Red Cell Production Research Articles

Incidence of deep vein thrombosis and pulmonary embolism in closed foot and ankle fractures treated with conservative versus surgical management: A nationwide analysis

ObjectivesThough there is extensive research on recovery and outcomes of proximal joint fractures, such as hip and knee, there is a paucity of such research in foot and ankle fractures and a lack of uniform data on the incidence of venous thromboembolism (VTE). This is a retrospective cohort study that seeks to investigate the incidence of VTE following surgically versus conservatively treated foot and ankle fractures MethodsThis study included all adult-patient hospitalizations with International Classification of Disease 10th Revision diagnosis codes related to closed foot and ankle fractures as a primary diagnosis in the National Inpatient Sample, an administrative database part of the Healthcare Cost and Utilization Project, for a total of 209,595 cases. Multivariate binary logistic regression was performed to determine the effect of age, gender, past medical history of venous thromboembolism (PMHVTE), Charlson Comorbidity Index, race, surgery, and transfusion of red cell products on the incidence of deep venous thrombosis (DVT) and pulmonary embolism (PE). ResultsOur study found an overall incidence of DVT and PE to be 0.4 % and 0.3 %, respectively, following foot or ankle fracture. Conservative management was found to have an increased relative risk of DVT and PE of 2.02 and 1.15, respectively. This difference persisted on performing inverse probability treatment weighting (IPTW) analysis to account for confounders. PMHVTE and requirement of blood transfusion was associated with an increased odds ratio (OR) of 3.41 and 3.217, respectively. Cases identified as Black and Asian/Pacific Islander also had an increased OR of DVT. ConclusionsThe results of this study demonstrate that surgical management of foot and ankle fractures is associated with a lower relative risk of VTE compared to medical management. PMHVTE and transfusion of red cell products increase the odds of VTE following either form of intervention.

A comparative study of two routinely used protocols for ex vivo erythroid differentiation

BackgroundErythropoiesis is a complex developmental process in which a hematopoietic stem cell undergoes serial divisions and differentiates through well-defined stages to give rise to red blood cells. Over the last decades, several protocols have been developed to perform ex vivo erythroid differentiation, allowing investigation into erythropoiesis and red cell production in health and disease. ResultsIn the current study, we compared the two commonly used protocols by assessing the differentiation kinetics, synchronisation, and cellular yield, using molecular and cellular approaches. Peripheral blood CD34+ cells were cultured in a two-phase (2P) or a four-phase (4P) liquid culture (LC) and monitored for 20 days. Both protocols could recapitulate all stages of erythropoiesis and generate reticulocytes, although to different extents. Higher proliferation and viability rates were achieved in the 4P-LC, with a higher degree of terminal differentiation and enucleation, associated with higher levels of the erythroid-specific transcription factors GATA-1, KLF-1, and TAL-1. Although the 2P-LC protocol was less efficient regarding terminal erythroid differentiation and maturation, it showed a higher yield of erythroid progenitors in the erythropoietin (EPO)-free expansion phase. ConclusionsWe provide data supporting the use of one protocol or the other to study the biological processes occurring in the early or late stages of erythroid differentiation, depending on the physiological process or pathological defect under investigation in a given study.

Appropriate use of blood components

Blood products are biological products derived from human blood. Japan currently meets its domestic needs with red cell products, platelet products, and plasma derivatives produced from blood, but this system could face challenges in the future. Blood transfusion therapy is only a replacement therapy that relieves symptoms, and should not worsen the patient's outcome. Blood transfusion therapy should also be based on evidence, and the Japan Society of Blood Transfusion and Cell Therapy has created "Blood Transfusion Guidelines Based on Scientific Evidence" and calls for its proper use. Although the transfusion triggers in this guideline are effective for considering the indication for blood transfusion, the necessity of blood transfusion is comprehensively judged by taking into account factors such as the pathology, severity of symptoms, and comorbidities in each patient. Hematologists and pediatricians who frequently use blood products need to be fully aware of the risks they pose and promote safer and more appropriate use.

Differential Effect of Transferrin Lobe Iron Occupancy on a Murine Model of Inflammation

Introduction: Iron status is both affected by and a determinant of the inflammatory response. Several lines of evidence support a role for transferrin as a signaling as well as an iron delivery molecule. We previously demonstrated that mice with transferrin mutations that prevent iron binding to the C-lobe of transferrin (C-blocked) have increased red cell production relative to erythropoietin and increased hepcidin expression relative to iron status compared to mice with transferrin mutations that prevent iron binding to the N-lobe of transferrin (N-blocked). Based on these observations, we hypothesize that two forms of transferrin would differentially modulate the hematopoietic and iron distribution changes that occur with inflammation. We explored this hypothesis by comparing the effects of lipopolysaccharide (LPS) administered either acutely or in repeated doses to wild-type, N-lobe blocked, or C-lobe blocked transferrin mutant mice. Methods: For a model of acute inflammation, 14 day old wild-type (wt), N-blocked and C-blocked Tf mutant mice were treated with 1mg/kg LPS or carrier i.p. and sacrificed 6 hours later. Repeated LPS dosing, seven daily i.p. injections of 0.3 mg/kg LPS or carrier beginning at seven days of age, served as a model of chronic inflammation. Results: While wild-type and C-blocked mice had the expected hypoferremia in response to acute LPS administration, there was no effect on serum iron in the N-blocked mice. These mice moreover demonstrated an unanticipated increase in liver iron concentrations with LPS. All strains manifested expected increases in liver hepcidin expression; however, these were attenuated in the N-blocked mice relative to the C-blocked. Moreover, N-blocked mice have higher serum C reactive protein concentrations compared to C-blocked and wt mice following acute LPS administration. The elevated Hamp1 expression was not sustained in the chronic model. Although serum iron returned to baseline in wt and C-blocked mice, it remained elevated in N-blocked mice. Hemoglobin concentrations were decreased in all strains after repeated endotoxin dosing; however, C-blocked mice demonstrated higher neutrophil to lymphocyte ratios than wt or N-blocked mice, and increased circulating RBC count relative to WBCs. Conclusions: We conclude that the specificity of transferrin lobe iron occupancy is an important modulator of the inflammatory response and speculate that this modulation includes an effect on hematopoietic cell lineage determination.

