An Optimized Human Erythroblast Differentiation System Reveals Cholesterol-Dependency of Robust Production of Cultured Red Blood Cells Ex Vivo.

The generation of cultured red blood cells (cRBCs) ex vivo represents a potentially unlimited source for RBC transfusion and other cell therapies. Human cRBCs can be generated from the terminal differentiation of proliferating erythroblasts derived from hematopoietic stem/progenitor cells or erythroid precursors in peripheral blood mononuclear cells. Efficient differentiation and maturation into cRBCs highly depend on replenishing human plasma, which exhibits variable potency across donors or batches and complicates the consistent cRBC production required for clinical translation. Hence, the role of human plasma in erythroblast terminal maturation is investigated and uncovered that 1) a newly developed cell culture basal medium mimicking the metabolic profile of human plasma enhances cell growth and increases cRBC yield upon erythroblast terminal differentiation and 2) LDL-carried cholesterol, as a substitute for human plasma, is sufficient to support erythroid survival and terminal differentiation ex vivo. Consequently, a chemically-defined optimized medium (COM) is developed, enabling robust generation of cRBCs from erythroblasts of multiple origins, with improved enucleation efficiency and higher reticulocyte yield, without the need for supplementing human plasma or serum. In addition, the results reveal the crucial role of lipid metabolism during human terminal erythropoiesis.

The non-canonical poly(A) polymerase FAM46C promotes erythropoiesis.

The post-transcriptional regulation of mRNA is a crucial component of gene expression. The disruption of this process can have detrimental effects on normal development and give rise to various diseases. The search for novel post-transcriptional regulators and the exploration of their roles are essential for understanding development and disease. Through a multimodal analysis of red blood cell trait genome-wide association studies (GWAS) and transcriptomes of erythropoiesis, we identified FAM46C, a non-canonical RNA poly(A) polymerase, as a necessary factor for proper red blood cell development. FAM46C is highly expressed in the late stages of the erythroid lineage, and its developmental upregulation is controlled by an erythroid-specific enhancer. We demonstrate that FAM46C stabilizes mRNA and regulates erythroid differentiation in a polymerase activity-dependent manner. Furthermore, we identified transcripts of lysosome and mitochondria components as highly confident in vivo targets of FAM46C, which aligns with the need of maturing red blood cells for substantial clearance of organelles and maintenance of cellular redox homeostasis. In conclusion, our study unveils a novel role of FAM46C in positively regulating lysosome and mitochondria components, thereby promoting erythropoiesis.

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

BACKGROUND: Erythropoiesis 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. RESULTS: In 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. CONCLUSIONS: We 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.

Murine Bone Marrow Erythroid Cells Have Two Branches of Differentiation Defined by the Presence of CD45 and a Different Immune Transcriptome Than Fetal Liver Erythroid Cells.

Mouse erythropoiesis is a multifaceted process involving the intricate interplay of proliferation, differentiation, and maturation of erythroid cells, leading to significant changes in their transcriptomic and proteomic profiles. While the immunoregulatory role of murine erythroid cells has been recognized historically, modern investigative techniques have been sparingly applied to decipher their functions. To address this gap, our study sought to comprehensively characterize mouse erythroid cells through contemporary transcriptomic and proteomic approaches. By evaluating CD71 and Ter-119 as sorting markers for murine erythroid cells and employing bulk NanoString transcriptomics, we discerned distinctive gene expression profiles between bone marrow and fetal liver-derived erythroid cells. Additionally, leveraging flow cytometry, we assessed the surface expression of CD44, CD45, CD71, and Ter-119 on normal and phenylhydrazine-induced hemolytic anemia mouse bone marrow and splenic erythroid cells. Key findings emerged: firstly, the utilization of CD71 for cell sorting yielded comparatively impure erythroid cell populations compared to Ter-119; secondly, discernible differences in immunoregulatory molecule expression were evident between erythroid cells from mouse bone marrow and fetal liver; thirdly, two discrete branches of mouse erythropoiesis were identified based on CD45 expression: CD45-negative and CD45-positive, which had been altered differently in response to phenylhydrazine. Our deductions underscore (1) Ter-119's superiority over CD71 as a murine erythroid cell sorting marker, (2) the potential of erythroid cells in murine antimicrobial immunity, and (3) the importance of investigating CD45-positive and CD45-negative murine erythroid cells separately and in further detail in future studies.

