Iron-restricted Erythropoiesis Research Articles

Utility of Automated Reticulocyte Research Parameters derived from Beckman Coulter DXH800 in Nutritional Deficiency Anaemias

Introduction: Reticulocyte parameters obtained from the automated analyzers have gained importance not only in the classification of anemias but also in therapeutic monitoring. Their role in nutritional anemia is by far limited to monitoring the hematinic response. The present study aims to describe the distribution of various reticulocyte parameters obtained from Beckman Coulter DXH 800 in cases of nutritional deficiency anemias. Method: Reticulocyte parameters of iron deficiency anemia (IDA) (n=146), megaloblastic anemia (MA) (n=119), and mixed deficiency anemia (n=32) were compared with a non-anemic healthy control group (n=68). Independent sample T-test and one-way ANOVA were used for comparisons. Results: The RET%, MRV, IRF, MSCV, HLR%, RSF, RDWr, and RDWr-SD were significantly higher in MA than in other groups and a significant inverse correlation of Hb was seen with MRV, RDWr, and RDWr-SD. In IDA there was a significant increase in RET%, ARC, IRF, HLR%, HLR#, and RDWr, and a reduction in MRV, MSCV, and RSF. The strongest positive correlation of Hb was with RSF. In the mixed deficiency anemia group, there was an increase in RET%, ARC, IRF, HLR%, HLR#, and RDWr and a reduction in MRV, MSCV, and RSF. Cases on treatment had significantly higher RET%, ARC, and HLR# compared to those without irrespective of etiology. Conclusion: The study strengthens the utility of RSF as a marker of iron-restricted erythropoiesis, and a low RSF and higher MRV in differentiating IDA from mixed deficiency anemia. Irrespective of the etiology, RET%, ARC, HLR%, and HLR# can be utilized for monitoring therapy in nutritional anemias.

Tmprss6 siRNA Treatment in Mice Stops Hepcidin Reduction across Pregnancy

Aim: Hepcidin levels are suppressed during human and murine pregnancy. It is unknown whether hepcidin levels are primarily the result of iron deficiency/utilisation or whether there is a pregnancy-specific mechanism for hepcidin suppression. We reasoned that Tmprss6 inhibition would cause constitutive BMP-SMAD signalling and help determine the mechanism of antenatal hepcidin suppression. Method: Female wildtype C57BL/6 mice were treated subcutaneously every 21 days with a GalNAc-siRNA conjugate targeting Tmprss6 (Silence Therapeutics GmbH, Berlin, Germany) or a non-targeting control (NTC) siRNA conjugate. Mice were mated and monitored for pregnancy. Samples were collected from pregnant mice humanely euthanised at E8.5 or E14.5, and unmated controls. Results: Treatment with Tmprss6 siRNA inhibited hepatic Tmprss6 mRNA expression (unmated fold change 0.077, p<0.0001). qPCR Hamp expression was significantly increased in Tmprss6 siRNA-treated mice, compared to NTC-treated mice, at all timepoints. Serum hepcidin, by ELISA, was higher in Tmprss6 siRNA-treated mice than in NTC-treated mice at E14.5 (1126ng/ml vs 178.5, p<0.0001). In unmated mice, serum hepcidin was not significantly elevated (640.9 vs 980.1ng/ml, p=0.224). Across NTC treated pregnancy, Hamp expression and serum hepcidin were reduced at E14.5 compared to unmated ( Hamp fold change E14.5 0.293, p=0.0367; serum hepcidin E14.5 178.5 vs 640.9, p=0.0247). In contrast, Tmprss6 siRNA treatment caused elevated hepcidin through pregnancy (E14.5 1126 vs unmated 980ng/ml, p=0.99); Fig 1a. Maternal liver iron was unchanged between groups. Tmprss6 siRNA reduced placental iron, fetal liver iron, and fetal weight (0.189 vs 0.226g, p<0.0001), Fig 1b. At E8.5/E14.5, Tmprss6 siRNA reduced Mean Cell Volume (E14.5: 41.42 vs 51.72fL, p<0.0001) and Mean Cell Haemoglobin (E14.5: 11.20 vs 15.68pg, p<0.0001); at E14.5 only, maternal anaemia was seen with Tmprss6 siRNA (haemoglobin 9.4 vs 13.85g/L, p=0.0004). Conclusion: Disruption of Tmprss6 increased hepcidin and ablated the hepcidin fall over pregnancy. This may suggest that any pregnancy-related factor which directly suppresses hepcidin cannot overcome Tmprss6 knockdown. Tmprss6 siRNA treatment resulted in iron-restricted erythropoiesis, reduced placental iron and impacted fetal development, despite unchanged liver iron stores.