Irf5 Expression in Macrophages Contributes to Iron Regulation within Erythromyeloblastic Islands

Erythroblastic islands (EBIs) classically consist of a central macrophage supporting surrounding erythroid precursors as they develop from CFU-Es to reticulocytes. More recently, it has been shown that, in the bone marrow, these structures also support granulopoiesis, expanding our understanding of the niche, and leading to the concept of erythromyeloblastic islands (EMBI). Under certain inflammatory conditions, there is an imbalance between erythropoiesis and granulopoiesis, eventually leading to anemia and increased neutrophil counts. The central macrophage has been proposed to play a role in regulating this balanced blood production; however, the molecular mechanisms remain unknown. Recently, we demonstrated that mice lacking the transcription factor interferon regulatory factor 5 ( Irf5 -/-) present iron deficiency anemia as they age, along with altered structures in the bone marrow EMBIs. Further, our CITEseq studies showed that Irf5 is specifically expressed by macrophages in the EMBIs. Therefore, we concluded that Irf5 is a regulator of the balance between erythropoiesis and granulopoiesis in the bone marrow erythromyeloblastic niche. To understand the mechanisms leading to iron deficiency anemia, as well as changes in the island, we first undertook a comprehensive analysis of hematopoietic progenitors. We measured the relative amounts of megakaryocytic-erythroid progenitors (MEP), granulocyte-monocyte progenitors (GMP) and common myeloid progenitors (CMP) in the bone marrow and observed decreased MEP (WT: 82.81% vs KO: 76.28%, p-value 0.011), increased GMP (WT: 14.94% vs KO: 22.16%, p-value 0.002) and unchanged CMP compared to wild-type (WT) as the mice aged. Given that Irf5 is expressed in both hematopoietic and non-hematopoietic cells, we generated Irf5fl/fl-Vav-iCre+ mice to evaluate the contribution of non-hematopoietic Irf5 to EMBI formation and composition. We observed a significant reduction in the number of EMBI, with overrepresentation of myeloid cells, suggesting that the phenotype observed in Irf5 -/- is mostly due to hematopoietic Irf5. However, when we measured the relative amounts of MEP and GMP in the Irf5fl/fl-Vav-iCre+ mice, these remained unchanged, suggesting that cell non-autonomous functions contribute to EMBI composition. scRNAseq analyses of EMBIs from 3 and 9 months-old littermate-matched WT and Irf5 -/-mice revealed that heme synthesis was the only downregulated pathway. In addition, central macrophages from Irf5 -/- EMBIs presented decreased expression of major iron regulatory proteins, including heme transporter Slc48a1 and Heme-Oxygenase-1 (HMOX1). Functional analyses using Calcein-AM staining and flow cytometry of freshly isolated central macrophages from enriched EMBIs of WT and Irf5 -/-mice revealed that the labile iron pool (LIP) of the central macrophage of Irf5 -/- EMBIs was significantly reduced (mean 14.0) compared to WT (mean 49.0, p-value 0.004). This apparent decrease in intracellular iron was concomitantly supported by significant decreases in serum iron (WT: 67.27 uM/L vs KO: 39.67 uM/L, p-value: 0.0059), increased serum EPO (WT: 108 pg/dL vs KO: 163.8 pg/dL, p-value: .0075), and Prussian blue staining of Irf5 -/-spleen indicating iron deposition in aging Irf5 -/- mice. Finally, these findings were conserved in Irf5fl/fl-Vav-iCre+ mice, highlighting a cell autonomous function for Irf5 in iron regulation within the island. Together, these data suggest that Irf5 may be a new regulator of iron scavenging, trafficking, and/or utilization within the central macrophage, potentially playing a major role in the red-cell production capacity of the EMBI.

Perioperative transfusion of red cell products in patients operated at general surgical units of National Hospital of Sri Lanka

Indications for red cell transfusion among general surgical patients have changed over time. Despite a scarcity in evidence, many international guidelines advocate a restrictive transfusion protocol.Objectives: To describe the red cell transfusion practices including transfusion thresholds in general surgical wards of a tertiary hospital in Sri Lanka.Methods: A descriptive cross-sectional study was done in all general surgical units of the National Hospital of Sri Lanka, Colombo. All patients transfused with red cell products for a 3-month period from 1/5/2017-1/8/2017 were included in the study. Indications for transfusion, haemoglobin at transfusion and the number of units transfused was the primary data recorded.Results: A total of 196 patients were included in the study. Most transfusions 79.08% (n=155) were for elective reasons. Most elective transfusions were to facilitate chronic extremity wound healing (52.2%, n=81). Most emergency transfusions were for intraoperative bleeding (31.7%, n=13). The majority (73.5%, n=114) of elective transfusions occurred at a Hb of 7-10 g/dL. The mean number of RBC units transfused in elective circumstances for a Hb of 7-10 g/dL was 1.59. A pre-transfusion blood picture was not available in over 85% (n=132) of elective transfusions. Only a single febrile non-haemolytic transfusion reaction was reported.Conclusions: The study revealed substantial divergence of red cell transfusion thresholds in perioperative practice when compared with international guidelines, nonadherence to rational practice such as obtaining a blood picture prior to elective transfusion and therefore the need to formulate local guidelines. It also highlighted the transfusion burden of diabetic foot disease.

