Erythropoiesis In Patients Research Articles

A Peptide-Based Erythropoietin-Receptor Agonist for Pure Red-Cell Aplasia

We investigated whether a novel, synthetic, peptide-based erythropoietin-receptor agonist (Hematide, Affymax) can stimulate erythropoiesis in patients with anemia that is caused by antierythropoietin antibodies. In this open-label, single-group trial, we enrolled patients with chronic kidney disease who had pure red-cell aplasia or hypoplasia due to antierythropoietin antibodies and treated them with a synthetic peptide-based erythropoietin-receptor agonist. The agonist was administered by subcutaneous injection at an initial dose of 0.05 mg per kilogram of body weight every 4 weeks. The primary end point was a hemoglobin concentration above 11 g per deciliter without the need for transfusions. We treated 14 patients with the peptide agonist for a median of 28 months. The median hemoglobin concentration increased from 9.0 g per deciliter (with transfusion support in the case of 12 patients) before treatment to 11.4 g per deciliter at the time of the last administration of the agonist; transfusion requirements diminished within 12 weeks after the first dose, after which 13 of the 14 patients no longer required regular transfusions. Peak reticulocyte counts increased from a median of 10x10(9) per liter before treatment to peak counts of greater than 100x10(9) per liter. The level of antierythropoietin antibodies declined over the course of the study and became undetectable in six patients. One patient who initially responded to treatment had a diminished hematologic response a few months later despite increased doses of the agonist and required transfusions again; this patient was found to have antibodies against the agonist. One patient died 4 months after the last dose of the agonist, and a grade 3 or 4 adverse event occurred in seven other patients during the study period. This novel agonist of the erythropoietin receptor can correct anemia in patients with pure red-cell aplasia caused by antierythropoietin antibodies. (ClinicalTrials.gov number, NCT00314795.).

Effect of lenalidomide therapy on hematopoiesis of patients with myelodysplastic syndrome associated with chromosome 5q deletion

Lenalidomide improves erythropoiesis in patients with low/intermediate-1 risk myelodysplastic syndrome and interstitial deletion of the long arm of chromosome 5 [del(5q)]. The aim of this study was to explore the effect of lenalidomide treatment on the reserves and functional characteristics of bone marrow hematopoietic progenitor/precursor cells, bone marrow stromal cells and peripheral blood lymphocytes in patients with low/intermediate-1 risk myelodysplastic syndrome with del(5q). We evaluated the number and clonogenic potential of bone marrow erythroid/myeloid/megakaryocytic progenitor cells using clonogenic assays, the apoptotic characteristics and adhesion molecule expression of CD34(+) cells by flow cytometry, the hematopoiesis-supporting capacity of bone marrow stromal cells using long-term bone marrow cultures and the number and activation status of peripheral blood lymphocytes in ten patients with low/intermediate-1 risk myelodysplastic syndrome with del(5q) receiving lenalidomide. Compared to baseline, lenalidomide treatment significantly decreased the proportion of bone marrow CD34+ cells, increased the proportion of CD36(+)/GlycoA(+) and CD36(-)/GlycoA(+) erythroid cells and the percentage of apoptotic cells within these cell compartments. Treatment significantly improved the clonogenic potential of bone marrow erythroid, myeloid, megakaryocytic colony-forming cells and increased the proportion of CD34(+) cells expressing the adhesion molecules CD11a, CD49d, CD54, CXCR4 and the SLAM antigen CD48. The hematopoiesis-supporting capacity of bone marrow stroma improved significantly following treatment, as demonstrated by the number of colony-forming cells and the level of stromal-derived factor-1 alpha and intercellular adhesion molecule-1 in long-term bone marrow culture supernatants. Lenalidomide treatment also increased the proportion of activated peripheral blood T lymphocytes. The beneficial effect of lenalidomide in patients with lower risk myelodysplastic syndrome with del(5q) is associated with significant increases in the proportion of bone marrow erythroid precursor cells and in the frequency of clonogenic progenitor cells, a substantial improvement in the hematopoiesis-supporting potential of bone marrow stroma and significant alterations in the adhesion profile of bone marrow CD34(+) cells.

Nucleated red blood cells and soluble transferrin receptor in thalassemia syndromes: relationship with global and ineffective erythropoiesis

The technology to recognize nucleated red blood cells (NRBC) automatically has only recently been developed. Modern hematology analyzers allow for rapid and accurate NRBC counts. The goal of our study was to evaluate NRBC counts and the concentrations of serum transferrin receptor (sTfR) in patients affected by different thalassemia syndromes and hereditary spherocytosis. We wished to gain a better understanding of the meaning of the presence of NRBC in peripheral blood and the relationship of the two parameters with effective and ineffective erythropoiesis in the different thalassemia syndromes. NRBC counts in peripheral blood were evaluated in a large group of patients with thalassemia (36 thalassemia major, 55 thalassemia intermedia and 61 Sbeta-thalassemia patients) and compared with data from 29 patients with hereditary spherocytosis; in all the patients the concentration of sTfR was evaluated as an index of global erythropoiesis. The NRBC count showed a good relationship with ineffective erythropoiesis: highest counts were observed in the thalassemia syndromes characterized by almost completely ineffective erythropoiesis. NRBCs were absent in patients affected by hereditary spherocitosis, a disease characterized by effective erythropoiesis. The NRBC count can be useful for better defining ineffective erythropoiesis in patients with thalassemia, and can help optimize transfusion therapy in severe thalassemia syndromes.