Novel Anti-TMPRSS6 Monoclonal Antibody Provides a Potential Therapeutic Approach for the Treatment of Polycythemia Vera

Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) triggered by activating mutations in JAK2, an important kinase in the principal signaling pathway of the erythropoietin receptor. Most of the clinical manifestations of PV are caused by increased erythrocyte count, leading to increased risk of pulmonary hypertension and thrombosis. The mainstay of therapy for PV is therapeutic phlebotomy to reduce the hematocrit (HCT) level and minimize the risk of thrombosis, however, the effect of phlebotomy is transient until patients become iron deficient. Therefore, systemic iron restriction could be effective in treating PV patients. Transmembrane serine protease 6 (TMPRSS6) is a negative regulator hepcidin, the master regulator of iron homeostasis. Increasing genetic, preclinical and clinical evidence has demonstrated that inhibition of TMPRSS6 can potently elevated hepcidin levels, thereby achieving systemic iron restriction. Previously, we reported the generation of anti-TMPRSS6 antibody MWTx-003 and its efficacy in reducing iron overload and improving ineffective erythropoiesis in a mouse model of β-thalassemia. In this study, we explored therapeutic application of MWTx-003 in PV using a Jak2 V617F bone marrow transplantation mouse model. The mouse model exhibits typical PV phenotypes of erythrocytosis and elevated HCT levels. In the PV mice, treatment with MWTx-003 (2-10 mg/kg, i.p., every 4 days for 3 weeks) led to significantly reduced HCT levels, red blood cell (RBC) counts and hemoglobin (HGB) concentration that were comparable to that of wildtype control mice. These effects were evident in a dose-dependent manner and observed as early as two weeks into treatment. It is worth noting that in the 10 mg/kg dose group, a worsening splenomegaly and expansion of early erythroid progenitors were observed after 3 weeks of treatment, and these findings are typical of iron-restricted erythropoiesis in mice. Taken together, the anti-TMPRSS6 antibody offers a promising therapeutic approach in the management of PV, where erythrocytosis and high HCT levels are associated with poor outcomes. A Phase I clinical trial with MWTx-003 in healthy volunteers has been initiated in China.

Re-examining poikilocytosis in goats: prevalence, type and association with age and disease.

Domestic goats (Capra aegagrus hircus) are a food, fiber and companion animal. Abnormal erythrocyte shapes (poikilocytes) are considered normal in young goats, but their association with disease is not well described. Likewise, there is little information on the significance of poikilocytosis in adult goats. The objective of this study was to investigate the prevalence, severity and type of poikilocytosis in young and adult goats and its association with age, sex, breed, laboratory results, and underlying disease. We retrospectively examined clinical and laboratory data from 1254 goats presented at the University of California-Davis Veterinary Medical Teaching Hospital from 1997 to 2019. We analyzed 313 blood smears from goats with moderate or marked (MOD-MKD) poikilocytosis on initial blood smear evaluation. Number and type of poikilocytes per 1000 red blood cells (RBCs) were enumerated. Laboratory values and primary disease categories were compared with the severity and type of poikilocytosis. Kids (<6 mos) and juveniles (>6 mos to <1 year) had a higher prevalence of MOD-MKD poikilocytosis (95/210, 45.2% kids; 27/59, 45.8% juveniles) than adult goats (≥1 year; 190/982, 19.3%) (p < 0.001). Kids had a higher percentage of elliptocytes, dacryocytes, and schistocytes and a lower percentage of polygonal and spiculated RBCs than juvenile and adult goats (p < 0.001). Of goats with MOD-MKD (vs NONE-SLIGHT) poikilocytosis, kids had lower HGB and MCH, and higher RDW (p ≤ 0.02); juveniles and adult goats had lower HCT, MCV, MCH, and albumin concentration (p ≤ 0.01), and all age groups had lower total CO2 concentration and higher PLT counts (p < 0.03). Adult goats with MOD-MKD poikilocytosis also had higher BUN:Cr ratios (p = 0.005). Gastrointestinal parasitism, Johne's disease, diarrhea/enteritis, lice, hepatic disease and renal disease (but not urolithiasis) were significantly associated with MOD-MKD poikilocytosis (p < 0.001). Goats with hepatic and renal disease had a higher prevalence and percentage of spiculated cells (p = 0.001). Goats with Johne's disease had a higher prevalence of polygonal cells (93.3%) and dacryocytes (66.7%) than other diseases, and elliptocytes predominated in a higher proportion (36.0%) of adult goats with GI parasitism vs other diseases (p < 0.05). These findings suggest that iron deficiency is an important pathophysiologic mechanism of poikilocytosis in juvenile and adult goats, and possibly in kids, whether due to iron-restricted erythropoiesis, chronic hemorrhage, functional iron deficiency, or a combination of these mechanisms. Further investigation into the detection and monitoring of iron deficiency and the value of poikilocytosis as a diagnostic marker of iron status in goats is warranted.