Preclinical Studies of the GlyT1 Inhibitor Bitopertin in Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) results from the haploinsufficiency of 1 of 20 different ribosomal proteins. While DBA patients have hematopoietic stem/progenitor cell defects which broadly impact hematopoiesis, the dominant phenotype is ineffective erythropoiesis and severe anemia. In ~60% of patients, the anemia responds to corticosteroid therapy, leaving an unmet need for safe and effective treatment. In DBA, the synthesis of heme (a chemical chelate) proceeds at a near normal rate while the ribosomal haploinsufficiency leads to slowed translation and delayed globin protein synthesis, resulting in a heme-globin imbalance. DBA patient colony forming unit-erythroid (CFU-E)/proerythroblasts and murine Rpl11 haploinsufficient CFU-E/proerythroblasts show increased heme- and reactive oxygen species (ROS)-mediated cell damage, apoptosis, and ferroptosis. Single cell RNAseq analyses also suggest that excessive heme is a major factor in DBA erythroid marrow failure. When patient marrow cells are cultured in the presence of succinylacetone to reduce heme synthesis or hemopexin to facilitate heme export, erythroid output significantly increases. Succinylacetone competitively inhibits the 2 nd step in heme synthesis, while hemopexin enhances heme export 100-fold and scavenges the extracellular heme. Neither would be a pragmatic and safe therapy for DBA patients. In the first step of heme biosynthesis, glycine and succinyl-CoA combine to form aminolevulinic acid. Erythroid precursors are unable to synthesize enough glycine to support their robust heme synthesis and must import glycine via glycine transporter 1 (GlyT1) (PMID 28495915). Bitopertin is an investigational oral small-molecule inhibitor of GlyT1. In Phase 3 clinical studies of schizophrenia, it was found to be ineffective but exhibited a favorable safety profile (PMID 27816567). To determine whether bitopertin could rebalance heme and globin synthesis, reduce ineffective erythropoiesis, and improve red cell production in DBA, we cultured marrow cells from patients with DBA without and with 10 nM bitopertin (n=3). Following 7 days of culture, bitopertin increased the mean number of cells (21%) and the number of CD71+ erythroid precursors (31%) without impacting erythroid maturation. We next made human cord blood CD34+ deficient for RPS19 (44±2% reduction) with RPS19 shRNA transduction (or control luciferase shRNA) to model RPS19 haploinsufficient DBA. Treatment with bitopertin improved erythroid expansion of RPS19-deficient CD34+ cells 60% (P<0.01) but not control CD34+ cells. Additionally, to assess erythroid differentiation in vitro in the context of an erythroblastic island (EBI) niche, we cultured the CD34+ cell-derived erythroblasts with cord blood-derived macrophages. Erythroid expansion is 7.9±1.3-fold higher (P<0.02) in EBI cultures compared to cultures without macrophages. Treatment of the EBI cultures with bitopertin increased the expansion of RPS19-deficient CD34+ cell-derived erythroblasts 3.9±0.3-fold (P<0.002) but did not increase the expansion of control CD34+ cell-derived erythroblasts. As a final study, Rpl11 haploinsufficient mice were treated with bitopertin (10 to 150 ppm in chow, 2-30 mg/kg/day) for 8 weeks. Bitopertin treatment resulted in a dose-dependent improvement in anemia. After treatment with an optimal dose of bitopertin (100 ppm in chow, 20 mg/kg/day), there was an 8.8% increase in the hemoglobin values, and there was significant improvement in red cell numbers (7.5±1.1 M/µL to 8.9±1.3 M/µL, p=0.005), hematocrit (41.6±4.9% to 45.5±4.2%, p<0.05), and mean corpuscular volume (55.7±3.5 fL to 51.8±4.2 fL, p=0.01). Additionally, bitopertin treatment diminished the developmental block at the CFU-E/proerythroblast stage and reduced erythroblast ROS. The improved in vitro erythroid differentiation of DBA patient marrow and RPS19-deficient CD34+ cells and the improved anemia of RPL11 haploinsufficient mice suggest that bitopertin might mitigate anemia in DBA patients. These encouraging preclinical data supported the initiation of a Phase 1/2 clinical trial (NCT05828108: Young et al, abstract this meeting).

The putatively high-altitude adaptation of macaque monkeys: Evidence from the fecal metabolome and gut microbiome.

Animals living in high-altitude environments, such as the Tibetan Plateau, must face harsh environmental conditions (e.g., hypoxia, cold, and strong UV radiation). These animals' physiological adaptations (e.g., increased red cell production and turnover rate) might also be associated with the gut microbial response. Bilirubin is a component of red blood cell turnover or destruction and is excreted into the intestine and reduced to urobilinoids and/or urobilinogen by gut bacteria. Here, we found that the feces of macaques living in high-altitude regions look significantly browner (with a high concentration of stercobilin, a component from urobilinoids) than those living in low-altitude regions. We also found that gut microbes involved in urobilinogen reduction (e.g., beta-glucuronidase) were enriched in the high-altitude mammal population compared to the low-altitude population. Moreover, the spatial-temporal change in gut microbial function was more profound in the low-altitude macaques than in the high-altitude population, which might be attributed to profound changes in food resources in the low-altitude regions. Therefore, we conclude that a high-altitude environment's stress influences living animals and their symbiotic microbiota.