Growth differentiation factor 15 production is necessary for normal erythroid differentiation and is increased in refractory anaemia with ring‐sideroblasts

The disturbed erythropoiesis in patients with refractory anaemia with ring-sideroblasts (RARS) is characterized by intramedullary apoptosis of erythroid precursors and increased iron accumulation in mitochondria. To gain insight into these pathophysiological mechanisms we compared the gene expression profile (GEP) of erythroid precursors from RARS patients to the GEP of normal erythroid precursors. Three hundred sixty four probe sets were up-, and 253 probe sets downregulated in RARS cells. Interestingly, Growth Differentiation factor 15 (GDF15), a cytokine from the TGFbeta family, was dramatically upregulated in all RARS patients. Measurement of GDF15 in the sera from twenty RARS patients confirmed this finding by showing significantly, 7.2-fold, increased protein levels (3254 +/- 1400 ng/ml vs. 451 +/- 87 ng/ml in normals). In vitro studies demonstrated erythroid-specific production of GDF15 and dependence on erythropoietin. Induction of apoptosis by arsenic trioxide, a drug which acts via reduction of the mitochondrial membrane potential, also stimulated GDF15 production. Downregulation of endogenous GDF15 production in erythoblasts by specific siRNA led to diminished erythroid differentiation. Taken together, our findings demonstrate a new role for GDF15 in normal erythropoiesis as well as in the ineffective erythropoiesis of RARS patients.

Transfusion Independence Achieved in a Case of Diamond Blackfan Anaemia by Essential Amino Acid Supplementation

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome usually presenting as pure red cell aplasia in the first few months of life, which can be associated with systemic features like short stature, skeletal, cardiac and renal abnormalities, and has an increased risk of aplasia and malignancy. The genetic defect is now known in 50% of the cases affecting ribosomal proteins and although the pathophysiological mechanism has not been elucidated, it is known that they have inefficient translation, which a recent report suggests that in some cases can be overcome by leucine supplementation both in vitro and in vivo.We report an 8 years old boy diagnosed at 2 years of age with an Hb 3.4 g/dL requiring monthly blood transfusions. During the first year following the diagnosis he received trials of both steroids and ciclosporin, which he failed to respond to and remained transfusion dependent at the same frequency. At 3 years and 10 months of age the parents sought the advice of a nutritionist because his height was below the 2nd centile (92 cm) and weight in the 9th centile (13.9 kg). High performance liquid chromatography measurement of amino acids in blood revealed deficiency of isoleucin 40 μM (NR >44) and threonine 34 μM (NR >31). He received an essential aminoacid supplementation formula containing the following daily amounts: L-5-Hydroxytryptophan 0.01669 g/day, L-Arginine 0.3373 g/day, L-Histidine 0.3625 g/day, L-Isoleucine 0.6043 g/day, L-Leucine 0.6964 g/day, L-Lysine 0.3539 g/day, L-Methionine 0.5265 g/day, L-Phenylalanine 0.4628 g/day, L-Taurine 0.1669 g/day, L-Threonine 0.4908 g/day, L-Valine 0.6652 g/day, Pyridoxal-5-phosphate 0.0106 g/day, Alpha-ketoglutaric acid 0.3049 g/day. Six months after the initiation of the essential amino acid supplementation the interval between transfusions started to become longer and he became transfusion independent at 12 months. The Hb has increased steadily from 10 g/dL to 13.6 g/dL. His height has steadily improved to the 9th centile and the weight to the 25th–50th centile.Whilst we cannot exclude that this child may have entered into spontaneous remission as it is known to happen in 5–10% of the patients with DBA, this case supports the recent report that essential amino acid supplementation may overcome the terminal differentiation defects found in the erythropoiesis of patients with DBA. A controlled trial of essential amino acid supplementation is warranted in patients with DBA.

Dysregulated Expression of MicroRNA-16 Contributes to Abnormal Erythropoiesis in Patients with Polycythemia Vera