Bone marrow iron scoring in healthy and clinically ill dogs with and without evidence of iron‐restricted erythropoiesis

There are few reports in dogs that have evaluated the utility of semi-quantitative scoring of bone marrow iron stores in conjunction with reticulocyte hemoglobin (CHr) to identify iron-restricted erythropoiesis due to absolute iron deficiency or iron sequestration. An established system for scoring iron stores in human bone marrow samples was applied to dogs. The objectives were to evaluate interobserver agreement (Κω ), determine marrow iron scores in dogs without detectable hematologic abnormalities, and assess combined interpretation of iron scores and CHr to evaluate for iron-restricted erythropoiesis. Four blinded observers independently scored iron in 139 Prussian blue-stained canine marrow samples from 0 (none) to 6 (very heavy), including healthy controls (n = 12), clinically ill dogs with (n = 100) and without (n = 16) detectable hematologic abnormalities, and dogs with experimental nutritional iron deficiency (n = 11). Additional medical record data were available for 118 dogs to evaluate for other evidence of iron deficiency (abnormal CHr, RBC indices, serum iron variables, external blood loss, or nutritional deficiencies). Mean Κω was 0.69 (substantial agreement) for all samples but was 0.44 (moderate agreement) for samples with iron scores <3, indicating distinguishing scores 0-2 may not be reliable. Dogs without detectable hematologic abnormalities had scores from 3-5. Dogs with scores <3 and decreased CHr often had more indicators of iron deficiency vs dogs only having low iron scores or low CHr. Evaluation of dogs with marrow iron score <3 for external blood loss or nutritional deficiencies is likely clinically worthwhile, particularly if there is also decreased CHr.

Momelotinib (JAK1/JAK2/ACVR1 inhibitor): mechanism of action, clinical trial reports, and therapeutic prospects beyond myelofibrosis.

Janus kinase (JAK) 2 inhibitors are now part of the therapeutic armamentarium for primary and secondary myelofibrosis (MF). Patients with MF endure shortened survival and poor quality of life. Allogeneic stem cell transplantation (ASCT) is currently the only treatment modality in MF with the potential to cure the disease or prolong survival. By contrast, current drug therapy in MF targets quality of life and does not modify the natural history of the disease. The discovery of JAK2 and other JAK-STAT activating mutations (i.e., CALR and MPL) in myeloproliferative neoplasms, including MF, has facilitated the development of several JAK inhibitors that are not necessarily specific to the oncogenic mutations themselves but have proven effective in countering JAK-STAT signaling, resulting in suppression of inflammatory cytokines and myeloproliferation. This non-specific activity resulted in clinically favorable effects on constitutional symptoms and splenomegaly and, consequently, approval by the Food and Drug Administration (FDA) of three small molecule JAK inhibitors: ruxolitinib, fedratinib, and pacritinib. A fourth JAK inhibitor, momelotinib, is poised for FDA approval soon and has been shown to provide additional benefit in alleviating transfusion-dependent anemia in MF. The salutary effect of momelotinib on anemia has been attributed to inhibition of activin A receptor, type 1 (ACVR1) and recent information suggests a similar effect from pacritinib. ACRV1 mediates SMAD2/3 signaling which contributes to upregulation of hepcidin production and iron-restricted erythropoiesis. Targeting ACRV1 raises therapeutic prospects in other myeloid neoplasms associated with ineffective erythropoiesis, such as myelodysplastic syndromes with ring sideroblasts or SF3B1 mutation, especially those with co-expression of a JAK2 mutation and thrombocytosis.

Iron status in chronic inflammatory disease: therapeutic implications.

Anemia of inflammation (AI) is a very frequent clinical condition affecting globally more than a billion people with chronic inflammatory disorders, such as chronic kidney disease, heart failure, and inflammatory bowel disease. It is usually associated with iron deficiency (ID), which imposes a severe additional burden on the recovery from the primary disease. The pathophysiology of iron dysregulation that may ultimately lead to absolute iron deficiency anemia (IDA) during inflammation is multifactorial and includes reduced iron absorption in the bowel, iron retention in macrophages of the reticuloendothelial system, reduction in circulatory half‑life of erythrocytes, inadequate production and activity of erythropoietin, and impaired proliferation and differentiation of erythroid progenitor cells. These result in hypoferremia and iron-restricted erythropoiesis. AI is mostly mild to moderate, normochromic and normocytic with normal or even increased ferritin levels. The current treatment options for AI include iron replacement therapy, treatment with erythropoiesis‑stimulating agents, and red blood cell transfusion. ID management is based on oral or intravenous iron preparations. Given the pathophysiology, treatment with oral iron, although widely used, presents several limitations that impact its effectiveness in patients with chronic inflammatory conditions. Instead, intravenous iron preparations are a valuable option for patients with chronic inflammatory diseases, as they overcome reduced bowel absorption. Novel therapeutic approaches include downregulation of hepcidin synthesis and function, and stabilization of the hypoxia‑inducible factor via inhibition of prolyl hydroxylase domain. Several studies in vitro and in vivo are ongoing; however, the results in humans are still elusive.

P399 Detection of iron restricted erythropoiesis in patients with IBD: How can we disentangle effects of inflammation?