PATHOPHYSIOLOGY OF PANCYTOPENIA: UNDERSTANDING BLOOD CELL PRODUCTION AND REGULATION IN THE BONE MARROW

Objective: The bone marrow is the primary site for blood cell formation, responsible for the production and export of red cells, platelets, and granulocytes. Pancytopenia is a condition characterized by a reduction in all three types of cellular components in peripheral blood, leading to symptoms of marrow failure. The management and prognosis of pancytopenia depend on identifying the underlying pathology.
 Methods: The pathophysiology of pancytopenia involves various factors such as infections, toxins, malignant cell infiltration, suppression, or ineffective hematopoiesis. Bone marrow examination, including aspiration and trephine biopsy, is crucial for diagnosing pancytopenia, especially when the cause is unclear or when malignancy is suspected. Geographic distribution and genetic disturbances contribute to variations in the incidence of disorders causing pancytopenia.
 Results: The study aims to evaluate the various causes of pancytopenia and correlate peripheral blood findings with bone marrow aspirate and trephine biopsy. The collected data will assist in planning the diagnostic and therapeutic approach for patients with pancytopenia.
 Conclusion: Pancytopenia involves disruptions in blood cell production and regulation. Bone marrow aspiration and trephine biopsy are crucial for diagnosis. Understanding blood cell regulation provides insights. This study aims to enhance diagnostic and therapeutic approaches for pancytopenia.

Silk Bone Marrow-like Niches for Red Cell Production Ex Vivo

Background Human adults produce up to 3 million red blood cells every second, equating to about 200 billion generated every day (Caulier and Sankaran Blood 2022). Leading studies point to the erythroblastic islands as the core of red blood cell production within the bone marrow microenvironment. Here, cellular, chemical, and physical elements define a specialized three-dimensional (3D) niche that orchestrates lineage commitment, cell maturation, and final enucleation. The extracorporeal generation of physiologically relevant models of the bone marrow is central to multiple healthcare challenges including studying the physio-pathological mechanisms of human erythropoiesis, personalized drug screening, and production of red blood cells on-demand. Different culture protocols have been developed, aiming at the mass production of erythrocytes in vitro, including bioengineering techniques for developing physiologically relevant models of the hematopoietic niche (Di Buduo et al. Haematologica 2021). Extrapolating the 3D structure and function of the erythroblastic islands is still challenging. Consequently, enucleation remains the main critical limiting step of efficient erythrocyte production ex vivo. Aim We have pioneered bone marrow tissue engineering by using silk fibroin, derived from Bombyx mori silkworm cocoons, to provide solutions for human platelet production (Di Buduo et al. Blood 2015). Here, we aimed at exploiting this technology by manufacturing innovative 3D silk-based bone marrow niches and flow chambers to direct the generation of compartmentalized erythroblastic islands and set effective protocols to produce fully mature human erythrocytes ex vivo. Methods We developed an all-natural tissue model consisting of silk fibroin and extracellular matrix proteins. The spongy scaffold was enclosed in a protective chamber providing a dynamic flow of nutrients and cytokines through an interconnected vascular-like network. The system was cultured for up to 3 weeks with hematopoietic stem and progenitor cells (HSPCs) from human mobilized peripheral blood, upon informed consent and in accordance with the Declaration of Helsinki. Results HSPCs could be efficiently differentiated into erythroblasts under the control of a bespoke cocktail of cytokines in combination with increasing concentrations of transferrin. After 1 week of culture, flow cytometry analysis demonstrated the gradual loss of stem-associated markers, such as CD34, CD105, and CD117, and the acquisition of the lineage-specific markers CD71 (transferrin receptor), CD36 (a transmembrane scavenger receptor), and CD235a (glycophorin-α). The analysis of the spatial distribution of cells within the silk scaffold, by real-time imaging, demonstrated the spontaneous development of cellular nests, resembling the physiological structure of the erythroblastic islands typically, but not exclusively, aggregating around and/or next to co-cultured CD68+ macrophages. After 2 weeks of differentiation, the population turned to a CD36-CD71lowCD235ahigh profile, indicative of terminal erythroblast maturation. Starting from week 3, fully mature CD36-C71-CD235ahigh erythrocytes were mainly localized at the periphery of the erythroblastic islands, in close contact with macrophages. Dynamic perfusion of the system at physiologically relevant shear stress allowed the collection of >90% enucleated CD235ahigh erythrocytes. Cells were fully hemoglobinized and expressed both α- and β-globin. May-Grunwald Giemsa staining revealed that the size, shape, and color of cultured erythrocytes were perfectively comparable to donor cells. The recovered erythrocytes showed significant downregulation of the expression of α4β1 integrin, which was retained by immature erythroblasts adhering to the silk scaffolds. Conclusion Our data demonstrated that the spatiotemporal control of the dynamics of cell-cell and cell-matrix interaction, together, with controllable shear forces, within a 3D physiologically relevant microenvironment, may be the key to guiding efficient erythropoiesis ex vivo. The silk bone marrow model represents a unique sustainable resource to gain insights into mechanisms of human erythropoiesis, and advance basic research to robust clinical translation.

