Adult Red Blood Cells Research Articles

Fetal Red Blood Cells: A Comprehensive Review of Biological Properties and Implications for Neonatal Transfusion.

Transfusion guidelines worldwide include recommendations regarding the storage length, irradiation, or even donor cytomegalovirus serostatus of red blood cell (RBC) units for anemic neonates. Nevertheless, it is totally overlooked that RBCs of these patients fundamentally differ from those of older children and adults. These differences vary from size, shape, hemoglobin composition, and oxygen transport to membrane characteristics, cellular metabolism, and lifespan. Due to these profound dissimilarities, repeated transfusions of adult RBCs in neonates deeply modify the physiology of circulating RBC populations. Unsurprisingly, the number of RBC transfusions in preterm neonates, particularly if born before 28 weeks of gestation, predicts morbidity and mortality. This review provides a comprehensive description of the biological properties of fetal, cord blood, and neonatal RBCs, including the implications that neonatal RBCs, and their replacement by adult RBCs, may have for perinatal disease pathophysiology.

Retinopathy of Prematurity in Eight Portuguese Neonatal Intensive Care Units: Incidence, Risk Factors, and Progression-A Prospective Multicenter Study.

Retinopathy of prematurity (ROP) is a retinal neovascular disease affecting preterm infants. Identifying risk factors for its development and progression is critical for effective screening and prevention. This study aimed to analyze the incidence of ROP and identify key risk factors for its development and progression. We conducted a prospective, observational cohort study on 455 neonates (gestational age [GA] < 32 weeks or birth weight < 1500 g) across eight Portuguese NICUs. ROP incidence was 37.8%, with 4.6% requiring treatment. Multivariate analysis identified low GA and the number of red blood cell (RBC) transfusions as significant factors for ROP development and progression. After adjusting for these variables, platelet transfusions, high maximum fraction of inspired oxygen (FiO2) in the second week, and surfactant use remained significantly associated with ROP development, while early and late sepsis, maternal chronic hypertension, and delayed enteral nutrition were associated with progression to ROP requiring treatment. These findings underscore the importance of addressing low GAs and adult RBC transfusions in ROP risk management and suggest that maximum FiO2, platelet transfusions, and sepsis also play crucial roles. Larger studies are needed to validate these results and explore preventive interventions, particularly regarding the impact of multiple adult RBC transfusions on fetal hemoglobin percentages.

A Transfusion Regimen With Same-donor Packed Red Blood Cells Reduces Exposure to Multiple Blood Donors in Craniosynostosis Surgery.

In major craniosynostosis surgery with moderate to severe blood loss, patients may be exposed to multiple donors. We have previously reported a method for reducing donor exposure using mixed pediatric units including plasma. To further reduce donor exposure, we used plasma-free divided pediatric units. The study aimed to investigate the feasibility of the new strategy for reducing donor exposure. This prospective observational study recruited children younger than 1 year who were scheduled for nonsyndromic craniosynostosis surgery. One adult red blood cell unit was divided into 4 equal units on the day before the operation for use intra- or postoperatively. Number of donor exposures, estimated blood loss, crystalloid, colloid, and blood product volumes, and coagulation parameters were evaluated. Nineteen infants were included. The mean estimated blood loss was 19 (3) mL/kg and the transfusion volume was 17 (7) mL/kg. The median donor exposure per patient was 1 (range, 1-3). During surgery, all infants received at least one DPU. Two infants received transfusions from more than one donor during the intraoperative period. In the first 24 hours postoperatively, 14 infants received transfusion; 10 received only DPUs, whereas 4 received from multiple donors. In all, multiple donor exposure was prevented in 14 of 19 infants. Postoperative Pk-INR was 1.33 (0.16); no plasma or platelets were transfused. The plasma-free DPU transfusion protocol may be useful to reduce donor exposure in open craniosynostosis surgery in infants.

Developing rainbow trout (Oncorhynchus mykiss) lose branchial plasma accessible carbonic anhydrase expression with hatch and the transition to pH-sensitive, adult hemoglobin polymorphs.

Salmonids possess a unique respiratory system comprised of three major components: highly pH-sensitive hemoglobins, red blood cell (RBC) intracellular pH (pHi) protection, and a heterogeneous distribution of plasma accessible carbonic anhydrase (paCA), specifically with absence of paCA at the gills. These characteristics are thought to have evolved to enhance oxygen unloading to the tissues while protecting uptake at the gills. Our knowledge of this system is detailed in adults, but little is known about it through development. Developing rainbow trout (Oncorhynchus mykiss) express embryonic RBCs containing hemoglobins that are relatively insensitive to pH; however, availability of gill paCA and RBC pHi protection is unknown. We show that pre-hatch rainbow trout express gill paCA, which is lost in correlation with the emergence of highly pH-sensitive adult hemoglobins and RBC pHi protection. Rainbow trout therefore exhibit a switch in respiratory strategy with hatch. We conclude that gill paCA likely represents an embryonic trait in rainbow trout and is constrained in adults due to their highly pH-sensitive hemoglobins.

