia in adult and pediatric patients through hematological parameters and bone marrow studies. Materials and Methods: In this study, 80 adult and 40 pediatric patients of pancytopenia/bicytopenia were evaluated. Detailed history, examination, Hb, TLC, platelet count, MCV, PBS examination, Bone marrow studies were performed. Statistical analysis was done using one-way anova. Result: The commonest presenting age in children was between 12-16 years of age and 19-40 years in adults. Male to female ratio in adults was 1.94:1, while that of pediatric patients was 1:0.74. On bone marrow examination, most common cause of pancytopenia/bicytopenia among pediatric subjects was aplastic anemia (32.5%) followed by megaloblastic anemia(20%), acute leukemia (17.5%) , erythroid hyperplasia (15%). Megaloblastic anemia was the commonest cause of pancytopenia/bicytopenia among adult cases (37.5%) followed by erythroid hyperplasia (20%) and aplastic anemia (10%). Conclusion: Our study concludes that there is statistically significant difference between the values of Hb, TLC, Platelet and MCV among adult and pediatric patients of pancytopenia; implying that these hematologic parameters vary with respect to mean values and range between the two groups. We also conclude nutritional deficiency to be a common cause of pancytopenia in both the age groups. Keywords: Aplastic anemia, Megaloblastic anemia, Pancytopenia.

Background and aimPeripheral cytopaenias and dyspoiesis are common in cirrhosis; however, the prevalence of dyspoiesis and its contribution in cirrhosis-related cytopaenias has not been studied. We aimed to study the bone marrow (BM) dyspoiesis and its impact on peripheral blood cell counts and refractory anaemia in patients with cirrhosis.Patients and methodsWe reviewed all the BM aspirates and biopsies of cirrhotic cases, done from 2011 to 2018 for clinical indications. Dyspoiesis was considered if >5% of the precursor cells of any of the three lineages showed dyspoietic changes. Primary haematological or non-haematological malignancies, chronic kidney disease, drug intake, acute and chronic hepatitis and granulomatous disease were excluded.ResultsOf 608 these, 82 cases (13.5%) showed dyspoiesis in the BM precursors. There was no difference in age (p=0.16), gender (p=0.58) and spleen size (p=0.35) in cases with or without dyspoiesis. Majority of the cases had dyspoiesis in erythroid series (62, 75.6%) and megakaryocytes (15, 18.2%). Dyspoiesis was more prominent in alcoholics 44 cases (53.6%) and autoimmune diseases 13 cases (15.8%). Erythroid hyperplasia (47.7±14.4 vs 40±11.1; pConclusionsBM dyspoiesis, especially dyserythropoiesis, is associated with severe refractory anaemia in patients with cirrhosis and requires new therapeutic approaches.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder that manifests with bone marrow failure, thrombosis and hemolysis. We present a 28-year-old male who presented with weakness, jaundice and transfusion dependence. On initial investigation, he was found to have anemia with jaundice with hemoglobin (Hb) capillary zone electrophoresis suggestive of Hb E (HBB: c.79G>A) trait. The same anomaly was also found in his mother. However, transfusion requirement was an unusual feature in the patient. As his corrected reticulocyte count was raised along with lactate dehydrogenase (LDH), which was suggestive of a hemolytic process, he was worked-up for the same. However, the direct Coombs test was negative. A bone marrow aspiration and biopsy was done to rule out hypersplenism but it revealed erythroid hyperplasia with reduced iron stores despite normal ferritin and iron studies. This was unusual as the patient had anemia requiring transfusions. He had no history of hemoglobinuria but a PNH by flowcytomety revealed a large clone of 81.2% in granulocytes and 88.5% in monocytes. The patient was started on Danazol and steroids for anemia which improved. He was counseled for matched sibling stem cell transplant. He had a full match with his brother. At the time of this study he awaits his transplant.

Anemia associated with alcoholism has numerous causes, most common being megaloblastic anemia and acquired sideroblastic anemia (SA). The bone marrow aspirate (BMA) and bone marrow iron (BMIr) findings and their correlation with peripheral blood smear (PBS) have not been extensively described in literature. We aim to study the spectrum of hematological abnormalities in chronic alcoholics. Complete blood count (CBC), PBS, BMA and BMIr of 71 chronic alcoholics were studied retrospectively over a period of 3 years. The slides were reviewed by 2 pathologists. The clinical history, CBC, PBS, BMA and BMIr findings were recorded. Out of 71 patients, 68 (95.77%) had anaemia. Red cell morphology varied from normocytic-normochromic, microcytic-hypochromic, macrocytic, to dimorphic anaemia. Principal findings seen on BMA were erythroid hyperplasia and megaloblastic maturation. BMIr was available in 41 patients; iron stores were decreased in 2 (4.88%), normal in 14 (34.15%), increased in 25 (60.97%). Seven (17.07%) cases showed presence of ring sideroblasts. Chronic alcoholics show a variety of abnormalities in BMA, which closely mimic many haematological disorders. A history of alcoholism should always be taken in these circumstances. SA should be ruled out in all chronic alcoholics with anaemia not responding to vitamin B(12)/folic acid, even with macrocytic picture on PBS.

CD71 is an immunohistochemical marker used in diagnosing acute myeloid leukemia (AML) M6-Er in humans; however, to our knowledge, it has not been reportedly used for immunohistochemistry in veterinary medicine. We evaluated the pathologic features of AML M6-Er in a retrovirus-negative cat and used CD71 to support the diagnosis. A 4-y-old spayed female Scottish Fold cat was presented with lethargy, anorexia, and fever. Whole-blood PCR assay results for pro feline leukemia virus/pro feline immunodeficiency virus and feline vector-borne diseases were negative. Early erythroid precursors were observed in the peripheral blood smear. Fine-needle aspiration of the enlarged spleen and splenic lymph node showed many early erythroid precursors. Bone marrow aspirate smears revealed erythroid hyperplasia with 68.4% erythroid lineage and 3.6% rubriblasts. Dysplastic cells infiltrated other organs. The patient was diagnosed with myelodysplastic syndrome, progressing to the early phase of AML M6-Er. The patient died on day 121 despite multidrug treatments. Postmortem examination revealed neoplastic erythroblasts infiltrating the bone marrow and other organs. Neoplastic cells were immunopositive for CD71 but immunonegative for CD3, CD20, granzyme B, von Willebrand factor, CD61, myeloperoxidase, and Iba-1. Although further studies are necessary for the application of CD71, our results supported the morphologic diagnosis of AML M6-Er.

Aims Hypoxia-inducible factors (HIFs) play important roles in the pathogenesis of erythrocytosis in chronic mountain sickness (CMS). von Hippel-Lindau (VHL) is a key regulator of hypoxia that can direct the poly-ubiquitylation and degradation of HIFs. Epigenetic mechanisms are believed to contribute toward adaption to chronic hypoxia. Here, we investigated the contribution and mechanism of VHL methylation in rats with erythrocytosis in CMS. Main methods The methylation status of VHL was measured via bisulfite sequencing PCR, while VHL, DNMT1, DNMT3α, and DNMT3β expression were assessed using real-time reverse transcription PCR and western blotting. HIF-2α and EPO expression levels in bone marrow were determined via immunohistochemical staining, and erythroid hyperplasia in bone marrow sections were observed with hematoxylin and eosin staining. Key findings We found that chronic hypoxia triggered erythroid hyperplasia in the bone marrow and increased the quantity of peripheral red blood cells in CMS rats. Chronic hypoxia significantly induced methylation at the CpG site in the VHL promoter, decreased VHL expression, and increased HIF-2α and EPO expression. Chronic hypoxia increased DNMT3α and DNMT3β expression, consistent with the decrease in VHL expression. The DNA methyltransferase inhibitor 5-azacytidine reduced chronic hypoxia-induced erythroid proliferation in the bone marrow of rats with CMS by suppressing VHL methylation and DNMTs expression. Significance Our study suggests that VHL methylation contributes toward excessive erythrocytosis in CMS by upregulating the HIF-2α/EPO pathway in the bone marrow of rats. We demonstrated that the DNMT inhibitor 5-azacytidine can attenuate erythroid hyperplasia in the bone marrow by demethylating the VHL promoter.