COVID-19 Outcomes at a Comprehensive Sickle Cell Center: A Two-Year Report

The Georgia Comprehensive Sickle Cell (SC) center at Grady health system is the largest adult comprehensive sickle cell center in the United States and has the first 24/7 acute care unit for management of sickle cell vaso-occlusive events (VOE). in 2019, the center provided around 3000 sickle cell clinic visits and 4000 acute care visits. When the COVID-19 pandemic reached the US, the SC center had seen a precipitous drop in the number of both clinic and acute care visits as local and state regulations for lockdown were passed. On the two-year anniversary for the COVID-19 pandemic, we are submitting this report of all the COVID-19 cases at a single adult center for sickle cell disease. Since the start of the pandemic, we have been tracking any COVID-19 cases reported in the patient population the SC center follows. Out of 1343 patients currently following in the center, 173 patients contracted COVID-19 between March 2020 and February 2022, and were tracked in the clinical database under IRB approval. The results show that of the 173 patients, 85 were female and 89 were male. By genotype, 62% of patients were SS, 29% were SC and 9% were Sβ+ thalassemia or Sβ0 thalassemia. 32% of patients were on hydroxyurea for disease modification with the majority of them being of the SS genotype and 45% had elevated fetal hemoglobin determined by a percentage fetal hemoglobin above 5% by hemoglobin electrophoresis. Chronic pain was defined in the patients experiencing daily pain episodes for more than 4 days a week for the last 3 months and calculation of daily morphine equivalents was reported based on the clinic follow-ups and the narcotic database utilization. 35% of the SC patients had chronic pain and the median morphine equivalents was 90 mg daily with a range between 45 and 225mg. On evaluating the rate of emergency department (ED) visits or hospitalizations for the sickle cell patients with COVID-19, 85% of patients had either an ED visit or hospitalization. 54% of the SC patients had the reason for the visit related to VOE and 29% had the reason related to COVID-19 primarily. 15% of SC patients were not seen in any emergency setting or required any hospitalization. 22% of the patients had lung findings on imaging. The treatment utilized for the patients was most commonly Remdesivir in 33, dexamethasone in 26, red cell products in 21 and monoclonal antibody in 13 patients. Of the 3 deaths reported, 2 happened early in the pandemic. One patient had presented in June 2020 with VOE and was found to have bilateral lung opacities but was asymptomatic and was discharged home to return few days later with clinical picture of multi-organ failure (MOF) for which a red cell erythrocytapheresis was attempted to no avail. The second patient had presented in July 2020 with COVID-19 pneumonia and was treated with Remdesivir and convalescent plasma to no avail after development of MOF and ARDS. The third death occurred in August 2021 during the delta variant wave and the patient was hospitalized for COVID-19 pneumonia and had initial improvement after initial treatment but after worsened lung findings, ARDS developed followed by MOF. Several reports from different centers have been published regarding the rate of COVID-19 related mortality and morbidity in sickle cell disease. The Grady comprehensive sickle cell center experience differs in the fact that 63 out of 173 (36%) patients who had contracted COVID-19 required treatment and 3 of those 63 had died. In fact, the deaths occurred early in the course of the pandemic in June and July 2020 respectively and since then, the rate of complicated COVID-19 hospitalizations has decreased with better treatment available. No deaths were registered during the omicron wave and 1 death was registered during the delta wave. In addition, the timeline for the COVID-19 cases reported fits the population timeline of peaks respectively happening in the summer of 2020, winter of 2021, summer of 2021 and winter of 2021. During the initial peak, we have noted a decrease in the number of clinic and acute care visits respectively. This was anticipated given the statewide lockdown that was implemented. To circumvent that, the SC center resorted to virtual visits utilization to deliver the needed healthcare visits to the patients. This measure has aided in protecting patients against COVID-19 during the multiple COVID-19 peaks occurring in the last 2 years and has accordingly reduced the mortality and morbidity of the patient population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DISC-0974, an Anti-Hemojuvelin (HJV) Monoclonal Antibody, Reduced Hepcidin and Improved Anemia in a Rat Model of Chronic Kidney Disease

Introduction DISC-0974 is a humanized monoclonal antibody against hemojuvelin (HJV) and is currently in Phase I clinical studies. HJV is a pathway-specific coreceptor for bone morphogenetic protein (BMP) signaling that regulates hepcidin expression and iron metabolism. Loss of function mutations of the HFE2 gene encoding HJV in humans cause profound reductions in hepcidin synthesis and severe iron overload. Consequently, it is hypothesized that pharmacological reduction of HJV activity will lead to reduction of hepcidin synthesis and increased iron availability. This hypothesis has been confirmed in studies of DISC-0974 in rodents and non-human primates and in healthy human volunteers. We have conducted studies to evaluate the effect of DISC-0974 in an animal model of CKD anemia. Anemia is a common complication in patients with CKD and has been associated with multiple adverse outcomes in this population. In CKD hepcidin is increased because of both reduced renal clearance and increased synthesis. This increase is believed to be a central contributor to the development of anemia by reducing the availability of iron from systemic iron stores and by reducing dietary iron absorption. Supporting this hypothesis, treatment with hepcidin lowering drugs has been demonstrated to increase serum iron and to increase hemoglobin (Sheetz et al, Br J Clin Pharmacol. 2019;85:935-948). We hypothesize that by downregulating hepcidin, DISC-0974 will have beneficial effects in treating anemia in the CKD patients because it will make iron available for improved erythropoiesis and increased hemoglobin synthesis as well as reducing erythropoietin resistance. Material and Methods The effect of DISC-0974 on improving anemia was evaluated in an adenine-induced rat model of CKD. In this study, renal impairment was induced by giving Sprague Dawley rats a diet containing 0.75% adenine for 3 weeks to induce kidney injury and then switched to a control diet for the remainder of the study (i.e., 3 more weeks). DISC-0974 (20 mg/kg) or vehicle (n-5/group) was administered intravenously once per week starting on Day 7 and continuing to Day 35. An additional group of rats (n=5/group) was fed with the control diet for the entire study and dosed intravenously with vehicle. Rats were euthanized on Day 42. Blood was collected on Days 0, 7, 21, 35, and 42 for evaluation of hematology parameters, as well as serum iron and hepcidin. Livers were also collected at study end for evaluation of hepcidin gene (HAMP) mRNA expression level. Results Consistent with a previous report (Sun et al, Nephrol Dial Transplant. 2013;28:1733-1743), the 0.75% adenine diet induced kidney dysfunction, as evidenced by marked increase in both serum creatinine and blood urea nitrogen. Rats on adenine diet developed reduced serum iron, reduced red cell production, and lowered hemoglobin with a hypochromic microcytic profile reflecting reduced iron availability. CKD rats appeared to tolerate DISC-0974 treatment well, as DISC-0974 @ 20mg/kg did not affect body weight. Consistent with the mechanism of action of blocking the formation of the BMP/BMPR/HJV complex, DISC-0974 reduced HAMP gene expression in the liver as measured at the end the study, which led to reduced serum hepcidin and increased serum iron concentration. Cellular hemoglobinization measured as reticulocyte Hemoglobin was increased. The maximum improvement in hemoglobin by DISC-0974 compared to the vehicle group was 17g/L in the rat CKD model. Conclusions We conclude that treatment with DISC-0974 at a dose that reduces hepcidin gene expression is able to increase iron availability and substantially prevents the reduction in hemoglobin that is seen in animals that develop adenine-induced renal impairment. This study supports the development of DISC-0974 for the treatment of patients with CKD anemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Verify: A Phase 3 Study of the Hepcidin Mimetic Rusfertide (PTG-300) in Patients with Polycythemia Vera