Transfusion of packed red blood cells in adults with sickle cell anemia treated at an emergency hospital.

The aim of this study was to analyze the prescription of packed red blood cells performed by emergency physicians for adults with sickle cell anemia. Transfusions performed in adults with sickle cell anemia treated at an emergency service in São Bernardo do Campo, São Paulo Brazil, between January 2018 and January 2022 were evaluated. For data comparison, the chi-square2 test was used. The significance level adopted was 5%. A total of 114 transfusions were performed. The mean age was 41.8±16.4 years, and pretransfusion hemoglobin was 6.1±1.23 g/dL. Regarding the indication, the adequacy of transfusions performed in symptomatic individuals was significantly higher compared to asymptomatic individuals (100% vs. 3.9%, p<0.001). Symptomatic individuals received excessive volumes of packed red blood cells less frequently than asymptomatic individuals (17.5% vs. 56.9%, p<0.001). The filtered subtype, indicated for sickle cell anemia, was prescribed in only a quarter of the patients. However, non-indicated subtypes were frequently prescribed. This study found low adequacy in the indication and calculation of the transfusion volume of packed red blood cells in asymptomatic individuals. Few patients received filtered red blood cells, resulting in increased risks of transfusion reactions. On the contrary, non-indicated subtypes were prescribed in a quarter of transfusions, which resulted in higher costs and delay in receiving packed red blood cells.

Classification of fetal and adult red blood cells based on hydrodynamic deformation and deep video recognition

Flow based deformation cytometry has shown potential for cell classification. We demonstrate the principle with an injection moulded microfluidic chip from which we capture videos of adult and fetal red blood cells, as they are being deformed in a microfluidic chip. Using a deep neural network - SlowFast - that takes the temporal behavior into account, we are able to discriminate between the cells with high accuracy. The accuracy was larger for adult blood cells than for fetal blood cells. However, no significant difference was observed between donors of the two types.

Differences in Cerebral Tissue Oxygenation in Preterm Neonates Receiving Adult or Cord Blood Red Blood Cell Transfusions

Repeated transfusions in preterm neonates with anemia of prematurity replace fetal hemoglobin (HbF) with adult Hb (HbA), which has a low oxygen affinity. The reduction of HbF is associated with a higher incidence of retinopathy of prematurity (ROP). To assess whether HbF and HbA are differently associated with cerebral tissue oxygenation in preterm neonates. This cohort study was a single-center, pilot study on cerebral oxygenation kinetics in preterm neonates with a gestational age between 24.0 weeks and 27.9 weeks who were admitted to the neonatal intensive care unit of Policlinico Universitario A. Gemelli IRCCS from December 27, 2021, to May 15, 2023. This study was ancillary to the ongoing, double-blind, multicenter Umbilical or Adult Donor Red Blood Cells in Extremely Low Gestational Age Neonates and Retinopathy of Prematurity (BORN) randomized clinical trial. The BORN trial outcome was ROP severity in neonates randomized to receive standard packed red blood cell (PRBC) transfusions obtained from RBCs of adult donors (A-RBCs) or from cord blood (CB-RBCs). According to standard procedures at the institute's neonatal intensive care unit, patients concurrently received continuous cerebral near-infrared spectroscopy (NIRS) monitoring. This cohort study was not prespecified in the trial protocol. Transfusion with A-RBCs or CB-RBCs. The main outcome was the kinetics of cerebral regional oxygen saturation (crSO2) and cerebral fraction of tissue oxygen extraction (cFTOE) associated with A-RBC or CB-RBC transfusions. Cerebral NIRS monitoring was performed by neonatologists and nurses, who were blinded to the PRBC type. The NIRS monitoring was conducted starting with the blood product order, during transfusion, and for the subsequent 24 hours after transfusion completion. The mean treatment effects of A-RBCs or CB-RBCs were quantified using a linear mixed model for repeated measures. Of 23 randomized neonates, 17 (11 male [64.7%]; median gestational age at birth, 25.6 weeks [IQR, 25.3-26.1 weeks]) with a median birth weight of 840 g (IQR, 580-900 g) were included in the study; NIRS was evaluated for 42 transfusion episodes, of which 22 were A-RBCs and 20 were CB-RBCs. Globally considering all posttransfusion time points, the overall crSO2 covariate-adjusted mean after CB-RBC transfusions was 5.27% lower (95% CI, 1.20%-9.34%; P = .01) than that after A-RBC transfusions, while the cFTOE after CB-RBC transfusions was 6.18% higher (95% CI, 1.66%-10.69%; P = .009) than that after A-RBCs. The findings of this cohort study suggest that A-RBC transfusions may be associated with more oxygen delivery to cerebral tissues of preterm neonates than transfusions from CB-RBCs. This finding may explain the previously observed association between low HbF and high ROP risk. It also suggests that use of CB to meet the RBC transfusion needs of neonates with a gestational age of less than 28 weeks may protect cerebral tissues from overexposure to oxygen.