Abstract Background Iron deficiency and anaemia are among the most common complications of IBD. While the distinction between functional and absolute iron deficiency (AID) is vital for therapeutic decision-making, inflammation is a frequent confounder in their diagnosis: An indirect indicator of body iron stores, serum ferritin is the most specific biomarker of AID. However, it is also an acute-phase reactant and can thus be obscured by IBD-related inflammation. Patients with active IBD often have anaemia of inflammation (AOI), i.e., reduced availability of iron for erythropoiesis (iron-restricted erythropoiesis, IRE) despite adequate stores. ECCO guidelines suggest adding biomarkers to distinguish functional from absolute ID in AOI. Here, we compared the utility of red cell indices and biochemical markers to detect IRE in IBD and define a decision threshold for the selection of biomarkers to discriminate AID and iron sequestration. Methods In an observational cross-sectional study, routine blood samples of patients with IBD were collected. CBC, iron status (serum ferritin, SF; transferrin saturation, TSAT; serum iron % of hypochromic red cells, %HYPO; reticulocyte haemoglobin content, CHr; soluble transferrin receptor, sTfR) and high sensitivity CRP (hsCRP) as inflammatory marker were determined by standard methods. sTfR-F index was calculated (sTfR (mg/L)/log10 SF(μg/L)). Patients with SF&amp;lt;30μg/L were assigned as AID-consistent controls to define decision thresholds for iron markers in patients with SF≥30μg/L which were then used to evaluate IRE in patients with SF≥30μg/L. The accuracy of the markers in the presence/absence of inflammation were assessed and contextual diagnostic thresholds determined. Results Samples were collected from 312 patients with IBD (159f/153m;163CD/149UC;42.7±14.5y), of whom 122(39.1%) had inflammation. 43 patients with SF&amp;lt;30μg/L were selected as AID-control group. Results in this group were: TSAT (cut-off 20%; AUCROC 0.730; sens.65%, spe.34%), %HYPO (3.5%; 0.733; 42%, 93%), CHr (32pg; 0.713; 51%, 86%), sTfR (2.70mg/L; 0.775; 81%, 63%), sTfR-F (2.01; 0.914; 88%, 88%). sTfR-F was used as reference for IRE in patients with SF ≥30μg/L. Accuracy of the markers as detectors of IRE is shown in Table 1. Conclusion sTfR was the most accurate sole marker of IRE in patients both with and without inflammation, its cut-off being higher in the presence of inflammation. In patients without inflammation, SF was a valuable additional parameter. Relative cost-effectiveness of the markers alone or in combination is under investigation.

Hepcidin and its multiple partners: Complex regulation of iron metabolism in health and disease.

The peptide hormone hepcidin is central to the regulation of iron metabolism, influencing the movement of iron into the circulation and determining total body iron stores. Its effect on a cellular level involves binding ferroportin, the main iron export protein, preventing iron egress and leading to iron sequestration within ferroportin-expressing cells. Hepcidin expression is enhanced by iron loading and inflammation and suppressed by erythropoietic stimulation. Aberrantly increased hepcidin leads to systemic iron deficiency and/or iron restricted erythropoiesis as occurs in anemia of chronic inflammation. Furthermore, insufficiently elevated hepcidin occurs in multiple diseases associated with iron overload such as hereditary hemochromatosis and iron loading anemias. Abnormal iron metabolism as a consequence of hepcidin dysregulation is an underlying factor resulting in pathophysiology of multiple diseases and several agents aimed at manipulating this pathway have been designed, with some already in clinical trials. In this chapter, we assess the complex regulation of hepcidin, delineate the many binding partners involved in its regulation, and present an update on the development of hepcidin agonists and antagonists in various clinical scenarios.

Benefits of pre-operative oral Sucrosomial® iron supplementation in cardiac surgery: influence of patient's baseline hemoglobin and gender.

The prevalence of low pre-operative hemoglobin (Hb) among cardiac surgery patients is high. As iron homeostasis is often impaired in these patients, restoration of iron availability might over-ride iron-restricted erythropoiesis. This post-hoc analysis of a previously published, large, randomized clinical trial (ClincalTrials.gov NCT03560687; n=1,000) assesses which sub-cohort of patients benefits the most from pre-operative Hb optimization with oral Sucrosomial® iron. Patients without baseline Hb (n=349) or receiving >5 red blood cell units (n=57) were excluded from the study. Data from the remaining 594 were reanalyzed according to treatment, baseline anemia (Hb <13 g/dL) or gender. Patients (pt) received a one-month course of 60 mg/day Sucrosomial® iron (Iron group, n=309) or routine care (Control group, n=285) prior to elective cardiac surgery. Main end-point variables were increase in Hb from randomization to hospital admission, transfusion requirements, and cost-effectiveness of Sucrosomial® iron administration. At hospital admission, Hb had increased 0.7 g/dL and 0.1 g/dL, for Iron and Control groups, respectively (p<0.001), with no gender-related differences, leading to a decrease in transfusion rate (30 vs 59%, respectively; p<0.001) and transfusion index (0.5 units/patient vs 1.2 units/pt, respectively; p<0.001). Sucrosomial® iron administration was well-tolerated, and yielded cost-savings of €92/pt (p<0.001), particularly in those presenting with baseline Hb <13 g/dL. This post-hoc analysis confirms pre-operative Sucrosomial® iron administration is a safe and cost-effective strategy to increase preoperative Hb and decrease transfusion requirements in elective cardiac surgery, especially in those anemic at baseline.