Background Patients with polycythemia vera (PV) are treated with periodic therapeutic phlebotomy (TP) alone or in combination with cytoreductive therapies to maintain hematocrit (HCT) levels below 45%, per NCCN and ELN guidelines. Since they are seen only periodically, patients with PV likely spend significant time with HCT levels above 45%, thereby increasing their risk of thrombosis [Marchioli NEJM 2013]. Indeed, a study of more than 4,000 US patients with PV in 2018 and 2019 found that only 22% maintained HCT<45% at all measurements and, of those with a prior thrombotic event, 36% had ≥1 additional thrombotic event within 5 years of treatment initiation, the most common being deep vein thrombosis (DVT) and stroke [Verstovsek, ASH 2020]. Excessive erythrocytosis in PV occurs despite iron deficiency, which is due to TP and utilization for red cell production. Hepcidin is also downregulated in PV. Patients report that fatigue is the most prevalent and severe systemic PV-associated symptom [Scherber, Cancer 2016]. This may be indicative of symptomatic iron deficiency, representing an unaddressed clinical challenge in the management of patients with PV. In the phase 2 REVIVE study (NCT04057040), we demonstrated the clinical utility of rusfertide (PTG-300), a potent hepcidin mimetic being assessed as a treatment for uncontrolled PV-associated erythrocytosis. In REVIVE, rusfertide demonstrated good tolerability, as well as consistent and durable HCT control with improvements in iron deficiency in patients with excessive TP needs despite standard of care PV therapy [Hoffman, ASH 2021]. Study Design and Methods VERIFY (NCT05210790) is a phase 3, multicenter, global, randomized trial comparing the efficacy and safety of rusfertide versus placebo when added to ongoing therapy for PV (Figure 1). The study population comprises PV patients who require frequent TP, with or without concurrent cytoreductive therapy, to control their HCT. The study is comprised of three parts: Part 1a: 1:1 randomized, double-blind, placebo-controlled, add-on phase with parallel-groups lasting 32 weeks.Part 1b: open-label treatment phase with cross-over for previous placebo-treated patients. During this phase, all patients who completed Part 1a successfully receive rusfertide for 20 weeks (week 32 to 52).Part 2: Long term extension phase during which all patients who complete Part 1b will continue to receive rusfertide for 104 weeks (week 52 to 156). Major inclusion criteria include: PV diagnosis (2016 WHO criteria) with either JAK2 V617F mutation or JAK2 exon 12 mutation; ≥3 TP in the previous 6 months or ≥5 TP in the previous 12 months due to inadequate HCT control; HCT <45%, white blood cells 4-20 x 109/L and platelets 100-1000 x 109/L at week 0 prior to randomization; a stable PV therapy regimen in patients receiving cytoreductive therapy at randomization; and cessation of cytoreductive therapy 2-6 months before screening in patients treated with TP alone. Major exclusion criteria include: clinically significant thrombosis, or active or chronic bleeding deemed clinically significant by the investigator, within 2 months before randomization, and a history of invasive malignancy within the previous 5 years. Patients who meet eligibility criteria are stratified by their ongoing PV therapy and randomized 1:1 to treatment with rusfertide or placebo in addition to the patient's ongoing PV therapy at week 0. The dose of cytoreductive therapy in Part 1a and Part 1b may be decreased for safety but may not be increased for efficacy, including control of HCT, thrombocytosis and/or leukocytosis. The primary endpoint is the proportion of patients achieving a response in part 1A from week 20 through week 32. A response is defined as absence of TP eligibility. TP eligibility is defined as either: a confirmed hematocrit ≥45% and that is at least 3% higher than the baseline hematocrit OR a HCT ≥48%. Secondary endpoints are: mean number of TPs from week 0 to 32; proportion of patients with HCT <45% from week 0 to 32; mean change from baseline to week 32 in total fatigue score as measured by the PROMIS Short Form, and mean change from baseline to week 32 in total symptom score as measured by the MFSAF. The VERIFY study was initiated in January 2022. Sites are being opened globally with an aim of enrolling approximately 250 patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PB2254: ASSOCIATION BETWEEN TNF-ALPHA LEVELS AND TNF-ALPHA 238 ALLELES POLYMORPHISMS AND SEVERITY OF ANEMIA AMONG SUDANESE CHILDREN WITH FALCIPARUM MALARIA