Genome-Scale CRISPR-Cas12a Screen Identifies Novel Fetal Hemoglobin Regulators

Reversal of the postnatal hemoglobin switch in red blood cells is a promising approach to therapies for sickle cell disease and β-thalassemia, as reactivating fetal hemoglobin (HbF, α2γ2) in adult red blood cells ameliorates disease complications. Studies over several decades have illuminated the paradigm of this fetal-to-adult hemoglobin transition, including central roles of transcriptional repressors BCL11A, LRF, and NFIA/X, which recruit repressive chromatin modifiers to the γ-globin locus. To elucidate undescribed regulatory mechanisms of these molecules and reveal new targets for therapies, we performed the first genome-wide genetic screen to pinpoint specific pathways or protein complexes that regulate HbF expression in adult erythroid cells. Because Cas9 technology is limiting at the genome-wide scale, we conducted a CRISPR-Cas12a genetic screen targeting all known coding genes in the human genome using a novel custom array directed towards 18,292 genes. The single CRISPR RNA (crRNA) library was cloned into a lentivirus scaffold and transduced into the adult-type erythroid cell line, HUDEP2, engineered to stably express Cas12a. HUDEP2 cells express little HbF, allowing for positive phenotypic selection (HbF+) by fluorescence-activated cell-sorting. By isolating the top 10% and bottom 10% of HbF-expressing cells and determining the representation of crRNAs via deep sequencing, we identified a crRNA subset enriched in the high-HbF population encompassing 211 candidate genes, representing potential regulators of HbF. To prioritize these genes for follow up studies, we conducted a domain-focused, CRISPR-Cas9 screen of all 211 candidate genes. Using 2,184 sgRNAs, this validation Cas9 screen in HUDEP2 cells nominated 18 high-confidence targets. Two of the top candidate genes were identified as modulators of the phosphatase PP2A, PPP2R4 and TIPRL. PP2A is a multi-subunit complex with diverse cellular functions as a serine/threonine phosphatase. To our knowledge, it has no previously known role on globin gene production. In validation experiments, disruption of either PPP2R4 or TIPRL in HUDEP2 cells significantly increased γ-globin mRNA and protein expression. Using sgRNA-Cas9 ribonucleoprotein complex nucleofection, PPP2R4 gene editing in primary human CD34+ cell derived erythroblasts significantly increased γ-globin mRNA and protein expression without overtly affecting erythroid maturation. Notably, the degree of effect is similar to that of BCL11A +58 enhancer disruption. Depletion of either PP2A regulator in HUDEP2 cells resulted in markedly reduced BCL11A protein levels. Furthermore, BCL11A overexpression in PPP2R4 or TIPRL-targeted cells reduced γ-globin mRNA commensurate to parental control cells, thereby demonstrating both TIPRL and PPP2R4 mediate γ-globin silencing predominantly via BCL11A. RNA-sequencing of TIPRL and PPP2R4-edited HUDEP2 cells revealed strong overlap of differentially expressed genes, consistent with convergent function. Altogether, our studies utilizing a novel Cas12a-based, genome-wide screen nominate a new pathway mediating γ-globin silencing via regulation of BCL11A.

Flickering of fetal erythrocytes membrane under gestational diabetes observed with dual time resolved membrane fluctuation spectroscopy

The membrane flickering of human fetal red blood cells (RBCs) affected by gestational diabetes mellitus (GDM) was studied with dual time resolved membrane fluctuation spectroscopy (D-TRMFS). This new technique is a modified version of the dual optical tweezers method that has been adapted to measure the mechanical properties of RBCs at two distant membrane points simultaneously. The micro-rheological parameters were obtained from direct membrane flickering measurements, followed by Fourier decomposition and cell membrane model adjustment. Our results show a significant decrease of 6.01 ± 1.19 nm in membrane fluctuations amplitude in healthy fetal, compared with healthy adult RBCs, meanwhile the amplitude in GDM cells increased 3.22 ± 1.10 nm compared with healthy fetal RBCs. Between GDM and healthy fetal RBCs, there are significant differences, especially in the bending modulus. Considering the mean of the two membrane points measured, the tension for GDM RBCs increased by 6.431 ± 3.57 (10−7 [N/m]) compared with healthy fetal RBCs, meanwhile, the bending was increased by 2.483 ± 0.58 (10−19 [J]) in GDM compared with healthy fetal RBCs. These results showed significant increment of 1.23 ± 0.07-fold and 3.29 ± 0.36-fold in tension and bending modulus in GDM, respectively. The strong impact of GDM on bending modulus could be associated with oxidative stress and lipid peroxidation, previously reported in fetal plasma of GDM cases.