Discovery of JAK2V617F mutation in myeloproliferative neoplasms (MPN) raised intriguing question on how a single mutation might underlie different clinical phenotypes. Micro-RNAs are important regulators of gene expression, intervening in normal and neoplastic hematopoiesis. An abnormal expression profile of miRNAs in primary myelofibrosis granulocytes (Guglielmelli P, Exp Hematol 2007) and in polycythemia vera (PV) erythroid cells has been described (Bruchova H, Exp Hematol 2007; Haematologica 2008); therefore, dysregulation of miRNAs might contribute to MPN pathogenesis. In a global analysis of miRNA expression in CD34+ cells from PV pts we found abnormally increased levels of miR-16; of note, defects in miR-16 causing reduced expression have been implicated in the pathogenesis of chronic lymphocytic leukemia (CLL). There are two miR-16 genes, miR-16-1 (in a cluster with miR-15a on chr 13, mutated/deleted in CLL) and miR-16-2 (in a cluster with miR-15b on chr 3), which differ each to the other in their pre-miR sequence while the mature sequence is conserved. Using stem-looped primer technology and real-time PCR, we determined that expression levels of miR-16 in CD34+ cells from 15 PV pts were 10 to 30-fold higher than in CD34+ cells from controls or reactive erythrocytosis (P<.0001); the increase was specific for miR-16 since levels of mature miR-15a and miR-15b were unchanged. miR-16 was also slightly increased in PV granulocytes (about 2-fold vs controls; P=.026). Levels of miR-16 were independent of the JAK2V671F allele burden (r=0.4). Sequencing the entire pre- and mature miR-16-1/ miR-15a and miR-16-2/miR15b regions disclosed no abnormality, in contrast with CLL for miR-16-1. We also determined gene copy number by RT-PCR, but found no evidence of miR-16-1/2 copy number changes. Since the mature sequence is shared by the two miR-16, we designed a RT-PCR approach to determine the levels of their respective pre-microRNAs. We found that levels of pre-miR-16-2 were significantly increased in PV CD34+ cells compared to controls, while pre-miR-16-1 did not show appreciable changes; there was also a significant correlation between the levels of pre-miR16-2 (unlike premiR- 16-1) and those of total mature miR-16 in PV CD34+ cells (r=0.7, P<.01). The preferential contribution of miR-16-2 to abnormally high total miR-16 levels in PV CD34+ cells was supported by knock-down experiments using siRNAs specific for either miR-16-1 or miR-16-2. In fact, while in normal CD34+ cells the two siRNAs produced a comparable reduction of total miR16 levels, in PV cells only the miR-16-2 siRNA significantly reduced the abnormally increased mature miR-16. It has been previously shown that miR-16 increases in latest stages of normal erythropoiesis in vitro; we determined the kinetics of miR-16 during cultures of CD34+ cells induced to differentiate along erythroid lineage from normal subjects and PV pts. In both control and PV cells there was a prompt down-regulation of miR-16 soon after starting culture; thereafter, miR-16 levels started to increase progressively from day 6 reaching a maximum on day 12–14; at any time point considered, the miR-16 levels measured in PV cells were significantly greater than controls. To address the function of miR-16 in erythroid differentiation, we over-expressed (Amaxa technology) mature miR16 in normal CD34+ cells both at the beginning of culture and at day 6, when progenitors were switched from a proliferative (in the presence of IL-3, IL-6, SCF) to a differentiative (plus Epo) culture phase. No significant modification compared to mock controls was observed in cells transfected at day 0, while in those transfected at day 6 the percentage of CD36+/GPA+ cells in the presence of EPO at least doubled; notably, also in the absence of EPO GPA+ cells, virtually absence in mocktreated cultures, were generated. Finally, preliminary evidences indicated that knockingdown miR-16-2 in PV CD34+ cells resulted in significant reduction of erythroid colony formation. Overall, these data indicate dysregulated expression of miR-16 in CD34+ and in in-vitro generated erythroid cells from PV patients, and point to miR-16-2 as being specifically involved; we also provided evidence for a role of miR-16 in normal erythroid differentiation and for its possible involvement in the abnormal erythropoiesis of PV.

Reticulocyte hemoglobin content

Under normal conditions, reticulocytes are the youngest erythrocytes released from the bone marrow into circulating blood. They mature for 1-3 days within the bone marrow and circulate for 1-2 days before becoming mature erythrocytes. Measurement of cellular hemoglobin concentration has long been reported by automated hematology analyzers as one of the red blood cell indices. The reticulocyte hemoglobin content (CHr or Ret-He) provides an indirect measure of the functional iron available for new red blood cell production over the previous 3-4 days. Measurement of reticulocyte hemoglobin content in peripheral blood samples is useful for diagnosis of iron deficiency in adults (Mast et al., Blood 2002;99:1489-1491) and children (Brugnara et al., JAMA 1999;281:2225-2230; Ullrich et al., JAMA 2005;294:924-930; Bakr and Sarette, Eur J Pediatr 2006;165:442-445). It provides an early measure of the response to iron therapy increasing within 2-4 days of the initiation of intravenous iron therapy (Brugnara et al., Blood 1994;83:3100-3101). Sequential measurements of reticulocyte hemoglobin content in patients with iron deficiency anemia provide a rapid means for assessing the erythropoietic response to iron replacement therapy (Brugnara et al., Blood 1994;83:3100-3101). It is also an early indicator or iron-restricted erythropoiesis in patients receiving erythropoietin therapy (Fishbane et al., Kidney Int 1997;52:217-222; Fishbane et al., Kidney Int 2001;60:2406-2411; Mittman et al., Am J Kidney Dis 1997;30:912-922; Tsuchiya et al., Clin Nephrol 2003;59:115-123; Chuang et al., Nephrol Dial Transplant 2003;18:370-377). Thus, reticulocyte hemoglobin content is a recent addition to an expanding list of biomarkers that can be used to differentiate iron deficiency from other causes of anemia.