ALK2 Inhibition and a Modified Activin Receptor Type Iia Ligand Trap Cotherapy Maximized Hematologic Improvements in a Mouse Model of Anemia of Inflammation

Introduction: Anemia of inflammation (AI) is commonly seen in patients with chronic diseases, including chronic kidney disease (CKD). Current therapeutic options to mitigate anemia in AI include treating the underlying inflammatory disease, oral or intravenous iron supplementation, erythropoietin, and blood transfusions. The persistent release of inflammatory cytokines in AI not only induces the iron regulator hepcidin to inhibit iron absorption and recycling, but also directly impacts the marrow to suppress erythropoiesis, further worsening the ineffective, iron-restricted erythropoiesis. Therefore, targeting both iron and erythropoiesis could comprehensively address anemia in AI. We previously showed that ALK2 inhibition was sufficient to reduce serum hepcidin and improve hemoglobin in a CKD mouse model. We also demonstrated that a modified activin receptor IIA ligand trap RKER-050 increased hemoglobin and red blood cell production by promoting erythroid differentiation in mice. Here, we explored the potential of additive improvements in hematologic parameters that could be achieved with a combination of hepcidin suppression by ALK2 inhibition and increased hematopoiesis with RKER-050 in a mouse model of AI. Methods: C57BL/6 mice were fed a control or 0.2% adenine with 40 ppm iron diet (Ade 40) for 5 weeks to induce CKD. After confirming anemia, CKD mice were subcutaneously treated with 3 mg/kg isotype control or KTI-m216, a neutralizing antibody against ALK2, twice a week for 4 weeks while continuing the Ade 40 diet or switching to a 0.2% adenine with 3 ppm low iron diet (Ade 3). Combination therapy was evaluated in CKD mice on Ade 40 diet receiving biweekly isotype or KTI-m216 and also received either weekly PBS or 7.5 mg/kg RKER-050 intraperitoneally for 4 weeks. Results: CKD mice developed impaired kidney function and characteristics of AI, including increased serum IL-6 and hepcidin, hypoferremia, tissue iron retention, and anemia compared to control mice. Serum hepcidin was reduced >95% in KTI-m216 treated mice on both Ade diets compared to mice receiving isotype control (CKD control). Transferrin saturation (TSAT) increased 65% in KTI-m216 treated mice on Ade 40 diet, with a similar trend in mice on Ade 3 diet. When compared to CKD control of the same diet, KTI-m216 treated mice had similar hematologic improvements: hemoglobin (+2.2 g/dL), hematocrit (+5.4%), red blood cells (+1.1 M/μl), and reticulocyte hemoglobin (CHr, +1.8 pg). These data support that increased circulating iron by KTI-m216 mediated hepcidin suppression can increase erythropoiesis in CKD mice. Data also suggest that dietary iron contribution is negligible in supporting erythropoiesis in this context. With combination therapy, mice receiving KTI-m216 alone (KTI-m216 and PBS) and a combined KTI-m216 and RKER-050 therapy had a similar reduction in serum hepcidin (>91%) and a similar increase in TSAT (>1.7-fold), compared to mice receiving vehicle (isotype and PBS) controls. Mice treated with KTI-m216 alone increased hemoglobin to 11.4 g/dL compared to vehicle treated mice with hemoglobin of 8.0 g/dL. Treatment with a combined KTI-m216 and RKER-050 therapy showed a further increase in hemoglobin to 13.5 g/dL. Additionally, red blood cell production was 6.4 M/μl in mice given vehicle controls and increased to 8.3 M/μl in KTI-m216 alone and 9.8 M/μl in combined KTI-m216 and RKER-050; hematocrit was 23.4% in vehicle control, 32.5% in KTI-m216 alone and 38.7% in combined KTI-m216 and RKER-050. Although hematocrit was not fully rescued, hemoglobin and red blood cells in mice receiving a combined KTI-m216 and RKER-050 therapy were not different from the non-CKD controls. Conclusion: These data illustrate that inhibition of ALK2 with KTI-m216 could potentially release sufficient iron from the recycling pathway to ameliorate anemia, but not restore RBC parameters to normal levels. Combined treatment with activin receptor ligand trap, RKER-050 and KTI-m216 normalized the RBC production in this preclinical model. These data support that increasing erythropoiesis with RKER-050 combined with iron mobilization with KTI-m216 has the potential to achieve the maximal amelioration of anemia in AI.

Iron deficiency anemia and thrombosis risk in children-revisiting an old hypothesis.

Iron deficiency anemia has a high prevalence in children and has repeatedly been implicated as a risk factor for arterial and venous thrombosis. As an effective therapy for iron deficiency anemia is available, understanding the association between this form of anemia and the potentially severe thrombosis phenotype is of major clinical interest. Recent findings shed light on pathophysiology of hypercoagulability resulting from iron-restricted erythropoiesis. Specifically, an animal model of induced iron deficiency allowed identifying multiple mechanisms, by which iron deficiency anemia results in increased thrombus formation and thrombus progression both in arterial and venous thrombosis. These findings complement and support conclusions derived from clinical data. The purpose of this mini review is to summarize current evidence on the association of iron deficiency anemia and thrombosis. We want to increase the awareness of iron deficiency as a risk factor for thrombosis in the pediatric population. We discuss how novel pathophysiological concepts can be translated into the clinical settings and suggest clinical studies on prevention and treatment strategies in high-risk patient groups.