Background: Falciparum malaria is the most leading cause of mortality and morbidity in the world especially in Africa among infants and young children due to severe malaria and malaria anemia. TNF-α is plays a central role in malaria pathogenicity either in the cure or complication of malaria. TNF-α levels overproduction and promoter polymorphisms at TNF-α 238 alleles may play a central role in reduced red cell production and malaria-related anemia through suppression of bone marrow erythropoiesis and dyserythropoiesis. Aims: The purpose of this study was to evaluate the TNF-α 238 alleles polymorphism and its association with TNF-α levels in the severe malaria anemia among Sudanese children in Gezira State. Methods: A cross-sectional hospital-based study was included 200 Sudanese children (mean age 8.43 ± 3.80 years; 53% boys; 47% girls). 100 children with severe falciparum malaria (mean age 8.63 ± 3.40 years; 61% boys; 39% girls) and 100 children with uncomplicated falciparum malaria (mean age 8.83 ± 4.20 years; 45% boys; 55% girls). RBCs parameters were determined using the Sysmex XP 300 N automated hematology analyzer. ELISA was further processed for TNF-α level using Human TNF-α ELISA MAX™ Deluxe Sets. DNA extraction was done using G-DEX™IIb Genomic DNA Extraction Kit. PCR and gel running system were used for detecting TNF-α 238 alleles polymorphism (A allele/ G allele). Obtained data were analyzed by SPSS (V 20.0) and Stat disk (V 13.0). Results: TNF-α 238A allele was a common allele (66.8%) among Sudanese children with falciparum malaria, and the G allele was rare (33.2%). While TNF-α 238 GA, AA, and GG account for (54.5, 39.5, and 6% respectively). Falciparum malaria-related anemia accounted for 32%, commonly in SM (55%) compared to UM (9%) giving highly statistically significant differences in TNF-α levels between falciparum malaria-related anemia and falciparum malaria without anemia (P value 0.000). Otherwise, The average of TNF-α levels in mild, moderate, and severe anemia were (190.75 ± 102.55, 189.70 ± 80.35, and 299.75 ± 82.27 pg/ml respectively) giving highly significant differences and strongly significant positive correlated with the severity of anemia (r + 0.309; P value 0.000). TNF-α 238 GA, AA, and GG account for (58, 36, and 6% respectively) in UM; while (51, 43, and 6% respectively) in SM. The TNF-α 238 A allele accounts for 83.6% of malaria anemia (P value 0.000) and 100% severe anemia (P value 0.000); children who have TNF-α 238 A allele, they have ability to acquire falciparum malaria-related anemia with susceptibility to 3.13 folds risk. Summary/Conclusion: Overproduction of TNF-α is essential for the elimination and clearance of falciparum parasite but may be associated with severity of malaria and malaria anemia. Overproduction of TNF-α in children with TNF-α 238 A allele may result in falciparum malaria-related anemia among children (mainly severe anemia). The findings of this study will assist clinicians in diagnosing and better managing severe malaria cases.

MO530: Daprodustat is not Associated With an Increased Risk of Cancer: Results From the ASCEND-D and ASCEND-ND Trials

Abstract BACKGROUND AND AIMS Increased HIF-1 levels are associated with mortality in some cancer patients, and levels of HIF-1 may also impact response to cancer treatments. Use of recombinant human erythropoietin or its analogs [erythropoiesis-stimulating agents (ESA)] in clinical trials in patients with cancer has been associated with increased risk of cancer-related morbidity and mortality, though the mechanism(s) for these effects is not clear. Daprodustat is an oral hypoxia-inducible factor (HIF)-prolyl hydroxylase inhibitor that increases the secretion of endogenous erythropoietin and the production of red cells. Cardiovascular outcome trials in patients with anemia of chronic kidney disease (CKD) requiring dialysis (ASCEND-D [1]; NCT02879305) and those not on dialysis (ASCEND-ND [2]; NCT028768355) have recently been published. Given the long latency with cancer events, we explored the effect of daprodustat as compared with ESA on the risk of cancer development utilizing a post-hoc modified intention-to-treat (mITT) approach, which includes events on and off treatment and provides greater protection to randomization than on-treatment approaches. METHOD Cancer-related adverse events (AEs) from the ASCEND studies were identified based on a predefined list of terms that included new cancer events or tumor progression and recurrence for patients receiving at least one dose of daprodustat or ESA (Safety Population). Patients were included into these trials as long as they had no history of cancer within 2 years prior to screening, were not currently receiving treatment for cancer and did not have a complicated kidney cyst. In addition to reporting the number and percentage of patients with cancer AEs, overall and by types, the rate per 100 person-year (PY) is also provided given differential follow-up for patients in these trials. RESULTS A total of 2964 patients were randomized in ASCEND-D and 3872 in ASCEND-ND, with ∼14 200 PY of follow-up. At baseline, patients reported a past history of cancer in 5.0% and 4.9% of patients receiving daprodustat or ESA, respectively, in ASCEND-D; and in 5.2% and 4.4% of patients receiving daprodustat or darbepoetin alfa, respectively, in ASCEND-ND. In the Safety Population, both the rate of cancer-related AEs and the rate per 100 PY were balanced for each study, with no pattern emerging regarding location or type (Table 1). Of the patients with overall cancer AEs in the Safety Population (Table 1), fatal AEs in ASCEND-D occurred in 11 patients receiving daprodustat and in 15 patients receiving ESA, which was &amp;lt;1% and 1% of the Safety Population, respectively. In ASCEND-ND, fatal AEs occurred in 6 patients receiving daprodustat and 11 patients receiving darbepoetin alfa, which was &amp;lt;1% of the Safety Population in both groups. Pooling of data from the D and ND studies also demonstrated balance in cancer events between the dapro and ESA arms. Further analysis of the characteristics of patients with cancer AEs demonstrated that the number of patients with multiple events, the time to first onset and outcome of the event and event seriousness and severity were also similar between treatment groups for both trials. CONCLUSION In cardiovascular outcome trials comprised of patients requiring dialysis (ASCEND-D) and not requiring dialysis (ASCEND-ND) with anemia of CKD, daprodustat was not associated with an increased risk of cancer, or cancer mortality, relative to ESA. Collectively, across these trials, cancer AEs are balanced.