Serum Level of Hepcidin and Erythropoietic Activity after Hematopoietic Stem Cell Transplantation.

Hepcidin, first identified in human urine as a bactericidal peptide, is now considered a central molecule that regulates iron metabolism. Synthesis of hepcidin is known to be up-regulated by at least two signals: iron signal and interleukin-6 (IL-6) signal. The existence of a third signal, an erythropoiesis-associated pathway, has recently been suggested by several researchers experimenting with a mouse model treated with either chemotherapy or irradiation. However, the finding has not yet been clarified in human clinical settings partly because of difficulty in quantitatively measuring the serum level of hepcidin. Therefore, we monitored and assessed the association between the serum level of hepcidin and erythropoietic activity before and after stem cell transplantation for hematological malignancies, because the conditioning regimen followed by SCT dramatically changes the status of erythropoiesis in patients. The serum level of hepcidin-25 was quantitatively measured using a liquid chromatography tandem mass spectrometry-based assay system with synthetic isotopic hepcidin as an internal control, which made our quantification much more reliable and reproducible with very small intra- and inter-assay CVs. (Intra-assay and inter-assay CVs were <6.7% and <8.8%, respectively.) We also measured serum levels of various iron-related parameters and IL-6 as well as bone morphogenetic protein (BMP)-2 and BMP-4. The level of serum hepcidin-25 at week -1 was high (mean ± SD; 74.9 ± 93.9 ng/ml), compared to that in control sera from 28 healthy volunteers (mean ± SD; 21.9 ± 12.3 ng/ml), and the level further increased and peaked at week+1 after SCT (mean ± SD; 255.9 ± 99.3 ng/ml), possibly due to the high level of IL-6 and suppression of erythropoiesis. The hepcidin level of patients at week+4 with delayed erythropoiesis (reticulocyte count less than 40,000 /μl) was significantly higher (p<0.02) than that in those with rapid erythropoiesis (reticulocyte count more than 80,000 /μl). The result did not change even if cases were limited to those without elevated IL-6 levels to exclude the influence of IL-6 on hepcidin expression. Transferrin saturation increased to almost 100% after conditioning therapy and slowly decreased concurrent with erythroid recovery. In contrast, the serum ferritin level tended to increase even after erythroid engraftment irrespective of serum IL-6 or hepcidin levels and the volume of blood cell transfusions after SCT. The level of serum BMP-2 was significantly elevated at all of these time points after SCT compared to that in control sera from 6 healthy volunteers. Among these findings, the level of serum hepcidin-25 was inversely and precisely correlated with reticulocyte counts during and after SCT, suggesting that serum hepcidin-25 may be a good indicator of erythropoietic activity as well as iron utilization by erythropoiesis. These findings indicated that hepcidin is regulated by erythropoiesis through a putative erythropoiesis-associated factor. High serum hepcidin levels observed in SCT patients might have contributed to the disturbance of iron homeostasis and increased serum ferritin levels. Further investigation is needed to clarify the clinical significance of hepcidin in SCT settings.

Clinical utility of serum soluble transferrin receptor levels and comparison with bone marrow iron stores as an index for iron-deficient erythropoiesis in a heterogeneous group of patients

To evaluate the correlation between raised soluble transferrin receptor (sTfR) and stainable marrow iron, and to define the utility of sTfR in discriminating between the presence or absence of iron-deficient erythropoiesis in patients with anaemia of chronic disease. Seventy-six consecutive adult patients without accelerated erythropoiesis who had undergone bone marrow (BM) aspiration/trephine for various clinical reasons during 2003-2006 were studied. All patients had serum iron studies (iron, transferrin and ferritin) and sTfR performed within 1 week of BM aspiration/trephine. These 76 patients were assigned to three groups based on the iron status of the BM and sTfR level: patients with normal sTfR and normal BM iron stores (n = 49), patients with an elevated sTfR and normal BM iron stores (n = 13) and patients reduced or absent BM iron stores (n = 14). Means (95% confidence interval) for mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), red blood cell haemoglobin (RBC Hb) content and median (5th and 95th percentiles) for haemoglobin were then calculated. All patients with absent BM iron stores had an elevated sTfR level. Patients with normal BM iron stores and elevated sTfR levels had significantly lower Hb, MCV, MCHC and RBC Hb content than patients with normal BM iron stores and normal sTfR levels. sTfR is the most sensitive serum biochemical marker for the identification of iron-deficient erythropoiesis. Normal BM iron stores can coexist with elevated sTfR and decreased MCV and MCHC. sTfR levels correlate better than BM iron stores with decreased MCV and MCHC. Therefore, sTfR is a useful marker of iron-deficient erythropoiesis, due to both absent iron stores, and restricted iron supply due to anaemia of chronic disease. As a single investigation, however, sTfR does not discriminate between these two causes of iron-deficient erythropoiesis.

Non-Transferrin-Bound Iron and Oxidative Stress Are Related to the Degree of Erythropoiesis in Patients with Thalassemia Intermedia.