Pure Red Cell Aplasia Encountered in a Tertiary Care Hematology Laboratory: A Series of Nine Distinctive Cases.

Pure red cell aplasia (PRCA) is characterized by severe anemia with reticulocytopenia and bone marrow erythroblastopenia. The early erythroblasts are markedly decreased; however, in rare instances, they may be normal or raised in number. There are varied etiologies, namely congenital or acquired and primary or secondary. The congenital PRCA is known as "Diamond-Blackfan anemia." Thymomas, autoimmune disease, lymphomas, infections, and drugs also may be familiar associates. However, the etiologies of PRCA are numerous, and many diseases/infections can be associated with PRCA. The diagnosis rests on clinical suspicion and appropriate laboratory workup. We evaluated nine cases of red cell aplasia, having severe anemia with reticulocytopenia. Nearly half of the cases showed adequate erythroid (> 5% of the differential count) but with a maturation arrest. The adequacy of the erythroid could confuse the hematologist and may even delay the diagnosis. Hence, it is empirical that PRCA could be considered a differential in every case of severe anemia with reticulocytopenia, even in the presence of adequate erythroid precursors in the bone marrow.

Murine Placental Erythroid Cells Are Mainly Represented by CD45+ Immunosuppressive Erythroid Cells and Secrete CXCL1, CCL2, CCL3 and CCL4 Chemokines.

Erythroid cells are emerging players in immunological regulation that have recently been shown to play a crucial role in fetomaternal tolerance in mice. In this work, we set ourselves the goal of discovering additional information about the molecular mechanisms of this process. We used flow cytometry to study placental erythroid cells' composition and BioPlex for the secretome profiling of 23 cytokines at E12.5 and E19.5 in both allogeneic and syngeneic pregnancies. We found that (1) placental erythroid cells are mainly represented by CD45+ erythroid cells; (2) the secretomes of CD71+ placental erythroid cells differ from the ones in syngeneic pregnancy; (3) CCL2, CCL3, CCL4 and CXCL1 chemokines were secreted on each day of embryonic development and in both types of pregnancy studied. We believe that these chemokines lure placental immune cells towards erythroid cells so that erythroid cells can induce anergy in those immune cells via cell-bound ligands such as PD-L1, enzymes such as ARG1, and secreted factors such as TGFß-1.

Immortalized erythroid cells as a novel frontier for in vitro blood production: current approaches and potential clinical application.

BACKGROUND: Blood transfusions represent common medical procedures, which provide essential supportive therapy. However, these procedures are notoriously expensive for healthcare services and not without risk. The potential threat of transfusion-related complications, such as the development of pathogenic infections and the occurring of alloimmunization events, alongside the donor's dependence, strongly limits the availability of transfusion units and represents significant concerns in transfusion medicine. Moreover, a further increase in the demand for donated blood and blood transfusion, combined with a reduction in blood donors, is expected as a consequence of the decrease in birth rates and increase in life expectancy in industrialized countries. MAIN BODY: An emerging and alternative strategy preferred over blood transfusion is the in vitro production of blood cells from immortalized erythroid cells. The high survival capacity alongside the stable and longest proliferation time of immortalized erythroid cells could allow the generation of a large number of cells over time, which are able to differentiate into blood cells. However, a large-scale, cost-effective production of blood cells is not yet a routine clinical procedure, as being dependent on the optimization of culture conditions of immortalized erythroid cells. CONCLUSION: In our review, we provide an overview of the most recent erythroid cell immortalization approaches, while also describing and discussing related advancements of establishing immortalized erythroid cell lines.

The oxygen dissociation curve of blood in COVID-19-An update.

An impressive effect of the infection with SARS-Co-19 is the impairment of oxygen uptake due to lung injury. The reduced oxygen diffusion may potentially be counteracted by an increase in oxygen affinity of hemoglobin. However, hypoxia and anemia associated with COVID-19 usually decrease oxygen affinity due to a rise in [2,3-bisphosphoglycerate]. As such, COVID-19 related changes in the oxygen dissociation curve may be critical for oxygen uptake and supply, but are hard to predict. A Pubmed search lists 14 publications on oxygen affinity in COVID-19. While some investigations show no changes, three large studies found an increased affinity that was related to a good prognosis. Exact causes remain unknown. The cause of the associated anemia in COVID-19 is under discussion. Erythrocytes with structural alterations of membrane and cytoskeleton have been observed, and virus binding to Band 3 and also to ACE2 receptors in erythroblasts has been proposed. COVID-19 presentation is moderate in many subjects suffering from sickle cell disease. A possible explanation is that COVID-19 counteracts the unfavorable large right shift of the oxygen dissociation curve in these patients. Under discussion for therapy are mainly affinity-increasing drugs.