Background and Objectives: Hemophagocytosis in bone marrow aspirates is fairly common and can be observed in a spectrum of clinical conditions. This study aimed at evaluating the clinicopathological profile of patients showing hemophagocytosis on bone marrow aspirates examined for varied clinical indications. Materials and Methods: In this study, all those bone marrow aspirate smears sent with various clinical indications and which showed hemophagocytosis were included. Finally, 25 bone marrow aspirates were studied. Results: Of 25 patients, 18 (72%) were males and 7 (28%) were females. The age of the patients ranged from 2 to 73 years. Two common clinical indications with which bone marrow aspirates were sent were fever of unknown origin 10 (40%) and HIV-positive patients with cytopenias 7 (24%). Anemia was the most common cytopenias observed followed by cytopenias in other combinations. Microcytic hypochromic anemia was the common morphological type of the anemia encountered in these patients 17 (68%). Twenty-three (92%) of the bone marrow aspirates showed cellular bone marrow with features of hemophagocytosis. Bone marrow aspirate from a HIV-positive patient showed erythroid hyperplasia (78% early erythroids) with giant erythroblasts and features suggestive of parvovirus B19 infection and hemophagocytosis. Conclusions: Hemophagocytosis in the bone marrow aspirates should not be overlooked and always be documented and reported. The presence of hemophagocytosis may be the only clue to the underlying conditions such as systemic infections or hemophagocytic lymphohistiocytosis (HLH). It is therefore recommended that every case which shows hemophagocytosis needs to be reported and further workup be done to confirm the diagnosis of HLH.

Wilms tumor gene 1 (WT1) is highly expressed in myelodysplastic syndrome (MDS) cells and is known to reflect the tumor burden in MDS. We evaluated the usefulness of WT1 mRNA levels for predicting the prognosis of MDS. At diagnosis, WT1 levels were strongly correlated with the percentage of blasts calculated based on non-erythroid cells, but not with that based on all nucleated cells (r = 0.57, p .05 vs r = 0.42, p = .13). Among the allogeneic transplant recipients, the presence of two consecutive WT1 levels ≥100 copies/μg RNA with a median interval of one month was associated with a 77.8% relapse rate at nine months from the first detection of a high WT1 level, and the median time to relapse was only 114 [36-257] days. WT1 levels at diagnosis were correlated with known prognostic factors. In addition, the presence of two consecutive high WT1 levels after allogeneic transplantation may predict early relapse of MDS.

Glucose-6-phosphate isomerase (GPI) deficiency, a genetic disorder responsible for chronic nonspherocytic hemolytic anemia, is the second most common red blood cell glycolytic enzymopathy. We report three patients from two unrelated families of Czech and Slovak origin with macrocytic hemolytic anemia due to GPI deficiency. The first patient had 15% of residual GPI activity resulting from two new heterozygous missense mutations c.478T > C and c.1414C > T leading to substitutions p.(Ser160Pro) and p.(Arg472Cys). Two other patients (siblings) inherited the same c.1414C > T p.(Arg472Cys) mutation in a homozygous constitution and lost approximately 89% of their GPI activity. Erythroid hyperplasia with dysplastic features was observed in the bone marrow of all three patients. Low hepcidin/ferritin ratio and elevated soluble transferrin receptor detected in our GPI-deficient patients suggest disturbed balance between erythropoiesis and iron metabolism contributing to iron overload.

BACKGROUND Pancytopenia is one of the pathological manifestation resulting from various underlying disease processes affecting the bone marrow. Hence, bone marrow examination is the diagnostic tool to detect the underlying aetiology of pancytopenia. Pancytopenia nowadays has become a relatively common haematological entity. The underlying cause is ranging from simple drug-induced bone marrow hypoplasia, megaloblastic anaemia to fatal bone marrow aplasias and leukaemias. Thus, identification of the correct cause will help in implementing appropriate therapy. MATERIALS AND METHODS This was a prospective study and 158 pancytopenic patients were evaluated clinically along with haematological parameters and bone marrow aspiration in Clinical Pathology, Department of Pathology, during the period of December 2016 to December 2017. RESULTS Among 158 cases, age of patients ranged from 3 to 88 years with a mean age of 45 years and male predominance. Most of the patients presented with fever and anaemia. The commonest physical finding was pallor followed by generalised weakness and splenomegaly. Dimorphic anaemia was the predominant blood picture. Among the 158 cases, 56 cases have undergone bone marrow aspiration in the department. The commonest bone marrow finding was erythroid hyperplasia with megaloblastic maturation (25%). CONCLUSION The present study shows the detailed primary haematological investigations along with bone marrow aspiration in pancytopenia patients, which is useful in understanding disease process. These are also helpful in planning further evaluation and management.

Bone marrow responses have been identified as key determinants of trypanotolerance in cattle as they determine the ability for hemopoietic cell regeneration and control of anemia. However, not much is known about such responses in Nigerian breed of goats known to show different susceptibilities to trypanosome infections. Early bone marrow events associated with pathogenesis of Sokoto Red goats to Trypanosoma vivax were investigated in six goats to assess their role in susceptibility of this goat breed to trypanosomiasis. A total of six Sokoto Red goats of mixed sexes were used. While four of the goats randomly selected were infected with Trypanosoma vivax, the remaining two served as control. The parameters examined included packed cell volume (PCV) and differential bone marrow cytology. Cytological changes in the bone marrow of goats with acute trypanosomosis were compared to the controls. T. vivax caused an acute disease course in the goats resulting to mild drop in PCV and death 2–3 weeks post infection (PI). Associated bone marrow (BM) cytological changes were characterized by moderate erythroid hyperplasia with a resultant lower myeloid:erythroid (M:E) ratio, while the granulocytic maturation rate was 3.15 ± 0.6 and 2.42 ± 0.0 for infected and control animals, respectively. However, significant macrophage (MC) numbers (hyperplasia) were detected in the BM of the infected group. Most of the MCs phagocytized mature red blood cells (RBC) and band or mature white blood cells (WBC) only while no MC phagocytized immature cells. The MCs in BM of control goats phagocytized no blood cells. The study confirmed that mature blood cells form the first cell types to be phagocytized by MCs of BM in the pathogenesis of anemia in T. vivax-infected Sokoto Red goats while early onset of MC hyperplasia and erythrophagocytosis are indicators of susceptibility to trypanosomosis in this breed of goats and perhaps other susceptible ruminants.