Using umbilical cord blood as a source of paediatric packed red blood cells: Processing and quality control.

Umbilical cord blood (UCB) has been used as a source of red blood cells (RBCs) for neonatal/paediatric transfusion purposes. This study adopted two different procedures to obtain umbilical RBC (U-RBC) to compare its quality control parameters to those of fractionated adult RBC (A-RBC), for paediatric purposes. UCB units (24) were filtered and processed based on two different methods, namely, conventional/manual (P1;n12) and automatic (P2;n12). They were compared to five fractionated A-RBCs. U-RBC and A-RBC were stored for 14 days and had their haematological, biochemical, haemolytic and microbiological parameters analysed at D1, D7 and D14. Cytokines and growth factors (GFs) in residual U-RBC plasma were measured. Mean volume of processed U-RBC units was 45 mL for P1 and 39 mL for P2; the mean haematocrit level reached 57% for P1 and 59% for P2. A-RBC recorded a mean volume of 44 mL. Haematologic and biochemical parameters analysed in U-RBC and A-RBC presented similar behaviours during storage time, except for parameter values, which differed between them. Pro-inflammatory and immunomodulatory cytokines, as well as GFs, were higher in U-RBC residual plasma than in that A-RBC. UCB can be processed into RBC based on either manual or automated protocols. U-RBC units met the referenced quality parameters defined for A-RBC. Some features, mainly the biochemical ones, should be further investigated to help improve quality parameters, with emphasis on differences found in, and particularities of, this material and on recipients of this new transfusion practice.

Engineering of the endogenous HBD promoter increases HbA2.

The β-hemoglobinopathies, such as sickle cell disease and β-thalassemia, are one of the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of β-globin subunits in adult hemoglobin. Many gene editing efforts to treat the β-hemoglobinopathies attempt to correct β-globin mutations or increase γ-globin for fetal hemoglobin production. δ-globin, the subunit of adult hemoglobin A2, has high homology to β-globin and is already pan-cellularly expressed at low levels in adult red blood cells. However, upregulation of δ-globin is a relatively unexplored avenue to increase the amount of functional hemoglobin. Here, we use CRISPR-Cas9 to repair non-functional transcriptional elements in the endogenous promoter region of δ-globin to increase overall expression of adult hemoglobin 2 (HbA2). We find that insertion of a KLF1 site alone is insufficient to upregulate δ-globin. Instead, multiple transcription factor elements are necessary for robust upregulation of δ-globin from the endogenous locus. Promoter edited HUDEP-2 immortalized erythroid progenitor cells exhibit striking increases of HBD transcript, from less than 5% to over 20% of total β-like globins in clonal populations. Edited CD34 +hematopoietic stem and progenitors (HSPCs) differentiated to primary human erythroblasts express up to 46% HBD in clonal populations. These findings add mechanistic insight to globin gene regulation and offer a new therapeutic avenue to treat β-hemoglobinopathies.

MicroRNA Molecular Profiling Reveals Increase Expression of hsa-miR-186-5p, hsa-miR-320a-3p, and hsa-miR-486-5p With Storage Time in Packed Blood Cells

BACKGROUND: Compared to other blood cells, adult red blood cells have a higher concentration of microRNAs (miRNAs). The effectiveness of preserved blood cells following transfusion is affected by a variety of factors, like changes in miRNA levels. One day, these small RNAs might help determine the efficacy and safety of blood products. AIMS: This study sought to identify the miRNA present in both fresh blood and stored blood because there is growing evidence that these cells are enriched with miRNAs. MATERIALS AND METHODS: Blood samples were taken from three healthy donors to detect the expression of miRNAs using illumina platform for RNA sequencing. Total RNA was isolated from stored units at 0, 14, 21, and 28 days, respectively. Bioinformatics analysis has been carried out to analyze the miRNAs. RESULTS: The majority of miRNA expression was decreased in time-dependent manner, particularly after day 14 of packed blood cells (PBCs) storage such as hsa-miR-20a-5p, hsa-miR-17-5p, and hsa-miR-423-3p. While other miRNAs such as hsa-miR-320a-3p, hsa-miR-186-5p, and hsa-miR-486-5p, showed a significant re-upregulated after day 21. CONCLUSION: In summary, the PBCs at days 14 and 21 had the lowest levels of miRNAs, which may indicate less of a relationship with storage lesions. However, older PBCs displayed significant levels of miRNAs, which could indicate storage lesions or cause a number of clinical issues for the recipients.