There is strong evidence that reactive oxygen species (ROS) are involved in the pathogenesis of thalassaemias. It has been shown that ROS are generated in increased amounts in thalassemic erythrocytes following the precipitation of excess unmatched globin chains and the deposition of non-heme iron and hemichromes. Continuous ROS production in thalassemic individuals may alter their overall redox status and cause tissue damage. Reduction in the levels of vitamin C, vitamin E, and carotenoids has been reported in beta-thalassemic patients receiving transfusion therapy. In this study we investigated the oxidative stress in relation to the degree of erythropoiesis in patients with beta-thalassemia Intermedia (TI). Forty patients with TI were included in the study. Sixteen patients had mild anemia and mild clinical phenotype. The remaining 24 patients had more severe phenotype, but rare transfusions had been used in only 8 of them. Non-transferrin-bound iron (NTBI) levels were determined using graphite furnace atomic absorption spectrometry. Lipid peroxidation expressed as malonyldialdehyde (MDA) concentration was measured by reverse-phase HPLC with fluorimetric detection. The erythroid marrow activity was estimated by measuring soluble transferrin receptors (sTfR) levels with a turbidimetric technique. The main results of the study showed thatNTBI and MDA levels were increased (normal controls <0.05micromol/L and <0.65 micromol/L respectively) in 32/40 patients, while sTfR was found 4- to 20-fold higher than normal in all patients,NTBI, MDA and sTFR levels were significantly higher in patients with the more severe phenotype compared with patients with the milder phenotype (p<0.01),NTBI correlated positively: with MDA (rho=0.502, p<0.003), with sTfR (rho=0.371, p<0.02) and Hb F (rho=0.464, p<0.005), while no correlation was found between NTBI and Hb levels (p>0.278) andMDA was correlated significantly with Hb F (rho=0.464, p<0.005), while the correlation with sTfR levels was poor (rho=0.313, p<0.05).Patients with thalassemia intermedia have increased MDA levels, suggestive of imbalanced oxidant/antioxidant equilibrium. This phenomenon seems to be related to altered iron homoestasis, especially with the increased levels of NTBI. NTBI induces ROS production mainly through the Fenton reaction. NTBI levels are affected by the severity of tissue hypoxia, as these were expressed by their positive correlations with levels of sTfR, MDA and Hb F. The following hypothesis may be explain our findings; increased Hb F leads to tissue hypoxia, which induces further erythropoetic activity, as this is expressed by increased sTfR levels. Increased erythropoetic activity, possibly through a negative effect on hepcidin production (Kattamis et al, Haematologica 91: 809–12, 2006) results in increased NTBI levels, which lead to increased ROS production and MDA levels. Further studies are required to validate this hypothesis that altered iron cycle contributes to the oxidant/antioxidant imbalance observed in thalassemia intermedia.

An Erythroid Differentiation Gene Expression Signature Predicts Response to Lenalidomide in Myelodysplasia.

Lenalidomide is an effective new agent for the treatment of Myelodysplastic Syndrome (MDS). In phase II clinical trials, patients treated with Lenalidomide achieved unprecedented rates of hematologic and cytogenetic responses, particularly in patients with interstitial deletions of chromosome 5q. More than 80% of patients with MDS do not have 5q deletions, though approximately 25% of these patients respond to Lenalidomide.3–5 Neutropenia and thrombocytopenia are the primary toxicities of Lenalidomide, and are severe in some cases. MDS is a highly heterogeneous disorder, and no diagnostic assays predict which patients without 5q deletions will respond to Lenalidomide. Using gene expression profiling, we identified a molecular signature that predicts Lenalidomide response. Biologic characterization of the predictive genes demonstrated that the response signature is comprised of a cohesive set of erythroid-specific genes: patients who respond have a lower expression of the erythroid signature in bone marrow mononuclear cells. To facilitate clinical implementation of a gene expression-based assay, we evaluated the signature using two methods, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and a bead-based detection technology. Using both methods, the erythroid gene expression signature was expressed at lower levels in patients who responded to Lenalidomide. The bead-based methodology employs ligation mediated amplification (LMA), a methodology that is capable of highly multiplexed reactions with preservation of the relative abundance of transcripts, with amplicon detection on Luminex microspheres, already in use for clinical diagnostic assays. This assay is capable of detecting a large gene expression signature (currently up to 100 genes) in a single, well-controlled assay. With further validation, LMA with fluorescent microsphere detection may become the superior technology for clinical diagnostic assays of multi-gene signatures. We tested the erythroid signature in an independent set of 12 samples using LMA with fluorescent microsphere detection. Again, all responders had a lower expression of the erythroid genes than responders. Validation in a larger cohort of patients is underway. These studies suggest that Lenalidomide-responsive patients without 5q deletions have a defect in erythroid differentiation analogous to the ineffective erythropoiesis in patients with 5q deletions, and that an erythroid gene expression signature predicts Lenalidomide activity in MDS.