The oral ferroportin inhibitor vamifeport improves hemodynamics in a mouse model of sickle cell disease

AbstractSickle cell disease (SCD) is an inherited hemolytic anemia caused by a single point mutation in the β-globin gene of hemoglobin that leads to synthesis of sickle hemoglobin (HbS) in red blood cells (RBCs). HbS polymerizes in hypoxic conditions, leading to intravascular hemolysis, release of free hemoglobin and heme, and increased adhesion of blood cells to the endothelial vasculature, which causes painful vaso-occlusion and organ damage. HbS polymerization kinetics are strongly dependent on the intracellular HbS concentration; a relatively small reduction in cellular HbS concentration may prevent HbS polymerization and its sequelae. We hypothesized that iron restriction via blocking ferroportin, the unique iron transporter in mammals, might reduce HbS concentration in RBCs, thereby decreasing hemolysis, improving blood flow, and preventing vaso-occlusive events. Indeed, vamifeport (also known as VIT-2763), a clinical-stage oral ferroportin inhibitor, reduced hemolysis markers in the Townes model of SCD. The RBC indices of vamifeport-treated male and female Townes mice exhibited changes attributable to iron-restricted erythropoiesis: decreased corpuscular hemoglobin concentration mean and mean corpuscular volume, as well as increased hypochromic and microcytic RBC fractions. Furthermore, vamifeport reduced plasma soluble VCAM-1 concentrations, which suggests lowered vascular inflammation. Accordingly, intravital video microscopy of fluorescently labeled blood cells in the microvasculature of Townes mice treated with vamifeport revealed diminished adhesion to the endothelium and improved hemodynamics. These preclinical data provide a strong proof-of-concept for vamifeport in the Townes model of SCD and support further development of this compound as a potential novel therapy in SCD.

Iron- and erythropoietin-resistant anemia in a spontaneous breast cancer mouse model.

Anemia of cancer (AoC) with its multifactorial etiology and complex pathology is a poor prognostic indicator for cancer patients. One of the main causes of AoC is cancer-associated inflammation that activates mechanisms, commonly observed in anemia of inflammation, whereby functional iron deficiency and iron-restricted erythropoiesis are induced by increased hepcidin levels in response to raised levels of interleukin-6. So far only a few AoC mouse models have been described, and most of them did not fully recapitulate the interplay of anemia, increased hepcidin levels and functional iron deficiency in human patients. To test if the selection and the complexity of AoC mouse models dictates the pathology or if AoC in mice per se develops independently of iron deficiency, we characterized AoC in Trp53floxWapCre mice that spontaneously develop breast cancer. These mice developed AoC associated with high levels of interleukin-6 and iron deficiency. However, hepcidin levels were not increased and hypoferremia coincided with anemia rather than causing it. Instead, an early shift in the commitment of common myeloid progenitors from the erythroid to the myeloid lineage resulted in increased myelopoiesis and in the excessive production of neutrophils that accumulate in necrotic tumor regions. This process could not be prevented by either iron or erythropoietin treatment. Trp53floxWapCre mice are the first mouse model in which erythropoietin-resistant anemia is described and may serve as a disease model to test therapeutic approaches for a subpopulation of human cancer patients with normal or corrected iron levels who do not respond to erythropoietin.

Role of hepcidin as a biomarker for iron status and its effect on anemia management in patients with chronic kidney disease (stage II-Iv) after HCV treatment

Abstract Background Anemia is a severe complication of chronic kidney disease (CKD) that is seen in more than 80% of patients with impaired renal function. Although there are many mechanisms involved in the pathogenesis of anemia of renal disease, the primary cause is the inadequate production of erythropoietin by the damaged kidneys. Aim of the work to assess hepcidin level in non dialysis patients (CKD stage 4 &amp;5) treated from Hepatitis C virus and its relation to iron parameters. Patients and Methods This study was conducted on 20 CKD patients (stage 4 and 5) treated from hepatitis C virus. All candidates included in this study subjected to careful history taking, full clinical examination and investigations (including complete blood count, renal chemistry, HCVAb, serum iron, total iron binding capacity, TSAT%, ferritin and hsCRP. Serum hepcidin was analyzed by ELISA technique. Results Serum hepcidin was 26.35±7.26; 40% in stage III, 37.8% in stage IV and 22.2% in stage V. There was statistically significant difference between GFR stages according to Hb., Drug intake ACE inhibitor/ARB, Plt., Creatinine, BUN, Iron, TIBC, Ferritin, T SAT%, CRP and Serum Hepcidin. We showed significant correlations between serum hepcidin and TIC, Iron, TIBC, Ferritin and TSAT%. Conclusion Median hepcidin value is elevated in nondialysis CKD patients due to increased inflammation and decreased clearance of hepcidin. Furthermore, iron status modifies serum hepcidin level and its association with Hb. Increased hepcidin level leads to iron-restricted erythropoiesis and recombinant human EPO (rhEPO) resistance by inhibiting iron absorption from gut and iron recycling from macrophages. Hence, elevated hepcidin can predict need for parenteral iron to overcome hepcidin-mediated iron-restricted erythropoiesis and need for relatively higher rhEPO doses to suppress hepcidin.