Bcl11a and the Correlated Key Genes Ascribable to Globin Switching: An In-silico Study.

BACKGROUND: Reactivation of HbF is a potential strategy to ameliorate symptoms of hemoglobinopathies such as sickle cell disease and b-thalassemia. After birth, there is a switch from fetal to adult hemoglobin, for which the molecular mechanisms and key regulators await further understanding in order to develop effective methods for HbF reactivation. Bcl11a, one of the major HbF reactivation regulators, demonstrates no significant changes at transcriptional levels in F erythroblasts compared to the non-HbF expressing cells. Therefore, it is possible that posttranscriptional regulation and epigenetic effects, for which the miRNAs play an important role, are the primary causes of the decreased Bcl11a protein level in adult erythroblasts. OBJECTIVE: This paper aims to determine the differentially expressed mRNAs and miRNAs of erythroblasts in HSCs from the fetal liver and bone marrow. METHODS: Raw high-throughput sequencing data (GSE110936, GSE90878) was downloaded from Gene Expression Omnibus (GEO) database. After RNAseq analysis, several data sets and tools were used to select key genes and examine selection validation. RESULTS: We selected 42 DEmRNAs and nine DEmiRs, including hsa-let-7f-5p, hsa-miR-21-5p, hsamiR- 22-3p, hsa-miR-126-5p, hsa-miR-146b-5p, hsa-miR-181a-5p, hsa-miR-92a-3p, hsa-miR-25-3p and hsa-miR-191-5p. Furthermore, hub genes including hist1h2bl, al133243.2, trim58, abcc13, bpgm, and fam210b were identified in the coexpression network, as well as RPS27A in the PPI network. Functional analysis revealed that these DEmRNAs and DEmiRs might play a role in gene expression regulation at multiple levels. Gene set enrichment analysis, in particular, revealed a possible role for genes in the globin switching process. CONCLUSION: According to our findings, a number of the DEmRNAs and DEmiRs may play significant roles in globin switching regulation and thus have the potential to be applied for HbF reactivation.

Metabolic sensor O-GlcNAcylation regulates erythroid differentiation and globin production via BCL11A.

BACKGROUND: Human erythropoiesis is a tightly regulated, multistep process encompassing the differentiation of hematopoietic stem cells (HSCs) toward mature erythrocytes. Cellular metabolism is an important regulator of cell fate determination during the differentiation of HSCs. However, how O-GlcNAcylation, a posttranslational modification of proteins that is an ideal metabolic sensor, contributes to the commitment of HSCs to the erythroid lineage and to the terminal erythroid differentiation has not been addressed. METHODS: Cellular O-GlcNAcylation was manipulated using small molecule inhibition or CRISPR/Cas9 manipulation of catalyzing enzyme O-GlcNAc transferase (OGT) and removing enzyme O-GlcNAcase (OGA) in two cell models of erythroid differentiation, starting from: (i) human umbilical cord blood-derived CD34+ hematopoietic stem/progenitor cells (HSPCs) to investigate the erythroid lineage specification and differentiation; and (ii) human-derived erythroblastic leukemia K562 cells to investigate the terminal differentiation. The functional and regulatory roles of O-GlcNAcylation in erythroid differentiation, maturation, and globin production were investigated, and downstream signaling was delineated. RESULTS: First, we observed that two-step inhibition of OGT and OGA, which were established from the observed dynamics of O-GlcNAc level along the course of differentiation, promotes HSPCs toward erythroid differentiation and enucleation, in agreement with an upregulation of a multitude of erythroid-associated genes. Further studies in the efficient K562 model of erythroid differentiation confirmed that OGA inhibition and subsequent hyper-O-GlcNAcylation enhance terminal erythroid differentiation and affect globin production. Mechanistically, we found that BCL11A is a key mediator of O-GlcNAc-driven erythroid differentiation and ß- and α-globin production herein. Additionally, analysis of biochemical contents using synchrotron-based Fourier transform infrared (FTIR) spectroscopy showed unique metabolic fingerprints upon OGA inhibition during erythroid differentiation, supporting that metabolic reprogramming plays a part in this process. CONCLUSIONS: The evidence presented here demonstrated the novel regulatory role of O-GlcNAc/BCL11A axis in erythroid differentiation, maturation, and globin production that could be important in understanding erythropoiesis and hematologic disorders whose etiology is related to impaired erythroid differentiation and hemoglobinopathies. Our findings may lay the groundwork for future clinical applications toward an ex vivo production of functional human reticulocytes for transfusion from renewable cell sources, i.e., HSPCs and pluripotent stem cells.