Is there a role for perioperative intravenous iron therapy in orthopedic and trauma surgery? Clinical experience in major lower limb surgery

SUMMARYPerioperative anemia is a common condition among orthopedic and trauma surgery patients. Preoperative anemia is most frequently caused by iron deficiency or chronic inflammatory disease and is one of the major predictive factors for perioperative blood transfusion. In addition to preoperative anemia, perioperative blood loss is a major cause of postoperative anemia although blunted erythropoiesis attributable to inflammation‐induced inhibition of erythropoietin (EPO) action and decreased iron availability may also play a role. We assessed the safety and effectiveness of perioperative intravenous iron administration, with or without EPO, to stimulate erythropoiesis in patients undergoing trauma (hip fracture) or elective orthopedic surgery (knee and hip replacement). Overall, no side effects were observed. The treatment resulted in lower transfusion requirements, decreased postoperative infection and mortality rates, and shorter length of hospital stay. If confirmed by large randomized clinical trials, these findings will provide a rationale to treat orthopedic and trauma patients with intravenous iron, alone or in combination with EPO, in order to attain higher postoperative hemoglobin concentrations, reduce postoperative complications and shorten hospital stay while limiting exposure to allogeneic blood.

Association between bone mineral density and erythropoiesis in Thai children and adolescents with thalassemia syndromes

Increased marrow erythropoiesis in patients with thalassemia syndromes results in the expansion of bone marrow cavities and consequently decreases bone tissues, leading to osteoporosis. Whether the soluble transferrin receptor (sTfR), a marker of erythropoietic activity, correlates with the bone mineral density (BMD) in thalassemic patients has not previously been addressed. Forty-six children and adolescents with thalassemia syndromes, who were either not transfused or suboptimally transfused, were studied. BMD was determined by dual-energy X-ray absorptiometry. Blood samples were obtained in order to determine sTfR and hemoglobin. The patients were categorized into four groups: 1, beta-thalassemia/hemoglobin E (beta-thal/E) with transfusion-dependency (TD) (n = 18); 2, beta-thal/E with transfusion-independency (TI) (n = 15); 3, beta-thalassemia major (beta-major) (n = 6); 4, hemoglobin H (HbH) (n = 7). All patients had normal serum free thyroxine (FT4) and thyroid-stimulating hormone (TSH), and intact parathyroid hormone (PTH), serum calcium (Ca), phosphate (P), and 25-OH-vitamin D levels. The BMD of patients in the beta-major and beta-thal/E with TD groups were not significantly different. In comparison with the beta-major and beta-thal/E with TD groups, the beta-thal/E with TI and HbH groups had significantly higher BMD of the total body (TB), femoral neck (FN), and lumbar spine (LS), as well as higher levels of hemoglobin. In contrast, the sTfR levels of the beta-major, beta-thal/E with TI, and HbH groups were significantly lower than those of the beta-thal/E with TD group. The BMD of TB, FN, and LS was negatively correlated with the sTfR level, but positively correlated with the hemoglobin level. In conclusion, increased marrow erythropoiesis is one of the major determinants of reduced bone mass in thalassemic patients with either no transfusion or suboptimal transfusion.

Inflammatory cytokine inhibition of erythropoiesis in patients implanted with a mechanical circulatory assist device

Mechanical circulatory assist devices (MCADs) are increasingly utilized independently of cardiac transplantation in the management of heart failure. Though MCAD use incorporates inherent mechanical risks, the inevitable onset of chronic anemia, with its associated morbidity and mortality, is also a significant concern. MCAD support has been correlated with elevated plasma levels of inflammatory cytokines TNF-alpha, IL-1beta, and IL-6, which have separately been found to inhibit erythropoietin (Epo)-induced erythrocyte (RBC) maturation. Previous analysis of hematological parameters for MCAD-supported patients concluded that an amplified inflammatory response impedes RBC proliferation and recovery from hemolytic anemia. Additional analysis may bolster this assertion. Hemoglobin concentration (HC), RBC distribution width (RDW), mean cell volume (MCV), and cardiac index were retrospectively analysed for 78 MCAD-supported patients implanted for greater than 30 days at the University of Arizona Health Sciences Center from 1996 to 2002. Analysis confirms that the HC, a conventional marker for anemia, declines with MCAD placement and remains below the clinically defined, minimum normal value. Inversely, the RDW rises above maximum normal measure, signifying an increased fraction of juvenile RBCs. The MCV remains unchanged and within normal limits, demonstrating adequate substrate for RBC formation. MCAD performance also stabilizes as adequate perfusion returns. These results further support our previously published conclusion that a sufficient response of erythropoiesis occurs in reaction to the onset of anemia by an increased production of immature RBCs. However, the cells never fully mature and join circulation. The patient's inflammatory cytokine response to the implanted device most likely mediates the chronic MCAD-induced anemia by inhibition of Epo effects.

Evidence for Ineffective Erythropoiesis in Patients with Severe Sickle Cell Anemia.