CRISPR/Cas9 Genome-Edited Autologous Hematopoietic Stem Cell Transplantation Therapies for Sickle Cell Disease

Hematopoietic stem cell transplantation is the only curative option for sickle cell disease (SCD), a life-threatening genetic disorder that impacts millions worldwide, yet the majority of patients do not have a suitable donor for an allogeneic transplantation. Increased levels of fetal hemoglobin (HbF) are protective against complications of SCD. BCL11A is a transcription factor that binds to the y-globin promoter and controls fetal-to-adult hemoglobin switching. Reducing BCL11A expression or inhibiting its activity can induce expression of HbF in adult red blood cells. We have developed 2 genome editing strategies targeting BCL11A for the induction of HbF levels and report the preclinical development of two potentially curative CRISPR/Cas9 ex-vivo genome-edited autologous hematopoietic stem cell therapies: HIX763 and OTQ923. HIX763 is an investigational cell therapy product harboring a genome modification in the erythroid-specific enhancer region of the BCL11A gene on chromosome 2 resulting in reduced BCL11A expression only in erythroid cells. OTQ923 is an investigational cell therapy product generated by targeted disruption of the γ-globin gene promoter on chromosome 11 to presumably delete BCL11A binding regions in the targeted region. Both HIX763 and OTQ923 are intended to durably increase HbF to therapeutically effective levels to ameliorate complications of SCD. Both products showed editing-dependent upregulation of γ-globin, HbF expression and number of F-cells upon erythroid differentiation of edited hematopoietic stem cells from healthy donors and SCD patients. No off-target editing was identified for both HIX763 and OTQ923 through a complementary suite of genome-wide biochemical and cell-based assays including in silico off-target prediction, SITE-Seq, PanHeme analysis of known oncogenes and tumor suppressors, karyotyping, UnIT evaluation of chromosomal rearrangements, and on-target long-range sequencing analysis. A six-month tumorigenicity study revealed no tumor formation or clonal outgrowth upon transplantation of full human doses of HIX763 and OTQ923 into immunocompromised NOD scid gamma mouse (NSG) mice. Finally, a comparability assessment of OTQ923 produced using the research process and the clinical manufacturing process showed comparability in the quality of cells, as measured by off-target editing, genomic rearrangement, cell viability, editing efficiency, HbF induction, and in vivo engraftment. Together these studies demonstrate that the preclinical data adequately reflects the behavior of the cells intended for clinical use and supports advancing these drug products into clinical trials for the treatment of sickle cell disease.

Treatment of Individuals with Severe Sickle Cell Disease with OTQ923, an Autologous, Ex Vivo, CRISPR/Cas9-Edited, CD34+ Hematopoietic Stem and Progenitor Cell Product, Leads to Durable Engraftment and Fetal Hemoglobin Induction