Introduction Congenital dyserythropoietic anemias are rare blood disorders characterized by congenital anemia and a wide range of morphological and functional abnormalities of erythroid precursors. Objectives To analyze the relative frequency of both light microscopic (LM) and electron microscopic (EM) morphological features of erythroblasts in a large group of patients with molecular proven congenital dyserythropoietic anemia type I (CDAI). Methods We retrospectively evaluated the LM and EM of bone marrow (BM) erythroblasts in 35 patients with CDAI. Thirty-four patients carried the CDAN1 Arg1042Trp founder mutation and one the p.Pro1130Leu mutation. BM slides of 24 patients were available for LM examination. EM studies were performed in all 35 patients. Results On LM, marked erythroid hyperplasia, binuclear erythroblasts and various non-specific dyserythropoietic features were documented in every case; internuclear chromatin bridges were detected in 19 patients (79%). In all EM of erythroblasts revealed a spongy appearance of heterochromatin, a widening of nuclear pores and invagination of cytoplasm into the nuclear region. Conclusions EM studies revealed high morphological frequency of specific ultrastructural changes in erythroblasts which facilitate prompt diagnosis of CDAI. Due to low specificity of BM LM findings, when BM EM is unavailable diagnostic approach should also include other inherited anemias. This article is protected by copyright. All rights reserved.

Background Precursor-targeted immune-mediated anemia (PIMA) has been suspected in dogs with nonregenerative anemia and bone marrow findings varying from erythroid hyperplasia to pure red cell aplasia. Phagocytosis of erythroid precursors/rubriphagocytosis (RP) reported in some affected dogs suggests a destructive component to the pathogenesis of PIMA. Objectives The purpose of the study was to characterize laboratory and clinical findings in dogs with suspected PIMA and RP, with emphasis on cytologic and histologic bone marrow findings. Methods Dogs with PIMA and RP were identified by review of paired bone marrow aspirate and core biopsy slides collected over a 4-year period. Samples were systematically assessed and characterized along with other pertinent laboratory data and clinical findings. Results Twenty-five dogs met criteria for PIMA and had RP that was relatively stage-selective. Erythropoiesis was expanded to the stage of erythroid precursors undergoing most prominent phagocytosis, yielding patterns characterized by a hypo-, normo-, or hypercellular erythroid lineage. A 4th pattern involved severe collagen myelofibrosis, and there was a spectrum of mild to severe collagen myelofibrosis overall. Evidence of immune-mediated hemolysis was rare. Immunosuppressive therapy was associated with remission in 77% of dogs treated for at least the median response time of 2 months. Conclusions Bone marrow patterns in dogs fulfilling criteria for PIMA were aligned with stage-selective phagocytosis of erythroid precursors and the development of collagen myelofibrosis, common in dogs with PIMA. Recognition of these patterns and detection of RP facilitates diagnosis of PIMA, and slow response to immunosuppressive therapy warrants further investigation into its pathogenesis.

Bone marrow histological features of sickle cell anaemia (SCA) patients during early stages and in the asymptomatic phase of the disease appear an interesting area of study, representing early-stage consequences of SCA with a close relation to its pathophysiology. Unfortunately, this field of research has never been specifically addressed before. Bone marrow biopsies from 26 consecutive Black African SCA patients (M:F=1.6:1; age 2-17 years), free of clinical signs of chronic bone marrow damage, with no recent history of symptomatic vaso-occlusive episodes, and waiting for haematopoietic stem cell transplantation (HSCT), underwent morphological, immunohistochemical and electron microscopy evaluation. Additional comparison with three bone marrow specimens from post-HSCT SCA patients and 10 bone marrow specimens from AS healthy carriers was performed. Bone marrow of SCA patients was normocellular or slighly hypercellular in all cases. Erythroid hyperplasia was a common feature. Myeloid lineage was slightly decreased with normal to slightly diminished neutrophilic granulocytes; CD68 positive monocytic-macrophagic cells appeared slightly increased, with a predominant CD163 positive M2/M(Hb) phenotype. A positive correlation was found between haemoglobin values and number of bone marrow erythroid cells (R2=0.15, p=0.05). Intravascular and interstitial clusters of erythroid sickle cells were found in bone marrow of pre-HSCT homozygous SS SCA patients, as well as heterozygous AS healthy carriers, and the single post-HSCT patient matched to an AS health carrier donor.

BACKGROUND Passenger lymphocyte syndrome occurs when donor lymphocytes are transplanted with a solid organ and produce alloantibodies that react with antigens on the recipient's red blood cells (RBCs). Typically, passenger lymphocyte syndrome presents as immunoglobulin G antibody-mediated, extravascular hemolytic anemia with reticulocytosis. Often, the donor was alloimmunized before transplantation. CASE REPORT A 34-year-old Group O, D+ man with a negative antibody screen received a liver transplant from a Group O, D− donor. Twenty Group O, D+ RBC units were transfused on Postoperative Days (PODs) 0 through 2. On POD 7, the patient developed anemia, a weakly positive antibody screen, and a positive direct antiglobulin test with anti-D in the eluate. After POD 8, a D− transfusion protocol was initiated. Despite laboratory evidence of hemolysis, two initial peripheral blood smears showed no increase in schistocytes or spherocytes, the reticulocyte count was depressed, and a marrow biopsy revealed erythroid hyperplasia. Eventually, anemia resolved after a period of medication non-compliance; however, a positive direct antiglobulin test persisted to the last follow-up date (POD 233). RESULTS Other potential causes of aplastic anemia were investigated, but no alternative cause was found. History excluded passive anti-D. D+, LW− cells were reactive, excluding anti-LW. Genotyping showed no evidence of a partial D genotype. Chart review revealed that the liver donor had a history of anti-D. A diagnosis of passenger lymphocyte syndrome was reached. CONCLUSION Although antibody-mediated hemolytic anemia has been reported to cause reticulocytopenia in the presence of marrow erythroid hyperplasia, this report of passenger lymphocyte syndrome causing a similar post-transplant anemia in association with reticulocytopenia is noteworthy.

Most of the patients with parvovirus B19 infection manifest with anemia and erythroid hypoplasia in bone marrow when the immunity of host is not competent enough to kill the virus like in posttransplant patients on immunosuppressive drugs and immunodeficiency syndromes. Herein, we present a case of parvovirus infection in an immunocompetent patient presenting with anemia, showing erythroid hyperplasia with hemophagocytosis in bone marrow, and also discuss the pathogenesis with review of literature in such a rare presentation.

Introduction: An unusual case of a 19 year old female, presenting with fever, pallor and hepatosplenomegaly for one month. She had microcytic anemia on peripheral smear examination but her bone marrow aspiration & biopsy revealed a hypercelluar marrow with megaloblastic erythroid hyperplasia. Resolution of fever within 48 hours of Vitamin B12 supplementation, initiated in view of the megaloblastic bone marrow picture & low serumVitamin B12 level, suggests a causal association. Conclusion: Vitamin B12 deficiency seems to be an unusual cause of PUO (Pyrexia of unkown origin) which should be ruled out in every case of PUO.

BACKGROUND Low-risk myelodysplastic syndromes (MDS) show several immunologic abnormalities, including increased frequency of autoimmune manifestations and/or overt autoimmune diseases, whose prognostic significance still remains controversial. STUDY DESIGN AND METHODS We studied the presence of erythroblast antibodies in mitogen-stimulated bone marrow (BM) cultures of 70 patients with early-stage MDS (refractory anemia and refractory anemia with ringed sideroblasts). RESULTS Sixty-six percent of patients showed positive erythroblast antibodies, along with BM erythroid hyperplasia and a hemolytic picture in the peripheral blood. Supernatants from positive cultures induced an increase of overall cellularity, the appearance of erythroblastic clustering, and dyserythropoietic signs in normal BM. We identified CD45dimGly-AdimCD71bright cells (red blood cell precursors at different maturation stage) as the target of the antibodies. Erythropoietin (EPO) levels were reduced and EPO receptors (EPO-R) increased in BM culture supernatants from positive patients. However, flow cytometric analysis showed that neither EPO nor EPO-R was involved in an abnormal stimulation driven by these autoantibodies. Values of the proapoptotic protein Bax were increased in positive patients and Bcl-2 levels were decreased, although not significantly. CONCLUSION MDS patients with anti-erythroblast autoimmunity showed increased BM apoptosis, suggesting that the autoimmune reaction may contribute to an unfavorable BM microenvironment for optimal erythropoiesis.