Ablation of Tmcc2 Gene Impairs Erythropoiesis in Mice.

(1) Background: Transcriptomic and proteomic studies provide a wealth of new genes potentially involved in red blood cell (RBC) maturation or implicated in the pathogenesis of anemias, necessitating validation of candidate genes in vivo; (2) Methods: We inactivated one such candidate, transmembrane and coiled-coil domain 2 (Tmcc2) in mice, and analyzed the erythropoietic phenotype by light microscopy, transmission electron microscopy (TEM), and flow cytometry of erythrocytes and erythroid precursors; (3) Results: Tmcc2-/- pups presented pallor and reduced body weight due to the profound neonatal macrocytic anemia with numerous nucleated RBCs (nRBCs) and occasional multinucleated RBCs. Tmcc2-/- nRBCs had cytoplasmic intrusions into the nucleus and double membranes. Significantly fewer erythroid cells were enucleated. Adult knockouts were normocytic, mildly polycythemic, with active extramedullary erythropoiesis in the spleen. Altered relative content of different stage CD71+TER119+ erythroid precursors in the bone marrow indicated a severe defect of erythroid maturation at the polychromatic to orthochromatic transition stage; (4) Conclusions: Tmcc2 is required for normal erythropoiesis in mice. While several phenotypic features resemble congenital dyserythropoietic anemias (CDA) types II, III, and IV, the involvement of TMCC2 in the pathogenesis of CDA in humans remains to be determined.

Environmental Nanoparticles Reach Human Fetal Brains.

Anthropogenic ultrafine particulate matter (UFPM) and industrial and natural nanoparticles (NPs) are ubiquitous. Normal term, preeclamptic, and postconceptional weeks(PCW) 8-15 human placentas and brains from polluted Mexican cities were analyzed by TEM and energy-dispersive X-ray spectroscopy. We documented NPs in maternal erythrocytes, early syncytiotrophoblast, Hofbauer cells, and fetal endothelium (ECs). Fetal ECs exhibited caveolar NP activity and widespread erythroblast contact. Brain ECs displayed micropodial extensions reaching luminal NP-loaded erythroblasts. Neurons and primitive glia displayed nuclear, organelle, and cytoplasmic NPs in both singles and conglomerates. Nanoscale Fe, Ti, and Al alloys, Hg, Cu, Ca, Sn, and Si were detected in placentas and fetal brains. Preeclamptic fetal blood NP vesicles are prospective neonate UFPM exposure biomarkers. NPs are reaching brain tissues at the early developmental PCW 8-15 stage, and NPs in maternal and fetal placental tissue compartments strongly suggests the placental barrier is not limiting the access of environmental NPs. Erythroblasts are the main early NP carriers to fetal tissues. The passage of UFPM/NPs from mothers to fetuses is documented and fingerprinting placental single particle composition could be useful for postnatal risk assessments. Fetal brain combustion and industrial NPs raise medical concerns about prenatal and postnatal health, including neurological and neurodegenerative lifelong consequences.