The role of hepcidin as a biomarker for iron status in patients with chronic kidney disease (stage IV and V) with negative virology

Abstract Background Anemia is a severe complication of chronic kidney disease (CKD) that is seen in more than 80% of patients with impaired renal function. Hepcidin, an acute phase reactant protein produced in the liver, is a key regulator of iron homeostasis. Aim of the Work to assess hepcidin level in 45 non-dialysis patients (CKD stage IV and V with negative virology) and its relation to iron parameters. Patients and Methods A cross sectional study was conducted at Nasser Institute for Treatment and Research on 45 patients with chronic kidney disease stage IV and V. All patients included in this study were subjected to the following: Careful history taking, full clinical examination and proper laboratory investigations. Results A statistically significant difference was found between CKD stage 4 and stage 5 according to Hb., iron, TIBC, Frerretin, serum and CRP. Also, there was a significant positive correlation of serum hepcidin with serum ferretin and hsCRP, while Hb and iron were significantly negatively correlated with hepcidin. We found statistically significant decrease in Hb level, serum Iron level, and TIBC in CKD stage 5 less than stage 4. We found statistically significant increase in Hepcidin level, serum ferritin, and hsCRP in CKD stage 5 more than stage 4. We found statistically significant Positive correlation between serum hepcidin with serum ferretin among patients with CKD stage 4 and 5. We found statistically significant Positive correlation between serum hepcidin with hsCRP among patients with CKD stage 4 and 5. We found statistically significant negative correlation between serum hepcidin with Hb among patients with CKD stage 4 and 5. A statistically significant Positive correlation between serum hepcidin with serum Iron among patients with CKD stage 4 and 5. Also we reported a statistically non-significant negative correlation between serum hepcidin and TIBC. Conclusion Elevated hepcidin can predict the need for parenteral iron to overcome hepcidin-mediated iron-restricted erythropoiesis and need for relatively higher rhEPO doses to suppress hepcidin in CKD patients with negative viral markers.

Role of Hepcidin as a biomarker for iron status in patients on regular hemodialysis

Abstract Background The etiology of anemia in End Stage Renal Disease is multifactorial. Importantly, ESRD patients also have several abnormalities in systemic homeostasis of iron, an essential component in the production of red blood cells. Aim of the Work to assess hepcidin level in negative virology End Stage Renal Disease patients &amp; its relation to iron level and erythropoiesis. Patients and Methods This study was conducted on 45 patients who are stage V chronic kidney disease on regular haemodialysis. Ten age and sex matched controls were included in the study. The study included 29 (64.4%) males and 16 (35.6%) females; their mean age was 53.40± 11.56 years. The prevalence of diabetes among the studied cases was 17.8%, while that of hypertensive was 42.2%. Mean of serum iron level was 64.23±19.53. Mean of TIBC was 409.96±67.85. Mean of Ferritin level 394.55±139.23 and mean of Hepcidin level was 218.51±127. Results Significant negative correlation between Hepcidin level and the Hemoglobin level, and highly significant positive correlation between Hepcidin level and serum Ferritin. Hepcidin up-regulation in the setting of CKD, with subsequent increased serum levels, results in impaired iron absorption from the intestine and decreased iron release from body storage sites. Ultimately, in the setting of such elevated levels, a state of functional iron deficiency may develop and lead to anemia due to iron-restricted erythropoiesis. Conclusion Based on current evidence, it seems likely that hepcidin represents a potentially modifiable mediator of anemia of CKD and is thus a potential target for future anemia therapy.

A simplified diagnostic pathway for the differential diagnosis of iron deficiency anaemia and anaemia of chronic disease.

Iron deficiency anaemia (IDA) and anaemia of chronic disease (ACD) are common causes of anaemia with similar clinical and laboratory features. IDA is caused by low iron stores while ACD is due to iron-restricted erythropoiesis occurring in inflammatory states. Differential diagnosis requires analysis of multiple biochemical and haematological parameters. IDA can occur simultaneously to ACD (mixed aetiology). It is essential that true iron deficiency is identified, as these patients will require iron therapy. This preliminary study investigated whether hepcidin, the master regulator of iron homeostasis, in conjunction with reticulocyte haemoglobin equivalent (RetHe) has the potential to differentiate IDA from ACD, and to exclude IDA in patients with mixed aetiology. Hepcidin concentration (measured using a commercially available ELISA method), RetHe, and iron parameters along with C-reactive protein (CRP) were analysed in 77 Gastroenterology patients with anaemia in a secondary care setting. Receiver operator characteristic (ROC) analysis showed that hepcidin at an optimal cut-off concentration of <6ng/ml could identify IDA with a sensitivity and specificity of 88.9% and 90.6% respectively and could distinguish ACD from IDA with both a sensitivity and specificity of 100% at a cut-off of >46ng/ml. Identifying true IDA in mixed aetiology patients could be achieved by RetHe analysis and applying an optimal cut-off of <30pg. Hepcidin, in conjunction with RetHe, offers a new simplified diagnostic pathway for differential diagnosis of IDA and ACD, thereby reducing the diagnostic turnaround time and allowing appropriate treatment of patients with a true iron deficiency.