Introduction: Increased levels of fetal hemoglobin (HbF; encoded by γ-globin [HBG1/HBG2]) are protective against complications of sickle cell disease (SCD). BCL11A is a transcription factor that binds to HBG1/HBG2 promoters and produces a γ-to-β globin (fetal-to-adult hemoglobin) switch. Inhibiting BCL11A binding to its target can reverse the switch and induce HbF expression in adult red blood cells (RBCs). OTQ923 is an autologous, ex vivo, CRISPR/Cas9-edited, CD34+ cellular product with a targeted disruption of the HBG1/HBG2 promoters on chromosome 11. Targeting this region in preclinical models led to HbF induction, phenotypically recapitulating hereditary persistence of fetal hemoglobin (a naturally occurring condition whose co-inheritance ameliorates SCD severity). This approach is more targeted than complete elimination of BCL11A expression in erythrocyte precursors for treating hemoglobinopathies. Additionally, unlike lentiviral vector gene addition, induction of endogenous γ-globin transcription could concur with reduction of βS-globin expression to avoid α- and β-like globin chain imbalance. Methods: Subjects with severe SCD, defined by a history of stroke, recurrent vaso-occlusive events (VOE), acute chest syndrome (ACS), chronic transfusions, recurrent priapism or red cell alloimmunization, were eligible to participate in the adult cohort (≥18-≤40 years old) of this clinical trial (NCT04443907). OTQ923 was manufactured from cryopreserved, peripheral blood derived CD34+ cells, collected after plerixafor mobilization. Prior to infusion, participants received myeloablative busulfan. Primary endpoints are to evaluate engraftment, long-term safety, and efficacy of HbF expression. Results: As of the data cut-off (July 8, 2022), 2 participants had received OTQ923, with follow-ups of 9 months (mos) and 3 mos, respectively. Here, we present initial clinical data from these 2 participants; additional data will be presented at the meeting. Both participants were homozygous for the sickle mutation. Participant 1 is a 22-year-old male with a history of silent cerebral infarcts, ACS, and priapism. At study entry, he was receiving chronic transfusions. He received 2.8x106 CD34+ cells/kg of OTQ923 (2 combined manufacturing batches). Participant 2 is a 21-year-old male with a history of ACS, and a silent cerebral infarct and was receiving hydroxyurea. He received 5.99x106 CD34+ cells/kg of OTQ923 (3 combined manufacturing batches). Time to neutrophil engraftment (the first of 3 consecutive days of absolute neutrophil count above 500 cells/mL after cell infusion) was 26 and 20 days for Participants 1 and 2, respectively. No OTQ923-related adverse events (AEs) were reported in either participant. All observed AEs were consistent with busulfan conditioning. Participants received RBC transfusions around conditioning and have received none after engraftment. For Participant 1, HbF level increased in the 6 mos after infusion and was stable at data cut-off (22.1% HbF at 9 mos after infusion). Induction of HbF was pancellular, with an F-cell percentage around 91% at 6 mos after infusion (Fig. 1A). Participant 2 had achieved an HbF level of 15.9% at 3 mos after infusion, the latest evaluable timepoint at data cut-off. Neither participant had any SCD-related VOEs after infusion of the cellular product. The proportion of edited alleles in the peripheral blood of Participant 1 was maintained at over 60% until Mo 6, which was the last evaluable timepoint (Fig. 1B). The indels were diverse, except for a predominant, large 5 kb deletion arising from simultaneous double-stranded DNA cleavage at HBG1 and HBG2 on-target sites, similar to previous reports. Conclusion: Preliminary data demonstrate clinically meaningful increases in HbF and evidence of benefit for both Participants 1 and 2 after OTQ923 infusion. Edited alleles were stable and maintained in the peripheral blood for >6 mos. The safety profile of OTQ923 was as expected after myeloablative busulfan and autologous hematopoietic stem cell transplantation. Taken together, these data suggest that this approach is safe and offers a potentially curative option for patients with severe SCD. Using cryopreserved, CD34+ cells as a starting material offers the advantage of centralizing manufacturing, scaling up the process to allow for improved patient access around the globe. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Red blood cells morphology and morphometry in adult, senior, and geriatricians dogs by optical and scanning electron microscopy.

Red blood cells (RBC) morphologic evaluation through microscopy optical (OM) and SEM, provides information to forecast, evaluate, and monitor the functioning of many organs. Factors, such aging and diseases affect RBC morphology in both, human and animals. SEM is useful to evaluate RBC morphology, although its use in diagnosis and evaluation in dogs is limited, due to the availability and cost. The aim of this research was to assess the normal RBC morphology in adult, senior and geriatrician dogs, clinically healthy by OM and SEM. In addition to evaluating the age effect, sex, body size, and their interaction on erythrocyte morphometry. To carry out the research 152 blood samples were evaluated from dogs of different sexes and body sizes (small, medium, and large). Three groups were made based on dogs age: group I adults (1-7.9 years old), group II senior (8-11.9 years old), and group III geriatricians (>12 years old). Erythrocyte parameters were evaluated by OM (diameter, height, and axial ratio). Per each dog, the parameters of 20 erythrocytes were measured. A total of 2,600 cells were scanned with the AmScope™ Software scale. In addition, the RBC morphology was evaluated by SEM. Statistical analyses used analysis of variance and a general linear model, which allows the comparison of multiple factors at two or more levels (p < 0.05). The results of this study showed that diameter and height were lower in adult dogs than in senior and geriatrician dogs (p < 0.05). Whereas, sex, body size, and the interaction did not show a significant effect (p > 0.05). Additionally, some images of anisocytosis, polychromasia, and poikilocytosis (echinocytes, acanthocytes, codocytes, spherocytes, stomatocytes, dacryocytes quatrefoil, and elliptocytes) were obtained by OM and SEM. Our study provides information about the morphological and morphometry alterations of adult, senior, and geriatrician dogs RBC. This work contributes to future investigations and the diagnosing diseases, where it is necessary to evaluate the morphology of RBC.

DEVELOPMENT OF A SPIKE-IN FLOW-CYTOMETRY METHOD FOR THE QUANTITATION OF FETAL HEMOGLOBIN (HBF) IN INDIVIDUAL RED BLOOD CELLS OF SICKLE CELL DISEASE (SCD) PATIENTS