Growth differentiation factor-15 (GDF-15) is a member of TGF-β superfamily. Among hematopoietic cells, this factor is mainly produced by erythroid series and is recently considered a biomarker of ineffective erythropoiesis (IE). Whether IE induces enhanced GDF-15 expression or is prompted by it, has remained elusive. In this study we investigated how high levels of GDF-15 contribute to IE-associated erythroid dysplasia. We assessed mRNA levels of GDF-15 during erythroid maturation as well as in patients with IE using qRT-PCR. Later, the erythroid colony-forming capacity of GDF-15-treated hematopoietic stem cells (HSCs) was evaluated by CFC assay. Any effect of elevated levels of GDF-15 on erythroid maturation was ultimately examined by expression analysis of erythroid-associated transcription factors and flow cytometry analysis of CD235a expression. GDF-15 mRNA expression increased during erythroid differentiation and also in β-thalassemia and MDS patients which was directly correlated with erythropoiesis severity. Treating the cells with high GDF-15 concentration (50 ng/ml) resulted in an approximate 30% decline in the capacity of erythroid colony formation of HSCs and CD235a positive cells. Additionally, erythroid-specific transcription factors showed significant down-regulation in the early stages of erythroid differentiation. According to the expression level of GDF-15 and the role it plays in the erythroid system, high-levels of this factor could be an auto-modulatory mechanism to control the excessive production of erythroid cells.

The generation of a functional erythrocyte from a committed progenitor requires significant changes in gene expression during a time of hemoglobin accumulation, rapid cell division, and nuclear condensation. Congenital Dyserythropoietic Anemia type I (CDA-I), is an autosomal recessive disease that presents with erythroid hyperplasia in the bone marrow. Erythroblasts in patients with CDA-I are frequently binucleate, have chromatin bridging, and defective chromatin condensation. CDA-1 is most commonly caused by mutations in Codanin-1 (CDAN1). The function of CDAN1 is poorly understood but it is thought to regulate histone incorporation into nascent DNA during cellular replication. The study of CDA-1 has been limited by lack of in vitro models that recapitulate key features of the disease and most studies on CDAN1 function have been done in non-erythroid cells. To model CDA-I we generated HUDEP2 mutant lines with deletion or mutation of R1042 of CDAN1, mirroring mutations found in CDA-1 patients. CDAN1 mutant cell lines had decreased viability, increased intercellular bridges, and binucleate cells. Further, they had alterations in histone acetylation associated with prematurely elevated erythroid gene expression, including gamma-globin. Together, these data imply a specific functional role for CDAN1, specifically R1042 on exon 24, in the regulation of DNA replication and organization during erythroid maturation. Most importantly, generation of models with specific patient mutations, such as R1042, will provide further mechanistic insight into CDA-I pathology.

Megaloblastic anemia is a general term used to describe a group of anemias caused by impaired DNA synthesis and characterized by macro-ovalocytes in peripheral blood smear and megaloblastic erythroid hyperplasia in the bone marrow.

Erythropoiesis in the bone marrow and spleen depends upon intricate interactions between the resident macrophages and erythroblasts. Our study focuses on identifying the role of the Nuclear Factor Erythroid 2-related factor 2 (Nrf2) during recovery from stress erythropoiesis. To that end, we induced stress erythropoiesis in Nrf2(+/+) and Nrf2 null mice and evaluated macrophage subsets known to support erythropoiesis and erythroid cell populations. Our results confirm macrophage and erythroid hypercellularity after acute blood loss. Importantly, Nrf2 depletion results in a marked numerical reduction of F4/80(+)/CD169(+)/CD11b(+) macrophages, which is more prominent under the induction of stress erythropoiesis. The observed macrophage deficiency is concomitant to a significantly impaired erythroid response to acute stress erythropoiesis in both the murine bone marrow and spleen. Additionally, peripheral blood reticulocyte counts as a response to acute blood loss is delayed in Nrf2 deficient mice compared to age-matched controls (11.0 ± 0.6% vs. 14.8 ± 0.6%, p≤0.001). Interestingly, we observe macrophage hypercellularity in conjunction with erythroid hyperplasia in the bone marrow during stress erythropoiesis in Nrf2(+/+) controls, with both impaired in Nrf2(−/−) mice. We further confirm the finding of macrophage hypercellularity in another model of erythroid hyperplasia, the transgenic sickle cell mouse, characterized by hemolytic anemia and chronic stress erythropoiesis. Our results demonstrate the role of Nrf2 in stress erythropoiesis in the bone marrow and that macrophage hypercellularity occurs concurrently with erythroid expansion during stress erythropoiesis. Macrophage hypercellularity is a previously underappreciated feature of stress erythropoiesis in sickle cell disease and recovery from blood loss.

Polycythemia vera (PV) is a chronic myeloproliferative neoplasm. Virtually all PV patients are iron deficient at presentation and/or during the course of their disease. The co-existence of iron deficiency and polycythemia presents a physiological disconnect. Hepcidin, the master regulator of iron metabolism, is regulated by circulating iron levels, erythroblast secretion of erythroferrone, and inflammation. Both decreased circulating iron and increased erythroferrone levels, which occur as a consequence of erythroid hyperplasia in PV, are anticipated to suppress hepcidin and enable recovery from iron deficiency. Inflammation which accompanies PV is likely to counteract hepcidin suppression, but the relatively low serum ferritin levels observed suggest that inflammation is not a major contributor to the dysregulated iron metabolism. Furthermore, potential defects in iron absorption, aberrant hypoxia sensing and signaling, and frequency of bleeding to account for iron deficiency in PV patients have not been fully elucidated. Insufficiently suppressed hepcidin given the degree of iron deficiency in PV patients strongly suggests that disordered iron metabolism is an important component of the pathobiology of PV. Normalization of hematocrit levels using therapeutic phlebotomy is the most common approach for reducing the incidence of thrombotic complications, a therapy which exacerbates iron deficiency, contributing to a variety of non-hematological symptoms. The use of cytoreductive therapy in high-risk PV patients frequently works more effectively to reverse PV-associated symptoms in iron-deficient relative to iron-replete patients. Lastly, differences in iron-related parameters between PV patients and mice with JAK2 V617F and JAK2 exon 12 mutations suggest that specific regions in JAK2 may influence iron metabolism by nuanced changes of erythropoietin receptor signaling. In this review, we comprehensively discuss the clinical consequences of iron deficiency in PV, provide a framework for understanding the potential dysregulation of iron metabolism, and present a rationale for additional therapeutic options for iron-deficient PV patients.

UNLABELLED Hereditary sequence variants in globin genes are usually silent and are rarer in α-globin chains than β-globin chains. Some may lead to an unstable protein with a hemolytic or thalassemic phenotype. Hb Taybe is an unstable α-chain hemoglobin variant caused by the deletion of a threonine residue at codon 38 or 39 of the α1 globin gene. This deletion results in a structural abnormality that affects the α1 β2 contact and the α1 β1 interface, producing a highly unstable Hb. OBJECTIVE We describe the clinical, laboratory, and morphological characteristics of 43 patients with Hb Taybe, sixteen of whom are heterozygous, eight are homozygous, and nineteen are double heterozygous for Hb Taybe and other α-gene mutations or deletions. RESULTS The clinical presentation is very variable from a mild hemolytic anemia to the need for red cell transfusion. Morphological characteristics include erythroid hyperplasia, defective hemoglobin production, and dyserythropoietic features. On electron microscopy dyserythropoiesis and cytoplasmic precipitation of globin compatible optical dense material is seen. CONCLUSIONS This is the largest report of Hb Taybe patients. Previous reported cohorts are not related to these cases. We conclude that patients carrying Hb Taybe have a unique hematological and clinical phenotype distinct from other hemoglobinopathies and from congenital dyserythropoietic anemia.