Validation of the mindray BC6000 hematology analyzer for erythroblast counting in peripheral blood / Validação do analisador hematológico Mindray BC6000 para a contagem de eritroblastos em sangue periférico

ABSTRACT Introduction Differential counting of erythroblasts in blood samples by hematology analyzers still has limitations. Technological advances in blood count equipment have proposed the fully automated counting of these cells, however, not without its validation. Objective Validate the automated count of erythroblasts in peripheral blood in the Mindray BC6000 hematology analyzer and verify the existence of correlation of the maturation stages of erythroblasts with the equipment's graphics. Material and Methods Prospective study with peripheral blood samples from the Clinical Pathology Laboratory Dr Paulo C. Azevedo, regardless of age and gender, to validate the erythroblast count in the Mindray BC6000 hematology analyzer compared to the manual method (gold standard), in the period of June 2019 to December 2020. Results Seventeen peripheral blood samples were analyzed from newborns (09/17 - 52.3%) and from patients older than 2 years (08/17 - 47.7%) who had more than 18% of erythroblasts after morphological analysis of the sample. Statistical analysis of erythroblast counts by the two methodologies showed that the Mindray BC6000 hematological counter has good reproducibility, precision and linearity. There was no correlation between the maturation stages of erythroblasts and the equipment graphics. Conclusion The proposed validation showed that the Mindray BC6000 hematological counter has good analytical performance for counting erythroblasts in peripheral blood. However, there is no correlation between the maturation stages of erythroblasts with the graphics generated by the equipment.

RESUMO Introdução A contagem diferencial de eritroblastos em amostras de sangue por analisadores de hematologia ainda apresenta limitações. Os avanços tecnológicos nos equipamentos de hemograma têm proposto a contagem totalmente automatizada dessas células, porém, não sem sua validação. Objetivo Validar a contagem automatizada de eritroblastos no sangue periférico no analisador hematológico Mindray BC6000 e verificar a existência de correlação dos estágios de maturação dos eritroblastos com os gráficos do equipamento. Material e Métodos Estudo prospectivo com amostras de sangue periférico do Laboratório de Patologia Clínica Dr. Paulo C. Azevedo, independente de idade e sexo, para validação da contagem de eritroblastos no analisador hematológico Mindray BC6000 em comparação ao método manual (padrão ouro), no período de junho de 2019 a dezembro de 2020. Resultados Foram analisadas 17 amostras de sangue periférico de recém-nascidos (17/09 - 52,3%) e de pacientes maiores de 2 anos (17/08 - 47,7%) que apresentavam mais de 18% de eritroblastos após análise morfológica da amostra. A análise estatística das contagens de eritroblastos pelas duas metodologias mostrou que o contador hematológico Mindray BC6000 tem boa reprodutibilidade, precisão e linearidade. Não houve correlação entre os estágios de maturação dos eritroblastos e os gráficos do equipamento. Conclusão A validação proposta mostrou que o contador hematológico Mindray BC6000 apresenta bom desempenho analítico para contagem de eritroblastos em sangue periférico. Porém, não há correlação entre os estágios de maturação dos eritroblastos com os gráficos gerados pelo equipamento.

Insertion/deletion and microsatellite alteration profiles in induced pluripotent stem cells.

We here demonstrate that microsatellite (MS) alterations are elevated in both mouse and human induced pluripotent stem cells (iPSCs), but importantly we have now identified a type of human iPSC in which these alterations are considerably reduced. We aimed in our present analyses to profile the InDels in iPSC/ntESC genomes, especially in MS regions. To detect somatic de novo mutations in particular, we generated 13 independent reprogramed stem cell lines (11 iPSC and 2 ntESC lines) from an identical parent somatic cell fraction of a C57BL/6 mouse. By using this cell set with an identical genetic background, we could comprehensively detect clone-specific alterations and, importantly, experimentally validate them. The effectiveness of employing sister clones for detecting somatic de novo mutations was thereby demonstrated. We then successfully applied this approach to human iPSCs. Our results require further careful genomic analysis but make an important inroad into solving the issue of genome abnormalities in iPSCs.

Generation of 'designer erythroblasts' lacking one or more blood group systems from CRISPR/Cas9 gene-edited human-induced pluripotent stem cells.