Preoperative Sucrosomial Iron Supplementation Increases Haemoglobin and Reduces Transfusion Requirements in Elective Heart Surgery Patients: A Prospective Randomized Study

Background: Low preoperative haemoglobin is frequently observed in heart surgery patients and is associated with a significant decrease in haemoglobin between post-operative days 2 and 3, known as haemoglobin drift. Overall, these patients tend to receive many RBC transfusions. Since iron homeostasis is often impaired in these patients, restoration of iron availability might override iron-restricted erythropoiesis. However, reduced tolerance to oral iron salts has limited this strategy to intravenous iron administration. Study Design and Methods: The purpose of this study was to assess whether preoperative supplementation with oral sucrosomial iron, a new iron-delivery technology with improved tolerance and bioavailability, might be an effective strategy for this patient population. One thousand consecutive patients were randomized and received either a one-month course of sucrosomial iron (60 mg/day) or no treatment prior to elective heart surgery at a single high-volume centre (ClinicalTrials.gov NCT03560687). Primary end-points were haemoglobin concentration on the day of hospital admittance and number of blood transfusions. Secondary end-points were haemoglobin drift, tolerance of treatment and cost-effectiveness of sucrosomial iron administration. Results: Baseline haemoglobin in the treatment group was higher (by 0.67 g/dL; p&lt;0.001) than that in the control group. The percentage of patients in the treatment group who required transfusion (35.4%) was half that in the control group (64.6%). The average number of transfused units per operation was 0.95 vs. 2.03 in the treatment and control groups, respectively. Haemoglobin drift was substantially similar in the two groups, and the tolerability of treatment was excellent (98%). The overall cost of treatment was 156 Euros less in the treatment group, expressed as a raw cost of transfusion. Conclusion: In elective heart surgery, routine preoperative sucrosomial iron administration seems to be a safe, well-tolerated and cost-effective strategy to increase preoperative haemoglobin and reduce the need for allogeneic blood transfusions.

P498 Diagnosing and differentiating iron deficiency in inflammatory disorders: still an unsolved mystery?

Abstract Background It is well established that inflammation can mimic some aspects of iron deficiency (ID) by impairing the utilisation of existing iron stores for red cell production and inducing an iron-sequestration syndrome and hypoferraemia. Diagnosis of ID is therefore complex and there is currently no standard clinical test capable of clearly distinguishing ID when inflammation is present. Often, a combination of parameters is necessary. The aim of this study was to investigate which sole parameter or combination of parameters can best predict ID in patients with IBD. Methods A cross-sectional study was conducted using routine blood samples from patients with IBD. Blood samples were analysed for standard blood count, iron status (serum ferritin; SF, transferrin saturation; TSAT), inflammation parameters (high sensitivity C-reactive protein; hsCRP) and soluble transferrin receptor (sTfR) using standard methodology. A multiparameter index test (MCV, TSAT, SF) was performed to define patients with iron restricted erythropoiesis (IRE) (IRE group) when at least 2 of the 3 parameters indicated IRE. Cut-off values for TSAT (&amp;lt;20%) and SF (&amp;lt;30/μL) were defined according to ECCO guidelines and for MCV (80 fL) according to the literature. Criteria taken to indicate absolute ID (AID) were hsCRP &amp;lt;5 mg/L, SF 30 μg/L and TSAT &amp;lt; 20%, while functional ID (FID) was denoted by hsCRP ≥ 5 mg/L, SF &amp;lt; 100 μg/L and TSAT &amp;lt; 20%. Results 168 IBD patients (88f; 85CD/83UC;43.4±14.9y) were enrolled. Of these, 107/168 had inflammation. 100/168 patients had IRE, of whom 70/100 had FID and 30/100 had AID. ROC analysis was performed to compare different parameters as detectors of IRE and to summarise the performance of each respective parameter (Table 1). When inflammation was absent, the best combination was SF and TSAT, whereas in the presence of inflammation, the best combination was found to be TSAT and sTfR. The addition of MCV, which showed high specificity regardless of inflammation, was found to add additional value to the diagnosis in the context of inflammation (p &amp;lt;0.001). Conclusion Of those parameters studied, none was suitable to diagnose IRE when taken alone. In the absence of inflammation, ID in IBD can be adequately determined by combining SF and TSAT. When inflammation is present, however, the combination of TSAT with sTfR measurement was found to be more accurate, while the additional inclusion of MCV added additional value to the diagnosis.