Senna occidentalis (S. occidentalis) is a toxic leguminous plant that contaminates crops and has been shown to be toxic to several animal species. All parts of the plant are toxic, but most of the plant's toxicity is due to its seeds. Despite its toxicity, S. occidentalis is widely used for therapeutic purposes in humans. The aim of the present work was to investigate, for the first time, the effects of the chronic administration of S. occidentalis seeds on hematopoietic organs, including the bone marrow and spleen. Fifty male Wistar rats were divided into five groups of 10 animals. Rats were treated with diets containing 0% (control), 0.5% (So0.5), 1% (So1), or 2% (So2) S. occidentalis seeds for a period of 90 days. Food and water were provided ad libitum, except to pair-fed (PF) group which received the same amount of ration to those of So2 group, however free of S. occidentalis seeds. It was verified that rats treated with 2% S. occidentalis seeds presented changes in hematological parameters. The blood evaluation also showed a significant decrease of the Myeloid/Erythroid (M/E) ratio. Chronic treatment with S. occidentalis promoted a reduction in the cellularity of both the bone marrow and spleen. Additionally, we observed changes in bone marrow smears, iron stores and spleen hemosiderin accumulation. Histological analyses of bone marrow revealed erythroid hyperplasia which was consistent with the increased reticulocyte count. These findings suggest that the long-term administration of S. occidentalis seeds can promote blood toxicity.

The clinical and biological manifestations of visceral leishmaniasis are often confusing, most particularly because it can mimic and lead to a variety of hematological disorders. The aim of this study was to investigate the hematologic abnormalities observed in infantile visceral leishmaniasis from January 2000 and December 2013. The study included 35 children with a mean age of 3.5 years. Clinical manifestations were dominated by splenomegaly, fever, and paleness, defining the classic triad in 16% of our patients. Anemia was present in all patients. Leukopenia was found in 51% of the cases. Thrombocytopenia was observed in 48% of our patients and 36% had pancytopenia. All cases were confirmed by the presence of Leishman bodies (amastigotes) in the bone marrow smears. Quantitative and qualitative megakaryocyte abnormalities were found. Similarly, dysgranulopoiesis was observed in 31% of the cases, eosinophilia was present in 6%, erythroid hypoplasia in 3%, and erythroid hyperplasia in 34%. Different features of dyserythropoiesis were revealed in 71% of the patients with images of hemophagocytosis in 6% and multiple dysplasias in 9%. The knowledge of these hematological abnormalities associated with infantile visceral leishmaniasis can assist us in searching for Leishman bodies in the bone marrow smears to provide a diagnosis more quickly without necessarily resorting to more sophisticated tests.

Introduction: Examination of peripheral blood film (PBF) and biopsy of the bone marrow are an indispensible adjunct to the study of diseases of the blood and may be the only way in which a correct diagnosis can be made. Marrow can be obtained by needle aspiration, percutaneous trephine biopsy or surgical biopsy. Morphological examination of the bone marrow by an experienced haematologist can provide very useful information important for many haematological and non-haematological disorders. Objective: The aim of the study was to diagnose both haematological and non-haematological disorders by only morphological bone marrow examination in a district town distant from the capital city where sophisticated diagnostic facilities are not available. Methods: This cross sectional type of descriptive study was carried out in Combined Military Hospital (CMH), Jessore and different private and public hospitals. Five hundred cases taken as a non-probability purposive sampling method were included in this study irrespective of age and sex from January 2009 to June 2011. Bone marrow was aspirated from posterior superior iliac spine and first piece of the body of the sternum taking aseptic precautions and after infiltrating local anaesthesia. Only two cases required percutaneous trephine biopsy. After aspiration bone marrow smears were stained and examined under microscope. 59 JAFMC Bangladesh. Vol 9, No 2 (December) 2013 Results: There were 294 (58.8%) male and 206 (41.2 %) female out of 500 cases. The age of the patients ranged from one year to 82 years. Haematological malignancy were 321 cases (64.2%), non-malignant haematological and non-haematological diseases were 112 (22.4%) and 56 (11.2%) cases respectively and normal active marrow were from 11 (2.2%) cases. Among 321 haematological malignancies, Acute Lymphoblastic Leukaemia (ALL) and Acute Myeloblastic Leukaemia (AML) were 126 (39.3%) and 99 (30.8%) respectively and Chronic Myeloid Leukaemia (CML) were 37 (11.5%). Out of 112 non-malignant haematological cases, erythroid hyperplasia was found in 42 (37.5%) cases in which micronormoblastic erythroid hyperplasia was 33 (29.5%) and megaloblastic erythroid hyperplasia was 9 (8.0%) cases. Aplastic anaemia/progressive marrow failure was diagnosed in 40 (35.7%) cases. Two (1.8%) cases were diagnosed as myelofibrosis. Non-haematological diseases were 56 of which 49 (87.5%) cases were secondary reactive marrow & only seven (12.5%) cases were secondary metastatic deposits in the bone marrow. Conclusion: Morphological examination of bone marrow aspirate is a key to the diagnosis of many diseases especially the haematological disorders. Microscopic examination of bone marrow aspirate by an experienced haematologist may solve many diagnostic difficulties faced in day to day clinical practice. Therefore, for the betterment of both patients and physicians more emphasis should be given to become well conversant in reporting a bone marrow aspirate by the haematologists. DOI: http://dx.doi.org/10.3329/jafmc.v9i2.21828 Journal of Armed Forces Medical College Bangladesh Vol.9(2) 2013

Background: Bone Marrow Aspiration plays a major role in the diagnosis of various hematolgical disorders which are very frequent in various age groups. The aim of this study was to analyze the causes of haematological disorders, and to interpret the bone marrow aspiration findings with various dyserythropoietic changes in it. Methods: This was a study carried out in the department of Pathology of Shri M P Shah Govt. Medical college over a period of two years from June 2008 to June 2010. Bone marrow examination of 100 cases of suspected haematological disorders was carried out. All details of the patients were the recorded in the department of pathology. Results: Out of 100 cases of bone marrow aspiration, erythroid hyperplasia and megaloblastic changes were commonest findings and Megaloblastic anemia was most common diagnosis given on bone marrow examination. Other dyserythropoietic changes were erythroidhypoplasia, micronormoblasts, dimorphic erythropoiesis, megaloblastoid changes and other like – cytoplasmic bleb, multinucleation,nuclear bridging. Conclusions: The sincere blood film examination and keen morphological evaluation of erythroid series for dyserythropiesis and leukopoesis and megakaryopiesis in bone marrow aspiration smear - supported with other investigation can navigate to etiological factors of anaemia and other haematological disorders.