Despite the recent advancements in transfusion medicine, red blood cell (RBC) alloimmunization remains a challenge for multiparous women and chronically transfused patients. At times, diagnostic laboratories depend on difficult-to-procure rare reagent RBCs for the identification of different alloantibodies in such subjects. We have addressed this issue by developing erythroblasts with custom phenotypes (Rh null, GPB null and Kx null/Kell low) using CRISPR/Cas9 gene-editing of a human induced pluripotent stem cell (hiPSC) parent line (OT1-1) for the blood group system genes: RHAG, GYPB and XK. Guide RNAs were cloned into Cas9-puromycin expression vector and transfected into OT1-1. Genotyping was performed to select puromycin-resistant hiPSC KOs. CRISPR/Cas9 gene-editing resulted in the successful generation of three KO lines, RHAG KO, GYPB KO and XK KO. The OT1-1 cell line, as well as the three KO hiPSC lines, were differentiated into CD34+ CD41+ CD235ab+ hematopoietic progenitor cells (HPCs) and subsequently to erythroblasts. Native OT1-1 erythroblasts were positive for the expression of Rh, MNS, Kell and H blood group systems. Differentiation of RHAG KO, GYPB KO and XK KO resulted in the formation of Rh null, GPB null and Kx null/Kell low erythroblasts, respectively. OT1-1 as well as the three KO erythroblasts remained positive for RBC markers-CD71 and BAND3. Erythroblasts were mostly at the polychromatic/ orthochromatic stage of differentiation. Up to ~400-fold increase in erythroblasts derived from HPCs was observed. The availability of custom erythroblasts generated from CRISPR/Cas9 gene-edited hiPSC should be a useful addition to the tools currently used for the detection of clinically important red cell alloantibodies.

Prognostic Value of Nucleated RBCs for Patients With Suspected Sepsis in the Emergency Department: A Single-Center Prospective Cohort Study.

OBJECTIVES: Increase of nucleated RBCs in peripheral blood has been shown to be predictive of mortality in ICU patients. The aim of this study was to explore the prognostic value of nucleated RBCs in the first blood sample taken at admission to the emergency department from patients with suspected sepsis. DESIGN: Single-center prospective cohort study. SETTING: Emergency department. PATIENTS: One-thousand two-hundred thirty-one consecutive adult patients with suspected sepsis were included in a prospective quality register-based cohort study. Inclusion criteria were as follows: patients received in rapid response team with blood cultures taken and immediate antibiotics given in the emergency department. INTERVENTION: Not applicable. MEASUREMENT AND MAIN RESULTS: Nucleated RBCs, Sequential Organ Failure Assessment score, Quick Sequential Organ Failure Assessment, Charlson Comorbidity Index, and commonly used laboratory tests measured in the emergency department were compared with 30-day mortality. Nvaucleated RBC counts were divided into five groups, called "Nucleated RBC score," according to nucleated RBC count levels and analyzed with logistic regression together with the Sequential Organ Failure Assessment score and Charlson Comorbidity Index. Of the 262 patients with nucleated RBCs equal to or higher than the detection limit (0.01 × 109/L), 26% died within 30 days, compared with 12% of the 969 patients with nucleated RBCs below the detection limit (p < 0.0001). Mortality was significantly higher for each increase in Nucleated RBC score, except from score 2 to 3, and was 62% in the highest group. In multivariate logistic regression, odds ratios for 30-day mortality were as follows: Nucleated RBC score: 1.33 (95% CI, 1.13-1.56), Sequential Organ Failure Assessment score: 1.32 (1.29-1.56), and Charlson Comorbidity Index: 1.17 (1.09-1.25). CONCLUSIONS: Most patients with suspected sepsis in emergency department had undetectable nucleated RBCs at admission to the emergency department. However, increased nucleated RBCs significantly predicted 30-day mortality. Nucleated RBCs may provide additional prognostic information to Sequential Organ Failure Assessment score and other laboratory tests.

Estimating biological reference intervals for cell blood counts in a dog population / Estimación de los intervalos biológicos de referencia para conteos celulares sanguíneos en una población de perros / Estimando intervalos de referência biológica para contagens de células sanguíneas em uma população de cães