HbF is the most relevant modulator of the severity of SCD, inhibiting the polymerization of the mutant sickle hemoglobin (HbS). Despite its clinical relevance, a straightforward method to quantify and identify protective levels of HbF in individual red blood cells (RBCs) is still lacking. Here, we sought to develop a fluorescent-labeled RBC “Spike-In”(Spk) control (Ctr), to be mixed with patient samples, to serve as an internal reference in a novel quantitative flow-cytometry method. The Spk-Ctr was generated by mixing equal numbers of CFSE-labeled and fixed HbF Neg adult and HbF Pos cord blood (CB) RBCs, whose mean corpuscular HbF (MCHbF) mass was pre-calculated, based on the %HbF (HPLC-derived) and the MCH (hematology analyzer), using the formula: MCHbF=(%HbFxMCH)/100. Spk-Ctr was mixed with a similarly-fixed Test Sample, composed of 10% HbF Pos (prepared with the same RBCs as the Spk-Ctr), permeabilized, stained with an anti-HbF PE conjugate, and acquired by a flow cytometer. The anti-HbF PE Median Intensity Fluorescence (MIF) was obtained for gated HbF Pos RBCs in both the Ctr CFSE Pos and Test CFSE Neg gated populations. A conversion factor (CF) was calculated by dividing the pre-calculated MCHbF by the MIF obtained for HbF Pos RBCs from the CFSE Pos Spk-Ctr. By multiplying the anti-HbF PE fluorescence intensity of each RBC in the test sample by the CF, an absolute HbF/RBC values (in pg of HbF per RBC) can be obtained, which can be used to generate a distribution histogram to calculate the percentage of RBCs above a protective HbF/RBC threshold. The Ctr CB used had 73.2% HbF and a MCH of 36.4pg, resulting in a MCHbF of 26.64pg. The anti-HbF PE MIF of the Spk-Ctr HbF Pos RBCs was 25,166a.u. (arbitrary units), resulting in a CF of 0.001058. The anti-HbF PE MIF of the HbF Pos RBCs of the Test Sample was 24,146a.u., leading to an estimated MCHbF of 25.56pg; a deviation of only 4% from the expected (known) MCHbF. In contrast, when samples were permeabilized and stained in separate tubes, this variation was 27%. Recently, a work described a laborious method requiring a standard curve, generated with rare samples from subjects with hereditary persistence of HbF, and calibration beads. This method correct only for experimental variations arising from flow-cytometry acquisition and bath-to-bath antibody affinity/fluorescence differences, but not from experimental variability associated with sample handling of the test patient samples and those used in the standard curve. Our method eliminates the complexity associated with the use of a cumbersome standard curve, as the Spike-In Ctr and patient cells are subjected to the exact same procedures (in the same tube). Thus, experimental variability (derived from differences in, pipetting, incubation time, temperature, affinity/fluorescence of antibody batches, instrument or acquisition settings, etc.) is not expected to interfere with the quantitation. Once fully developed and validated, our method may serve as an auxiliary tool for the clinical management of SCD patients, contributing to the prediction of complications (allowing preventive measures to be adopted), or allowing the monitoring of the response to therapeutic approaches based on the induction of HbF (including hydroxyurea).

Thyroid hormone-dependent and independent processes of red blood cell transition from larval to adult type during metamorphosis in Xenopus laevis.

Amphibian metamorphosis results in drastic whole-body remodeling. Thyroid hormone (TH) drives most of these metamorphic changes. A prominent event during this remodeling is the red blood cell (RBC) transition from larval to adult forms, which exclusively contain larval and adult hemoglobin, respectively. However, the role of TH in RBC transition remains unclear. Here we reconfirmed that RBC transition of Xenopus laevis is completed much later than morphological metamorphosis. Further, larval and adult RBCs/erythroblasts proliferated both in the erythropoietic liver and in circulation during metamorphic climax. RBC transition was also confirmed in Rana ornativentris, but in contrast to X. laevis, adult RBC-specific proliferation was observed from the early climax stages. We also revealed in either species that RBC transition occurs in the liver prior to circulating RBCs. Moreover, anemia induction using phenylhydrazine during the prometamorphosis of X. laevis caused precocious RBC transition even when TH synthesis was blocked, resulting in metamorphosis-arrested larvae in which most of RBCs were of adult type. These results indicate that a decline in larval RBCs facilitates RBC transition during metamorphosis in a TH-independent manner. Further, combined administration of phenylhydrazine and TH induced precocious appearance of adult RBCs in early prometamorphic X. laevis tadpoles, whereas individual treatment with phenylhydrazine or TH did not cause precocious RBC transition; this suggests that TH is required to initiate RBC transition by promoting the differentiation of adult erythroblasts during early prometamorphosis in X. laevis. These results show that TH-dependent and independent processes are present in RBC transition in X. laevis.

Gene Editing for Inherited Red Blood Cell Diseases.