This review focuses on anemia resulting from production defects generally associated with marrow erythroid hyperplasia and ineffective erythropoiesis. The definition, epidemiology, etiology, pathogenesis, diagnosis, differential diagnosis, management, complications, and prognosis of the following production defects are discussed: Megaloblastic anemias, megaloblastic anemia caused by cobalamin deficiency, megaloblastic anemia caused by folic acid deficiency, copper deficiency, and sideroblastic anemias. Figures depict intracellular interdependent cofactor activity of cobalamin and folic acid, synthesis of succinyl–coenzyme A from methylmalonyl–coenzyme A, folic acid functions as a coenzyme in single-carbon transfer reactions, cobalamin assimilation, peripheral blood smear in folic acid or cobalamin deficiency, and a Prussian blue stain showing ring sideroblasts. Tables list causes of cobalamin deficiency and folic acid deficiency. This review contains 5 figures; 2 tables; 95 references.

TET2 mutations have been frequently observed in myeloid malignancies including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN) and MDS/MPN [1–4]. TET...

Objectives E-cadherin, epithelial calcium-dependent cell adhesion protein, has been identified as a marker of immature erythroid precursors in recent years. However, the specificity of E-cadherin in bone marrow specimens for erythroblasts vs myeloblasts or other early hematopoietic precursors in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) has not been fully elucidated. Methods We analyzed 105 cases of AML and MDS to evaluate the specificity of E-cadherin. Results Of 84 cases of AML, including cases with megakaryocytic, erythroid, monocytic, and granulocytic differentiation, all five acute erythroleukemia cases were positive, as well as one case of megakaryoblastic leukemia that showed coexpression of glycophorin A. In addition, we demonstrate that a panel of three markers, E-cadherin, CD117, and CD34, is effective in identifying lineage-specific myeloblasts in cases of MDS where left-shifted erythroid hyperplasia may complicate morphologic assessment of myeloblasts. Conclusions In marrow specimens, E-cadherin is a useful marker for erythroid differentation.

Red blood cell (RBC) homeostasis tightly balances production of new erythroid cells and clearance of damaged or senescent RBCs. Macrophages play a central role in this process in the specialized niches termed erythroblastic island. Macrophages expressing CD169 (Sialoadhesin or Siglec-1), F4/80, CD11b, VCAM-1, ER-HR3 and Ly-6G play important roles in physiological and pathological erythropoiesis. Sickle cell disease (SCD) is characterized by chronic stress erythropoiesis as a compensatory mechanism for anemia. Recent publications have detected macrophage hypercellularity correlated with erythroid hyperplasia in genetic models of erythrocytosis and monocyte-derived macrophage proliferation in the spleen. Because macrophages are integral part of the erythropoietic niche, our objective was to assess the macrophage compartment in transgenic sickle cell mice (SS) at steady state compared to AA controls. Furthermore, we used acute blood loss in C57BL/6J wild type (WT) to confirm if macrophage hypercellularity accompanies erythroid hyperplasia in a mouse model without erythrocytosis or sickling. Using flow cytometry, macrophages were identified with F4/80, CD45, CD11b and CD169, while erythroid progenitor cells were identified with Ter119 and CD71 in the BM and spleen of SS mice and age-matched control (AA) mice. Each subset of macrophages is 3.8 to 6.8-fold higher in SS mice bone marrow compared to AA at steady state (p≤0.05, Fig 1a). Findings were similar for other CD169 subsets in the spleens of SS mice. This macrophage hypercellularity was accompanied by expected erythroid hyperplasia, with 12-fold higher immature erythroid progenitor cells (CD71hiTer119hi) in SS mice than AA controls (p≤0.001). To confirm the increase in macrophage numbers that accompanied erythroid hyperplasia in SS mice as a response to erythroid stress, we induce stress with acute blood loss in WT mice by phlebotomy (once). Interestingly, we found macrophage hypercellularity accompanies erythroid hyperplasia in WT mice during recovery from acute blood loss. WT mice showed about 47-61% increases in CD169 macrophages subsets in the marrow and 47-83% increase in the spleen during recovery from acute blood loss (5-7 days) compared to untreated mice (Fig. 1b). This macrophage hypercellularity was accompanied with 88% increase in immature erythroid progenitor cell. Similarly, we investigated if there was age dependence in macrophage hypercellularity that accompanies erythroid hyperplasia in SS mice. In SS mice, there was no significant differences in macrophage numbers in the BM juvenile mice (5 weeks old) compared to young adults (14 weeks old), presumably because the higher demand for erythroid production to compensate for anemia had already started in the juvenile animals. However, there was a significantly higher macrophage hypercellularity in the spleen at 14 weeks compared to juvenile animals (p≤0.001). There was also a reduction in hematocrit (p≤0.05) and hemoglobin (p≤0.01) from 5 to 14 weeks in SS mice, although this does not correlate to either macrophage hypercellularity or erythroid hyperplasia. We also quantified mRNA expression of heme oxygenase 1 (Hmox1), a critical regulator of erythroid stress response, in the BM of untreated SS mice, and untreated and stressed WT mice. As expected, BM cells from SS mice at steady state expressed significantly higher Hmox1 expression than in WT mice (p≤0.001). Acute blood loss induced-stress raised the Hmox1 expression in WT mice to comparable levels to that of SS mice at steady state. Our investigation is the first to our knowledge to detect BM macrophage hypercellularity is associated with erythroid hyperplasia in response to acute blood loss and in congenital hemolytic anemia. Our data of macrophage hypercellularity in SCD opens up further research opportunity to elucidate the role of these cells in multiorgan complication in SCD. Figure 1: Macrophage hypercellularity accompanies erythroid hyperplasia in sickle cell disease and during recovery from acute blood loss. (a) BM macrophage quantification (per femur) in untreated sickle cell mice compared to AA controls (n = 5). Student's Ttest * SS mice compared to AA mice. (b) Acute blood loss induced macrophage hypercellularity in the BM of WT mice (n = 4 - 5). Student's Ttest * Untreated compared to stressed WT mice. Figure 1 Disclosures Ofori-Acquah: Shire Human Genetic Therapies Inc: Other: Financial Relationship. Kato:Novartis, Global Blood Therapeutics: Consultancy, Research Funding; Bayer: Research Funding.

Background: Pancytopenia is not a disease entity but a triad of findings that result from a number of disease processes. These disorders may affect bone marrow either primarily or secondarily, resulting in the manifestation of pancytopenia. Objective : To identify the various causes of pancytopenia in patients attending to Star hospital, Kathmandu, Nepal. Methods: Fifty two patients with pancytopenia were included in this study from June 2011 to June 2012. Complete blood count, peripheral blood smear, bone marrow aspiration and trephine biopsies were performed according to standard methods. Results: Out of 52 cases, there were 26 cases(50%) of aplastic anaemia, 18 cases(34.61%) of megaloblastic anaemia, 4 cases(7.69%) of acute leukemia, 2cases(3.84%) of erythroid hyperplasia, 1 case(1.92%) of metastatic tumor and 1 case(1.92%) of multiple myeloma. Conclusion: Aplastic anaemia and megaloblastic anaemia were the most common causes of pancytopenia in this study. Health Renaissance, January-April 2013; Vol. 11 No.1; 134-137 DOI: http://dx.doi.org/10.3126/hren.v11i2.8220

During erythropoiesis, hemoglobin (Hb) synthesis increases from early progenitors to mature enucleated erythrocytes. Although Hb is one of the most extensively studied proteins, the role of Hb in erythroid lineage commitment, differentiation, and maturation remains unclear. In this study, we generate mouse embryos and embryonic stem (ES) cells with all of the adult α and β globin genes deleted (Hb Null). While Hb Null embryos die in midgestation, adult globin genes are not required for primitive or definitive erythroid lineage commitment. In vitro differentiation of Hb Null ES cells generates viable definitive proerythroblasts that undergo apoptosis upon terminal differentiation. Surprisingly, all stages of Hb Null-derived definitive erythroblasts develop normally in vivo in chimeric mice, and Hb Null erythroid cells undergo enucleation to form reticulocytes. Free heme toxicity is not observed in Hb Null-derived erythroblasts. Transplantation of Hb Null-derived bone marrow cells provides short-term radioprotection of lethally irradiated recipients, whose progressive anemia results in an erythroid hyperplasia composed entirely of Hb Null-derived erythroblasts. This novel experimental model system enables the role played by Hb in erythroid cell enucleation, cytoskeleton maturation, and heme and iron regulation to be studied.