Abstract Background: The results from automated equipment and peripheral blood smears allow correlating clinical data with cellular blood counts (CBC), generating information on pathologies of hematological and non-hematological origin in dogs. Objective: To describe qualitative and quantitative magnitudes of CBC in healthy donor dogs of a blood- bank in Medellín (Colombia). Methods: A descriptive-prospective study was carried out from 146 records of CBC results from a blood-bank. The samples were processed by automated equipment, and trained personnel performed the observation of peripheral blood smears to collect qualitative information. Variables such as age, sex, breed, quantitative results, and observations of the peripheral blood smears were considered. For the definition of biological intervals (BIs), the mean and two standard deviations were used for the data with a normal distribution. Otherwise, the 2.5th and 97.5th percentiles were used. Results: The size, granularity, and cytoplasmic vacuoles in monocytes and polymorphonuclear eosinophils, as well as the presence of Barr chromatin and occasional circulating erythroblasts, were remarkable findings to the peripheral blood smear. With the standardization of data obtained from dogs of large and giant breeds, the present work provides BIs for some of the CBC data in the studied population. Conclusion: The findings of the present study allow approximations to the definition of alterations in blood cells and their counts, which can guide the veterinarian towards an early diagnosis in dogs.

Resumen Antecedentes: Los datos obtenidos de equipos automatizados y extendidos de sangre periférica permiten correlacionar la clínica y los resultados del hemograma, generando información de interés sobre patologías de origen hematológico y no hematológico en perros. Objetivo: Describir las magnitudes cualitativas y cuantitativas del hemograma en perros sanos, donantes de un banco de sangre en Medellín (Colombia). Métodos: Se realizó un estudio descriptivo-prospectivo a partir de 146 registros de resultados de hemograma remitidos por un banco de sangre. Las muestras fueron procesadas por un equipo automatizado y personal entrenado realizó la observación del extendido de sangre periférica para la colección de información de corte cualitativo. Variables como edad, sexo, raza, resultados cuantitativos de las muestras y observaciones al extendido de sangre periférica fueron consideradas. Para la definición de intervalos biológicos (IBs) se utilizaron la media y dos desviaciones estándar para los datos con distribución normal. De lo contrario, se utilizaron los percentiles 2,5 y 97,5. Resultados: El tamaño, granularidad y vacuolas citoplasmáticas en monocitos y polimorfonucleares eosinófilos, así como la presencia de Cromatina de Barr y eritroblastos circulantes ocasionales fueron hallazgos llamativos al extendido de sangre periférica. Con la normalización de los datos para caninos de razas grandes y gigantes, el presente trabajo aporta IBs para algunos de los datos del hemograma en la población estudiada. Conclusión: Los hallazgos del presente estudio permiten aproximaciones a la definición de alteraciones en las células hemáticas y sus conteos, que pueden orientar al médico veterinario hacia un diagnóstico temprano en perros.

Resumo Antecedentes: As informações obtidas de equipamentos automatizados e esfregaços de sangue periférico permitem correlacionar os resultados clínicos e de hemogramas, gerando informações interessantes sobre patologias de origem hematológica e não hematológica em cães. Objetivo: Descrever as magnitudes qualitativa e quantitativa do hemograma em cães saudáveis, doadores de um banco de sangue em Medellín (Colômbia). Métodos: Foi realizado um estudo descritivo-prospectivo a partir de 146 registros de resultados de hemograma encaminhados por um banco de sangue. As amostras foram processadas por uma equipe automatizada e pessoal treinado observou o esfregaço de sangue periférico para a coleta de informações qualitativas. Foram consideradas variáveis como idade, sexo, raça, resultados quantitativos das amostras e observações do esfregaço de sangue periférico. Para a definição de intervalos biológicos (IBs), foram utilizados a média e dois desvios-padrão para os dados com distribuição normal. Caso contrário, foram utilizados os percentis 2,5 e 97,5. Resultados: O tamanho, a granularidade e os vacúolos citoplasmáticos dos monócitos e das células polimorfonucleares dos eosinófilos, bem como a presença da cromatina de Barr e ocasionais eritroblastos circulantes foram achados marcantes na disseminação do sangue periférico. Com a normalização dos dados para cães de raças grandes e gigantes, o presente trabalho fornece IBs para alguns dos dados de hemograma na população estudada. Conclusão: Os achados do presente estudo permitem aproximações à definição de alterações nas células sanguíneas e suas contagens, o que pode orientar o médico veterinário responsável pelo diagnóstico precoce em cães.

Cannibalized erythroblasts accelerate developmental neurogenesis by regulating mitochondrial dynamics.