Today gene therapy is a real therapeutic option to address inherited hematological diseases that could be beneficial for thousands of patients worldwide. Currently, gene therapy is used to treat different monogenic hematological pathologies, including several red blood cell diseases such as β-thalassemia, sickle cell disease and pyruvate kinase deficiency. This approach is based on addition gene therapy, which consists of the correction of hematopoietic stem cells (HSCs) using lentiviral vectors, which integrate a corrected version of the altered gene. Lentivirally-corrected HSCs generate healthy cells that compensate for the deficiency caused by genetic mutations. Despite its successful results, this approach lacks both control of the integration of the transgene into the genome and endogenous regulation of the therapeutic gene, both of which are important aspects that might be a cause for concern. To overcome these limitations, gene editing is able to correct the altered gene through more precise and safer approaches. Cheap and easy-to-design gene editing tools, such as the CRISPR/Cas9 system, allow the specific correction of the altered gene without affecting the rest of the genome. Inherited erythroid diseases, such as thalassemia, sickle cell disease and Pyruvate Kinase Deficiency, have been the test bed for these gene editing strategies, and promising results are currently being seen. CRISPR/Cas9 system has been successfully used to manipulate globin regulation to re-activate fetal globin chains in adult red blood cells and to compensate for hemoglobin defects. Knock-in at the mutated locus to express the therapeutic gene under the endogenous gene regulatory region has also been accomplished successfully. Thanks to the lessons learned from previous lentiviral gene therapy research and trials, gene editing for red blood cell diseases is rapidly moving from its proof-of-concept to its first exciting results in the clinic. Indeed, patients suffering from β-thalassemia and sickle cell disease have already been successfully treated with gene editing, which will hopefully inspire the use of gene editing to cure erythroid disorders and many other inherited diseases in the near future.

Identification of novel γ-globin inducers among all potential erythroid druggable targets

AbstractHuman γ-globin is predominantly expressed in fetal liver erythroid cells during gestation from 2 nearly identical genes, HBG1 and HBG2, that are both perinatally silenced. Reactivation of these fetal genes in adult red blood cells can ameliorate many symptoms associated with the inherited β-globinopathies, sickle cell disease, and Cooley anemia. Although promising genetic strategies to reactivate the γ-globin genes to treat these diseases have been explored, there are significant barriers to their effective implementation worldwide; alternatively, pharmacological induction of γ-globin synthesis could readily reach the majority of affected individuals. In this study, we generated a CRISPR knockout library that targeted all erythroid genes for which prospective or actual therapeutic compounds already exist. By probing this library for genes that repress fetal hemoglobin (HbF), we identified several novel, potentially druggable, γ-globin repressors, including VHL and PTEN. We demonstrate that deletion of VHL induces HbF through activation of the HIF1α pathway and that deletion of PTEN induces HbF through AKT pathway stimulation. Finally, we show that small-molecule inhibitors of PTEN and EZH induce HbF in both healthy and β-thalassemic human primary erythroid cells.

Quantitation of residual D positive red cells in D negative recipients of D positive solid organ transplants

AbstractBackgroundSolid organ transplants (SOT) from D positive donors are potentially sensitising events for D negative recipients. For this reason, it is important to quantify the presence of residual D positive red blood cells (RBCs) in the recipient's circulation and calculate the correct dose of prophylactic anti‐D (PAD) required to prevent sensitisation. This is especially important in females of child‐bearing potential where the presence of allo anti‐D can, at worst, cause the death of the fetus in future pregnancies.ObjectiveThis study aimed to identify the patient characteristics of D positive SOT cases referred to Red Cell Immunohaematology, NHSBT for flow cytometry investigation. This information could indicate improvements required in the current testing methodology, as well as to the calculations used to prescribe PAD for this patient group.MethodsSamples were investigated using a Beckman Coulter Navios Flow Cytometer using BRAD3–FITC (anti‐D), AEVZ5.3‐FITC (isotype matched negative control) and BIRMA17C‐PE (granulocyte exclusion reagent). Mollison's calculation was used to estimate the dose of PAD required to prevent sensitisation in the D negative recipients. The calculation was adapted to consider the presence of organ donor D positive adult RBCs in the circulation of recipients instead of, larger, fetal RBCs.ResultsSamples from 20 patients, all female, aged 14–53 years (one 2‐year‐old outlier) were referred from 2016 to September 2020. The transplants were—liver (n = 6), kidney (n = 6) and lung (n = 8). D positive cell populations were identified in 11 cases (0.1–8.0 ml); and required PAD (500–1500 IU). From these 20 patients, 10 sent a follow‐up sample, where 8 required PAD top‐up due to the detection of residual D positive cells (0.1–2 ml)—liver (n = 1), kidney (n = 1) and lung transplant (n = 6).ConclusionAll patients in the study were D negative females, in which 18 were considered by guidelines to be of childbearing potential (2–42 years old) and 2 were &gt;50 years old. Referrals demonstrate an awareness for the correct calculation of PAD to prevent D sensitisation. The sample size is small, but top up requirement in 8/20 of cases demonstrates accurate quantification is clearly needed to ensure the appropriate dose of PAD is provided.