Objective: The major pathophysiological features of β-thalassemia are anemia and ineffective erythropoiesis. Ineffective erythropoiesis has been shown by an intense marrow erythroid hyperplasia and increased apoptosis during basophilic to orthochromatic normoblast stages. Some cytokines like interferon-γ (IFN-γ), tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1) have been found to involved in apoptosis. Although the pro-apoptotic activity of IFN-γ and TNF-α is well documented, there are only few studies on IL-1, especially on erythroid lineage. In this in vitro study, the role of cytokine IL-1α and IL-1β in apoptosis of erythroid progenitor cells from β- thalassemia/HbE patients was assessed. Methods: Erythroid progenitor cells were isolated from peripheral blood of healthy subjects and β-thalassemia/HbE patients. Cells were then cultured, with and without 20 ng/ml IL-1α and IL-1β. Total cells and percent cell viability were performed by using trypan blue staining. Percent cell apoptosis was analyzed by using flow cytometer. Results: Both IL-1α and IL-1β were significantly decreased erythroid progenitor cells. IL-1 at 20 ng/ml reduced the glycophorin A positive cells and percent cell viability of erythroid progenitor cells from β-thalassemia/HbE patients, while there was increased apoptosis in this group. The highest percent apoptosis was observed in 20 ng/ml IL-1β treated β-thalassemia/HbE erythroid progenitor cells. Conclusion: IL-1β could be involved in apoptosis of erythroid progenitor cells from β-thalassemia/HbE patients which might be related with ineffective erythropoiesis of the disease. DOI: http://dx.doi.org/10.3126/ajms.v3i4.6805 Asian Journal of Medical Science Vol.3(4) 2012 pp.8-14

Background: Pancytopenia refers to a reduction in all the three cellular elements of blood. The aim of this study was to identify the various causes of pancytopenia in patients attending to Manipal teaching hospital in Pokhara. Materials and Methods: This was a cross-sectional study, carried out in Manipal teaching hospital from August 2008 to August 2010. Fifty-four patients with pancytopenia were included in the study. Complete blood count, bone marrow aspirations and trephine biopsies were performed. Data were analyzed using SPSS 11.0 version. Results: Out of 54 cases, there were 16 cases (29.60%) of hypoplastic bone marrow, 15 cases (27.78%) of hematological malignancies, 13 cases (24.10%) of megaloblastic anemia, 4 cases (11.11%) of erythroid hyperplasia and 6 cases (7.41%) of normcellular bone marrow. Acute myeloid leukemia was the commonest hematological malignancy. Conclusion: The commonest cause of pancytopenia in our study was hypoplastic bone marrow followed by hematological malignancies and megaloblastic anemia. DOI: http://dx.doi.org/10.3126/jpn.v2i3.6023 JPN 2012; 2(3): 207-210

Infection of erythroid cells by Friend spleen focus-forming virus (SFFV) leads to acute erythroid hyperplasia in mice, due to expression of its unique envelope glycoprotein, gp55. Erythroid cells expressing SFFV gp55 proliferate in the absence of their normal regulator, erythropoietin, because of the interaction among the viral envelope protein, the erythropoietin receptor, and a short form of the receptor tyrosine kinase Stk (sf-Stk). This leads to constitutive activation of several signal transduction pathways. Our previous studies showed that sf-Stk interacts with SFFV gp55, forming disulfide-linked complexes. This covalent interaction, along with other noncovalent interactions with SFFV-gp55, results in constitutive tyrosine phosphorylation of sf-Stk and rodent fibroblast transformation. Here, we determined the precise amino acid region within sf-Stk that contributes to fibroblast transformation by the polycythemia-inducing (SFFV-P) and the anemia-inducing (SFFV-A) strains of SFFV. Sf-Stk deletion mutants showed different transforming abilities in fibroblasts infected with SFFV-P and SFFV-A, although the N-terminal extracellular domain of sf-Stk was essential for fibroblast transformation by both viruses. Point mutations of sf-Stk indicated that cysteine 19 was critical for fibroblast transformation by SFFV-P, although all four cysteines (8, 19, 37 and 42) appeared to be important for fibroblast transformation by both SFFV-P and SFFV-A. Mutation of sf-Stk cysteine 19 abolished its ability to form dimers with SFFV-P and SFFV-A gp55. These results suggest that the interaction between sf-Stk and the envelope proteins of the polycythemia- and anemia-inducing variants of SFFV is architecturally different.

A female patient of Portuguese origin, who was born to consanguineous parents, presented with moderate anemia, mild jaundice and splenomegaly. Bone marrow aspiration showed an erythroid hyperplasia and binucleate erythroblasts, evoking a congenital dyserythropoietic anemia, type II (CDA II). Although microcytosis cast some doubt on the diagnosis, investigation was pursued along this line. The CDA II was finally ruled out as underglycosylation of band 3, remnants of endoplasmic reticulum cisternae and mutations in the SEC23B gene were all missing. On the other hand, analysis of the α-globin genes showed a base substitution at codon 125 (CTG→CGG) of the α2-globin gene, ascertaining a homozygosity for Hb Plasencia (HBA2:c.377T>G). This variant has an unstable α chain. In the absence of a thorough work-up, dyserythropoietic anemia associated with hemoglobin (Hb) variants having a moderately unstable α chain, may be mistaken for CDA II.

Pulmonary hypertension is associated with reduced nitric oxide bioavailability and early mortality in sickle cell disease (SCD). We previously demonstrated that placenta growth factor (PlGF), an angiogenic factor produced by erythroid cells, induces hypoxia-independent expression of the pulmonary vasoconstrictor endothelin-1 in pulmonary endothelial cells. Using a lentivirus vector, we simulated erythroid expression of PlGF in normal mice up to the levels seen in sickle mice. Consequently, endothelin-1 production increased, right ventricle pressures increased, and right ventricle hypertrophy and pulmonary changes occurred in the mice within 8 weeks. These findings were corroborated in 123 patients with SCD, in whom plasma PlGF levels were significantly associated with anemia, endothelin-1, and tricuspid regurgitant velocity; the latter is reflective of peak pulmonary artery pressure. These results illuminate a novel mechanistic pathway linking hemolysis and erythroid hyperplasia to increased PlGF, endothelin-1, and pulmonary hypertension in SCD, and suggest that strategies that block PlGF signaling may be therapeutically beneficial. This trial was registered at http://clinicaltrials.gov as #NCT00011648.

The diagnosis of polycythemia vera (PV) requires the integration of clinical and laboratory findings, bone marrow morphologic features, and JAK2 analysis. JAK2(V617F) (exon 14) mutation is found in 95% of PV cases. Functionally similar mutations in JAK2 exon 12 have also been described, but a thorough bone marrow study has not been done. We identified 7 PV cases with exon 12 mutations; all had hypercellular bone marrow with erythroid hyperplasia. Small, atypical megakaryocytes predominated; atypical megakaryocyte lobation and abnormal chromatin distribution was identified in all cases. Rare clusters of megakaryocytes could be found but were typically subtle. Because JAK2 exon 12-positive PV cases lack the classic myeloproliferative morphologic features, bone marrow samples from the patients may be difficult to classify as myeloproliferative neoplasms. Clinically suspected PV with low serum erythropoietin and absent JAK2(V617F), together with the bone marrow findings of erythroid hyperplasia and subtle megakaryocytic atypia, should prompt an evaluation for an exon 12 mutation.