Red Blood Cell Function and Disorders of Iron Metabolism

The red blood cell, or erythrocyte, is an anucleate biconcave disk, approximately 7 µm in diameter. The principal function of the red blood cell is to exchange carbon dioxide for oxygen while in the pulmonary circulation, exchange that oxygen for carbon dioxide in the peripheral tissue, and carry that carbon dioxide back to the lungs. The physicochemical composition of the red cell is aligned to optimize that function. The state of tissue oxygenation, in turn, regulates the production of red cells. This review covers red blood cell function, iron metabolism, iron deficiency, iron overload, and primary iron overload. Figures show a model of the hemoglobin molecule showing the relative alignment of the α chains and β chains; the normal oxygen-hemoglobin dissociation curve shifted by changes in temperature, pH, and the intracellular concentration of 2,3-diphosphoglycerate; body iron supply and storage; regulation of hepcidin expression and its role in disease; blood smear from a patient with iron deficiency; and mechanisms contributing to iron overload in iron-loading anemias. Tables list laboratory results associated with decreased iron balance, causes of iron deficiency, oral iron replacement therapy, parenteral iron replacement therapy, primary iron overload syndromes, secondary iron overload syndromes, laboratory results associated with increased iron stores, and other rare disorders of iron overload. This review contains 6 figures, 20 tables, and 111 references Keywords: Red blood cell, iron deficiency anemia, iron overload, hemochromatosis, hemoglobin, iron balance

Red Blood Cell Function and Disorders of Iron Metabolism

The red blood cell, or erythrocyte, is an anucleate biconcave disk, approximately 7 µm in diameter. The principal function of the red blood cell is to exchange carbon dioxide for oxygen while in the pulmonary circulation, exchange that oxygen for carbon dioxide in the peripheral tissue, and carry that carbon dioxide back to the lungs. The physicochemical composition of the red cell is aligned to optimize that function. The state of tissue oxygenation, in turn, regulates the production of red cells. This review covers red blood cell function, iron metabolism, iron deficiency, iron overload, and primary iron overload. Figures show a model of the hemoglobin molecule showing the relative alignment of the α chains and β chains; the normal oxygen-hemoglobin dissociation curve shifted by changes in temperature, pH, and the intracellular concentration of 2,3-diphosphoglycerate; body iron supply and storage; regulation of hepcidin expression and its role in disease; blood smear from a patient with iron deficiency; and mechanisms contributing to iron overload in iron-loading anemias. Tables list laboratory results associated with decreased iron balance, causes of iron deficiency, oral iron replacement therapy, parenteral iron replacement therapy, primary iron overload syndromes, secondary iron overload syndromes, laboratory results associated with increased iron stores, and other rare disorders of iron overload. This review contains 6 figures, 20 tables, and 111 references Keywords: Red blood cell, iron deficiency anemia, iron overload, hemochromatosis, hemoglobin, iron balance

ATG4A regulates human erythroid maturation and mitochondrial clearance

AbstractAutophagy is a self-degradation pathway that is essential for erythropoiesis. During erythroid differentiation, autophagy facilitates the degradation of macromolecules and the programmed clearance of mitochondria. Impaired mitochondrial clearance results in anemia and alters the lifespan of red blood cells in vivo. While several essential autophagy genes contribute to autophagy in erythropoiesis, little is known about erythroid-specific mediators of this pathway. Genetic analysis of primary human erythroid and nonerythroid cells revealed the selective upregulation of the core autophagy gene ATG4A in maturing human erythroid cells. Because the function of ATG4A in erythropoiesis is unknown, we evaluated its role using an ex vivo model of human erythropoiesis. Depletion of ATG4A in primary human hematopoietic stem and progenitor cells selectively impaired erythroid but not myeloid lineage differentiation, resulting in reduced red cell production, delayed terminal differentiation, and impaired enucleation. Loss of ATG4A impaired autophagy and mitochondrial clearance, giving rise to reticulocytes with retained mitochondria and autophagic vesicles. In summary, our study identifies ATG4A as a cell type–specific regulator of autophagy in erythroid development.

Congenital anemia reveals distinct targeting mechanisms for master transcription factor GATA1

AbstractMaster regulators, such as the hematopoietic transcription factor (TF) GATA1, play an essential role in orchestrating lineage commitment and differentiation. However, the precise mechanisms by which such TFs regulate transcription through interactions with specific cis-regulatory elements remain incompletely understood. Here, we describe a form of congenital hemolytic anemia caused by missense mutations in an intrinsically disordered region of GATA1, with a poorly understood role in transcriptional regulation. Through integrative functional approaches, we demonstrate that these mutations perturb GATA1 transcriptional activity by partially impairing nuclear localization and selectively altering precise chromatin occupancy by GATA1. These alterations in chromatin occupancy and concordant chromatin accessibility changes alter faithful gene expression, with failure to both effectively silence and activate select genes necessary for effective terminal red cell production. We demonstrate how disease-causing mutations can reveal regulatory mechanisms that enable the faithful genomic targeting of master TFs during cellular differentiation.

Causes of Anemia Due to Diminished Red Blood Cell Production in Pediatrics

The critical feature that distinguishes the anemias discussed in this article is the presence of an inappropriately low reticulocyte count for the degree of anemia. This is consistent with nutritional deficiencies, decreased erythropoietin levels, aplastic anemia, or inherited bone marrow failure syndromes. In addition, bone marrow replacement by a benign or malignant process, including those associated with ineffective erythropoiesis (eg, congenital dyserythropoietic anemias), comprise the differential diagnosis. Anemia causes increased morbidity and mortality in pediatric population; the absolute reticulocyte count (ARC) can be calculated by multiplying the percent reticulocytes by the RBC count/L. In a patient with anemia, ARC values within the “normal range” (generally 50-100 × 109/L) indicate an inappropriate response and suggest that there is an underlying red cell production issue due to intrinsic or extrinsic factors, or a combination of the two. The mean corpuscular volume (MCV) is another valuable red cell index that narrows the differential diagnosis of anemia due to diminished production. It is critical, however, to check age-specific normal values for MCV and to recognize that certain diseases can present with low, normal, and/or high MCVs. Finally, the red cell distribution width (RDW) assists in differentiating whether the anemia is likely due to a mixed process (wide RDW) or from a single cause (normal RDW). However, the management of anemia depends on the etiology. Early diagnosis and prompt treatment improve the quality of life of patients.