Peripheral destruction of sickled erythrocytes is recognized as the central pathologic mechanism of SCD. Less well established is the contribution of ineffective erythropoiesis to the pathophysiology of SCD. Patients with SCD frequently develop mixed hematopoietic chimerism after nonmyeloablative stem cell transplantation (NMA-SCT). This provides an opportunity to directly compare the differentiation and survival of normal donor and SCD erythropoiesis in vivo. To this end, total genomic DNA (gDNA) and erythroid-lineage specific chimerism in peripheral blood (PB) and marrow (BM) were analyzed in 5 patients (3 with SCD, 2 with non-SCD) following NMA-SCT with HLA-matched donors. RBC chimerism was measured by beta-globin RNA pyrosequencing, which directly and quantitatively sequences coding region polymorphisms in beta-globin RNA that distinguish recipient and donor-derived erythroid precursors. The clinical characteristics of the 5 patients are shown in the table below. All patients developed mixed gDNA chimerism, ranging from 18–50%, between days 60–180, with no differences between PB and BM levels. BM erythroid precursor chimerism, determined by Y chromosome FISH and ABO staining in 4 of 5 patients, were identical to total BM gDNA chimerism. PB from all 3 SCD patients had a ~2 fold greater level of donor-derived beta-globin RNA compared with total gDNA. These levels were similar for the 2 non-SCD patients. The presence of ineffective SS erythropoiesis was directly revealed by analysis of BM, which similarly revealed higher expression of donor-derived beta-globin RNA relative to the degree of donor erythroid progenitor engraftment in Pts 1–3. These findings were not present in Pts 4–5. Chimerism was determined in purified marrow populations of immature (pro-and basophilic –– glycophorin [GYPA+, CD71+ hi]) versus mature (polychromatophilic, orthochromatophilic [GYPA+, CD71+dim] ) erythroblasts to determine the stage of maturation at which SS erythroblasts are lost. While non-SCD patients demonstrated similar rates of donor and host erythropoiesis, all 3 SCD patients demonstrated progressive intramedullary loss of SS erythroblasts with maturation, with enrichment of donor erythroid precursor chimerism appearing from the earliest stages of hemoglobinization. The presence of ineffective erythropoiesis in SCD explains the maturation advantage of AA or SA donor erythroid precursor cells over SS cells that allows for greater donor contribution to overall erythropoiesis following SCT. This is the first definitive in vivo demonstration of ineffective erythropoiesis in SCD, and supports the notion that NMA-SCT can be curative for SCD in the setting of stable donor engraftment. Further studies examining the underlying mechanism of ineffective erythropoiesis in SCD are under investigation.Patient clinical characteristicsPt 1Pt 2Pt 3Pt 4Pt 5Pt age/sex34/F52/M5/M45/M45/FPt dxSCDSCD+ myelomaSCDCLLHDDnr sickle genotype/sexSA/MSA/MSA/M––/F––/Mstem cell sourcePBSCPBSCBMPBSCCB% donor at d60–180PB gDNA2545505410BM gDNA2652504920Cells: erythroid progenitors2349––5022PB beta-globin RNA661001005018BM beta-globin RNA551001005415

The outcomes of hypertransfusion in major ABO incompatible allogeneic stem sell transplantation.

Major ABO incompatibility may be potentially associated with immediate or delayed hemolysis and delayed onset of erythropoiesis in patients receiving allogeneic hematopoietic stem cell transplantation (HSCT). To determine if hemolysis can be prevented by the inhibition of graft erythropoiesis, we performed hypertransfusion and assessed red cell transfusion requirement and independence. Between October 1995 and December 2001, 28 consecutive patients receiving major ABO incompatible HSCT at Samsung Medical Center were hypertransfused to maintain their hemoglobin levels at 15 g/dL or more. We retrospectively compared the outcomes of these patients with those of 47 patients at Asan Medical Center whose target hemoglobin levels were 10 g/dL. Reticulocyte engraftment was significantly delayed in hypertransfused group (51 days vs. 23 days; p=.001). There was no significant difference in the total amount of red cells transfused within 90 days post-HSCT (25 units vs. 26 units; p=.631). No significant difference in the time to red cell transfusion independence was observed between the two groups (63 days vs. 56 days; p=.165). In conclusion, we failed to improve red cell transfusion requirement and independence in major ABO incompatible HSCT with hypertransfusion.

Supplemented very low protein diet ameliorates responsiveness to erythropoietin in chronic renal failure

The aim of this study was to evaluate the relationship between uremic state and erythropoiesis in patients with predialytic chronic renal failure (CRF). We monitored for 2 years the erythropoietin (EPO) requirement in patients with advanced CRF (creatinine clearance < or =25 mL/min), randomized to either low protein diet (LPD) group (0.6 g/kg body weight/day, N = 10) or very low protein diet (VLPD) group (0.3 g/kg body weight/day, N = 10) supplemented with a mixture of ketoanalogs and essential amino acids, both kept at target hemoglobin levels. The achieved protein intake after 6 months was 0.79 +/- 0.02 g/kg body weight/day and 0.50 +/- 0.02 g/kg body weight/day in LPD and VLPD, respectively; such a difference was maintained up to the end of follow up. The final hemoglobin values did not differ from the basal values in either group (11.5 +/- 0.2 g/dL and 11.5 +/- 0.3 g/dL). EPO dose, that was similar at baseline (62.4 +/- 9.6 UI/kg body weight/week and 61.8 +/- 8.8 UI/kg body weight/week subcutaneously), remained unchanged in LPD but progressively decreased in VLPD down to the final value of 41.2 +/- 7.0 UI/kg body weight/week (P < 0.0001 vs. basal and LPD). VLPD was associated with a decrease of urinary excretion and serum levels of urea nitrogen and phosphate; however, EPO requirement was not correlated with the changes of these parameters. On the contrary, the variation of EPO dose directly correlated with the modification of parathyroid hormone (PTH) levels, that diminished from 229 +/- 55 pg/mL to 118 +/- 16 pg/mL (P < 0.0001) in VLPD and did not change in LPD. In patients with advanced CRF, an effective decrease of protein intake of 0.3 g/kg body weight/day induces a reduction of about 35% of the EPO dose required to maintain the target hemoglobin levels. This effect appears dependent on the correction of a moderate secondary hyperparathyroidism.