Acute erythroid leukemia (AEL) is a rare type of acute myeloid leukemia (AML) for which diagnostic criteria have been refined in the 2008 World Health Organization (WHO) classification of AML. The relationship of AEL to myelodysplastic syndromes (MDSs) and to AML with myelodysplasia-related changes (AML-MRC) is not clearly defined. We conducted a retrospective, multi-institutional study of patients with AEL and compared them with patients with MDS or AML-MRC with erythroid hyperplasia (≥ 50% erythroid cells). Among a total of 124 patients with AEL, 32% had a history of MDS or chronic cytopenia, 32% had therapy-related disease, and 35% had de novo disease. Sixty-four percent of patients had unfavorable AML risk-group karyotypes. FLT3 and RAS mutations were infrequent, occurring in 6% and 2%, respectively. The median overall survival (OS) of all AEL patients was 8 months, comparable with that of patients with MDS or AML-MRC with erythroid hyperplasia. The OS was related to cytogenetic risk group, but not blast count or morphologic dysplasia. Our findings suggest that AEL is in the continuum of MDS and AML with erythroid hyperplasia, where karyotype rather than an arbitrary blast cutoff represents the most important prognostic factor.

Objective: To describe the bone marrow aspiration cytologic findings in visceral leishmaniasis. Study design: Bone marrow aspiration of 204 documented cases of kala-azar were reviewed in order to find aspiration clues other than typical intrahistiocytic and free Leishman-Donovan bodies. Aspiration findings were divided into different groups of common, uncommon as well as atypical/unusual findings. Results: Common findings were granulomas, intrahistiocytic and free typical organisms, plasma cells with or without inclusions, eosinophilia, free floating cytoplasmic bodies with or without Leishman bodies, granular bodies and erythroid hyperplasia. Uncommon findings were: intracellular (non-histiocytic) organisms, hemophagocytosis, plasma cells with abnormal crystalline inclusions, leukemic blasts and necrosis. Atypical/unusual findings were: spore-like organisms, regular or irregular shape of aggregates of organisms (flower-like, ball-like, rosette-like, doughnut-like and platelet-like), intracytoplasmic granule-like organisms (kinetoplast only), pseudo-Pelger Huet, increased vessels, fibroblasts, Reed-Sternberg-like cells, multinucleated giant cells, tart cells and foamy cells. Conclusion: Knowledge of common, uncommon and unusual/atypical bone marrow aspiration findings of kala-azar may help arrive at a correct diagnosis; avoid unnecessary workups, such as sophisticated molecular techniques; and avoid fatal outcomes in untreated or non-diagnosed cases.

A family with congenital dyserythropoietic anaemia type III was studied. Twenty patients and 10 of their healthy siblings were clinically examined and questioned about their medical history. Blood sampling and bone marrow aspirations were also performed. Forty-five percent of the patients reported symptoms of anaemia and 35% regularly felt weakness, fatigue, or headache. However, the majority of the patients regarded themselves as healthy. The bone marrow showed a uniform picture of erythroid hyperplasia with multinuclear erythroblasts and gigantoblasts with up to 12 nuclei. There was laboratory evidence of intravascular haemolysis and mild anaemia. We also observed a high prevalence of monoclonal gammopathy of undetermined significance (3 cases) and myeloma (1 case) among the patients.

Groups of mice have been autopsied at regular intervals during the period of lymphomyeloid tissue regeneration which follows the phase of hypocellularity induced by the i.v. injection of 100 mug (4 mg/kg bodyweight) nitrogen mustard. The marrow cellularity recovered to levels in the normal range by the 8th day and remained in this range up to the 40th day. Subsequently, the marrow showed a slight degree of hypocellularity up to day 120. Granulocytes were predominant during the initial phase of marrow regeneration during the second week post-injection. The thymus exhibited periodic size variations such that it was substantially larger than the thymus of age-matched controls from 20-30 d, 46-73 d, and 75-100 d after injection. The lymph nodes and lymphoid tissue of the spleen regenerated only slowly to reach control values by 40-50 d. Superimposed on the recovering lymphoid tissue of the spleen was a phase of erythroid hyperplasia lasting from 10-18 d post-injection. This coincided with a shift from granulocytosis to erythroid hyperplasia in the marrow. This erythroid hyperplasia lasted until day 30 when the cellular composition of the marrow and spleen returned to normal. A possible explanation of these results is that nitrogen mustard introduces a degree of synchrony into stem cell proliferation and differentiation. Additionally, these results emphasize the role of the haematopoietic microenvironment in the control of stem cell differentiation.

To investigate the possibility that a proliferative non-neoplastic process influences extracellular cyclic nucleotide concentrations, we measured plasma cyclic AMP and cyclic GMP levels in 38 patients with homozygous beta-thalassaemia. This group consisted of 20 patients with thalassaemia major transfused regularly (mean pre transfusion Hb levels, 11 g/dl), and 18 patients with thalassaemia intermedia who did not require regular blood transfusion (mean Hb levels, 8.7 g/dl). In the patient group, plasma cyclic AMP levels were similar to those of 37 normal subjects matched for age and sex, whereas plasma cyclic GMP levels were markedly higher. Moreover, in the thalassaemic patients there was a significant negative correlation between plasma cyclic GMP levels and haemoglobin concentrations, suggesting that their marked erythroid hyperplasia may play a role in determining alterations in extracellular cyclic GMP levels.

Purpose WHO classification of myeloid malignancies is based mainly on the percentage of bone marrow (BM) blasts. This is considered from total nucleated cells (TNCs), unless there is erythroid-hyperplasia (erythroblasts ≥ 50%), calculated from nonerythroid cells (NECs). In these instances, when BM blasts are ≥ 20%, the disorder is classified as erythroleukemia, and when BM blasts are < 20%, as myelodysplastic syndrome (MDS). In the latter, the percentage of blasts is considered from TNCs. Patients and Methods We assessed the percentage of BM blasts from TNCs and NECs in 3,692 patients with MDS from the Grupo Español de Síndromes Mielodisplásicos, 465 patients with erythroid hyperplasia (MDS-E) and 3,227 patients without erythroid hyperplasia. We evaluated the relevance of both quantifications on classification and prognostication. Results By enumerating blasts systematically from NECs, 22% of patients with MDS-E and 12% with MDS from the whole series diagnosed within WHO categories with < 5% BM blasts, were reclassified into higher-risk categories and showed a poorer overall survival than did those who remained in initial categories (P = .006 and P = .001, respectively). Following WHO recommendations, refractory anemia with excess blasts (RAEB)-2 diagnosis is not possible in MDS-E, as patients with 10% to < 20% BM blasts from TNCs fulfill erythroleukemia criteria; however, by considering blasts from NECs, 72 patients were recoded as RAEB-2 and showed an inferior overall survival than did patients with RAEB-1 without erythroid hyperplasia. Recalculating the International Prognostic Scoring System by enumerating blasts from NECs in MDS-E and in the overall MDS population reclassified approximately 9% of lower-risk patients into higher-risk categories, which indicated the survival expected for higher-risk patients. Conclusion Regardless of the presence of erythroid hyperplasia, calculating the percentage of BM blasts from NECs improves prognostic assessment of MDS. This fact should be considered in future WHO classification reviews.