Metabolic support was long considered to be the only developmental function of hematopoiesis, a view that is gradually changing. Here, we disclose a mechanism triggered during neurulation that programs brain development by donation of sacrificial yolk sac erythroblasts to neuroepithelial cells. At embryonic day (E) 8.5, neuroepithelial cells transiently integrate with the endothelium of yolk sac blood vessels and cannibalize intravascular erythroblasts as transient heme-rich endosymbionts. This cannibalistic behavior instructs precocious neuronal differentiation of neuroepithelial cells in the proximity of blood vessels. By experiments in vitro, we show that access to erythroblastic heme accelerates the pace of neurogenesis by induction of a truncated neurogenic differentiation program from a poised state. Mechanistically, the poised state is invoked by activation of the mitochondrial electron transport chain that leads to amplified production of reactive oxygen species in addition to omnipresent guanosine triphosphate (GTP) with consequential upregulation of pro-differentiation ß-catenin.

Leukoerythroblastic reaction associated with COVID-19 infection. Case report / [Reacción leucoeritroblástica asociada a infección por COVID-19. Reporte de caso]

Introduction: COVID-19 disease is an acute viral pneumonia with multiple extrapulmonary manifestations, including certain hematological alterations. The leukoerythroblastic reaction or leukoerythroblastosis is defined by the presence in peripheral blood of nucleated erythroid cells and immature myeloids. Among its most frequent causes is medullary invasion by solid tumors and hematological neoplasms, so the recognition and consequent study of this reaction is extremely important. To date, only a single case of COVID-19 disease associated with a leukoerythroblastic reaction has been reported worldwide in adult patients. Methodology: In the following manuscript we describe the presence of leukoerythroblastosis in a male hospitalized for COVID-19, in which multiple causes were ruled out. Results: The patient had resolution of the leukoerythroblatosis as his respiratory symptoms resolved. Conclusion: The COVID-19 disease could be a new known cause of a leukoerythroblastic reaction, although we believe that a greater number of reported cases is necessary to affirm that the association was not random.

Introducción: La enfermedad COVID-19 es una neumonía viral aguda con múltiples manifestaciones extrapulmonares dentro de las cuales se incluyen ciertas alteraciones hematológicas. La reacción leucoeritroblástica o leucoeritroblastosis se define por la presencia en sangre periférica de células eritroides nucleadas y mieloides inmaduras. Dentro de sus causas más frecuentes se encuentra la invasión medular por tumores sólidos y neoplasias hematológicas, por lo que el reconocimiento y consecuente estudio de dicha reacción es sumamente importante. Hasta el momento sólo se ha reportado a nivel mundial un único caso de enfermedad COVID-19 asociado a reacción leucoeritroblástica en pacientes adultos. Metodología: En el siguiente manuscrito describimos la presencia de leucoeritroblastosis en un masculino internado por COVID-19, en el cual se descartaron múltiples causas secundarias.. Resultados: El paciente presentó resolución de la leucoeritroblatosis a medida que sus síntomas respiratorios se resolvieron. Conclusión: La enfermedad COVID-19 podría ser una nueva causa conocida de reacción leucoeritroblástica aunque creemos necesario un mayor número de casos reportados para afirmar que la asociación no fue al azar

BMI1 enables extensive expansion of functional erythroblasts from human peripheral blood mononuclear cells.

Transfusion of red blood cells (RBCs) from ABO-matched but genetically unrelated donors is commonly used for treating anemia and acute blood loss. Increasing demand and insufficient supply for donor RBCs, especially those of universal blood types or free of known and unknown pathogens, has called for ex vivo generation of functional RBCs by large-scale cell culture. However, generating physiological numbers of transfusable cultured RBCs (cRBCs) ex vivo remains challenging, due to our inability to either extensively expand primary RBC precursors (erythroblasts) or achieve efficient enucleation once erythroblasts have been expanded and induced to differentiation and maturation. Here, we report that ectopic expression of the human BMI1 gene confers extensive expansion of human erythroblasts, which can be derived readily from adult peripheral blood mononuclear cells of either healthy donors or sickle cell patients. These extensively expanded erythroblasts (E3s) are able to proliferate exponentially (>1 trillion-fold in 2 months) in a defined culture medium. Expanded E3 cells are karyotypically normal and capable of terminal maturation with approximately 50% enucleation. Additionally, E3-derived cRBCs can circulate in a mouse model following transfusion similar to primary human RBCs. Therefore, we provide a facile approach of generating physiological numbers of human functional erythroblasts ex vivo.