The safety and efficacy of an accelerated iron sucrose dosing regimen in patients with chronic kidney disease: Management of comorbidities in kidney disease in the 21st century: Anemia and bone disease

Provision of adequate iron to support erythropoiesis in patients with chronic kidney disease (CKD) is time consuming and may present adherence problems for patients in the outpatient setting. We studied an accelerated regimen of high-dose intravenous iron sucrose therapy in a cohort of iron-deficient, anemic CKD patients. Intravenous iron sucrose 500 mg was infused over three hours on two consecutive days in 107 CKD patients (glomerular filtration rate, 32.3 +/- 19.6 mL/min/1.73m2, baseline hemoglobin 10.2 +/- 1.7 g/dL). Iron indices (transferrin saturation, ferritin) were measured at baseline and at two and seven days after completion of the iron regimen. Blood pressures were monitored immediately prior to, and hourly throughout the iron sucrose infusions. Transferrin saturation and serum ferritin increased from 18.5 +/- 8.5% and 177 +/- 123.8 ng/mL at baseline to 40.2 +/- 22.3% and 811 +/- 294.1 ng/mL in 102 evaluated patients (P < 0.015). In 55 patients with additional measurements at 7 days post-dosing, the transferrin saturation and ferritin had fallen to 26.3 +/- 10.6% and 691 +/- 261.8 ng/mL (P < 0.015 compared to two days' post-dose). Blood pressure rose slightly, but not significantly, throughout the infusions, and altering the infusion rate was not necessary. Two patients had seven adverse events that were considered related to iron sucrose. An accelerated regimen of high-dose intravenous iron sucrose therapy in CKD patients is safe and effective in restoring iron stores, and may potentially save time and improve patient adherence.

Mechanism of inhibitory effect of 3-hydroxykynurenine on erythropoiesis in patients with renal insufficiency.

Increase of 3-hydroxykynurenine (3-HKYN) concentration was observed in plasma patients with chronic renal insufficiency (CRI). These changes positively correlated with the concentration of creatinine, but negatively correlated with haematological parameters: haematocrit, haemoglobin and red blood cells count. The changes were accompanied by a slight decrease of the concentration of erythropoietin (EPO) in plasma of patients with CRI. The "in vitro" study in the conditions of hypoxia showed that 3-HKYN inhibited the release of EPO from HepG2 cells to the culture base. In conclusion, the erythropoiesis in CRI could be attributed to the influence of 3-HKYN on EPO synthesis. Thus, we propose that 3-HKYN can be an uraemic of toxin partially responsible for anaemia patients with CRI.

Anemia of chronic disease in rheumatoid arthritis is associated with increased apoptosis of bone marrow erythroid cells: improvement following anti–tumor necrosis factor-α antibody therapy

AbstractCircumstantial evidence has implicated tumor necrosis factor α (TNF-α) in the pathogenesis of anemia of chronic disease (ACD) in rheumatoid arthritis (RA). We investigated the role of TNF-α in erythropoiesis of patients with active RA (n = 40) and the effect of anti–TNF-α antibody administration (cA2). Patients with RA had lower numbers of CD34+/CD71+ and CD36−/glycophorin A+ (glycoA+) bone marrow (BM) cells and increased proportions of apoptotic cells within the CD34+/CD71+ and CD36+/glycoA+ cell compartments, compared to healthy controls (n = 24). Erythroid burst-forming units (BFU-Es) obtained by BM mononuclear or purified CD34+ cells were significantly lower in RA patients compared to controls. These abnormalities were more pronounced among patients with ACD. Increased TNF-α levels in patient long-term BM culture supernatants inversely correlated with BFU-Es and hemoglobin levels and positively with the percentage of apoptotic CD34+/CD71+ and CD36+/glycoA+ cells. Following cA2 therapy, a normalization was documented in the number of CD34+/CD71+ and CD36−/glycoA+ cells, the number of BFU-Es, and the proportion of apoptotic CD34+/CD71+ and CD36+/glycoA+ cells, which was associated with a significant increase in hemoglobin levels compared to baseline. Recovery from anemia was more prominent in patients with ACD. The exogenous addition of an anti–TNF-α antibody in the cultures increased BFU-E number in patients prior to cA2 treatment but not after treatment, further substantiating the inhibitory role of TNF-α on patients' erythropoiesis. We conclude that TNF-α–mediated apoptotic depletion of BM erythroid cells may account for ACD in RA and that cA2 administration may ameliorate ACD in these patients by down-regulating the apoptotic mechanisms involved in erythropoiesis.