Expansion of Red Blood Cells for Transfusion

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. SCI-46-SCI-46
Author(s):  
Anna Rita F Migliaccio ◽  
Carolyn Whitsett ◽  
Giovanni Migliaccio
Keyword(s):  
Stem Cells ◽  
Blood Transfusion ◽  
Blood Supply ◽  
Ex Vivo ◽  
Red Cells ◽  
Blood Group Antigens ◽  
Red Cell ◽  
Erythroid Cells ◽  
Red Cell Transfusion ◽  
Safety Considerations

Abstract Abstract SCI-46 Blood transfusion, the earliest form of cell replacement therapy, has become indispensable for modern medicine making the safety and adequacy of the blood supply a national priority. The US blood supply is adequate overall because in 2006 the number of blood units collected exceed by 7.8% the number of those transfused. However, issues surrounding blood transfusion, such as sporadic shortages and potential adverse events to recipients (related to changes in red cell physiology during storage and alloimmunization in chronically transfused patients) prompted past and current efforts to develop alternative transfusion products. Recently, the culture conditions to generate erythroid cells have greatly improved making the production of a transfusion product ex-vivo a theoretically possible, although expensive, proposition. This recognition is inspiring several investigators to develop production processes for ex-vivo generation of red cell transfusion products. A proof-of-concept demonstrating that ex-vivo generated red cells protect mice from experimentally induced lethal anemia has been obtained. Alternative sources of stem cells which include human embryonic stem cells (hESC) and induced pluripotency stem cells (iPS), are being explored. Since red cells do not have a nucleus, safety considerations suggest that they may represent the first cell therapy product to be generated from hESC and iPS. In addition, discarded hematopoietic stem cells present in adult and cord blood donations may theoretically generate numbers of red cells ex-vivo sufficient for transfusion. Affordable clinical grade humanized culture media have also been developed. Possible differences in immunological and biological properties of erythroid cells from different sources are under investigation. These differences include size, levels of activity of glycolytic enzymes and carbonic anhydrase, expression of different isozymes, hemoglobin and antigenic profiles (HLA class II antigens). This last aspect is particularly important because ex-vivo expanded red cells pose the same risk for infection and incompatibility as any transfusion product but pose unique antigenic risks. Since expression of blood group antigens is susceptible to post-transcriptional modifications, the ex-vivo expansion process itself may induce antigenic variability. Therefore, even cells generated from completely matched stem cell sources may induce auto-immunity and/or appear incompatible. Regarding the identity of ex-vivo generated red cell transfusion products, a conservative approach would be to define them as “enucleated red cells”. In principle, however, ex-vivo generated erythroblasts may also serve as transfusion product. Since they undergo 4–64 further divisions and reduce iron overload, they may represent a more potent transfusion product for patients that require chronic transfusion. The clinical use of these cells, however, may involve development of specific procedures to facilitate their homing/maturation in the erythroid niches of the recipients. In summary, on the basis of these cost, logistic and safety considerations we hypothesize that the clinical application of ex-vivo expanded erythroblasts will involve in sequence, drug discovery for personalized therapy, systemic drug delivery, genotypically matched transfusion for alloimmunized patients and then transfusion in the general population. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Future of Red Cell Transfusion Lies in Cultured Red Cells

2021 ◽  
Author(s):  
Rizwan Javed ◽  
Lorraine Flores ◽  
Saurabh Jayant Bhave ◽  
Asheer Jawed ◽  
Deepak Kumar Mishra
Keyword(s):  
Stem Cells ◽  
Large Scale ◽  
Ex Vivo ◽  
Blood Groups ◽  
Red Cells ◽  
Controlled Clinical Trial ◽  
Scale Production ◽  
Red Cell ◽  
Potential Cost ◽  
Hematopoietic Stem

AbstractBlood is a very important resource for healthcare-based services and there has been a consistently increasing demand for it in most parts of the world. Poor volunteer-based collection system, high-risk of transfusion-transmitted infections, and emergence of new pathogens as evident from the ongoing Coronavirus Disease 2019 (COVID-19) pandemic are potential challenges to the global healthcare systems. It is imperative to explore safe and reliable alternatives to red cell transfusions. Ex vivo culture of red cells (cRBCs) from different sources such as hematopoietic stem cells (HSCs), pluripotent stem cells, and immortalized progenitors (e.g., BELA-2 cells) could revolutionize transfusion medicine. cRBC could be of great diagnostic and therapeutic utility. It may provide a backup in times of acute shortages in patients with rare blood groups, and in cases with multiple antibodies or sickle cell anemia. The CRISP-Cas9 system has been used to develop personalized, multi-compatible RBCs for diagnostic reagents and patients with multiple allo-antibodies. cRBC could be practically feasible for pediatric patients, who require small quantities of red cell transfusions. cRBC produced under good manufacturing practice (GMP) conditions has been reported to survive in human blood circulation for more than 26 days. Recently, a phase I randomized controlled clinical trial called RESTORE was initiated to assess the survival and recovery of cRBCs. However, feasible technological advancement is required to produce enough cRBCs for clinical use. It is crucial to identify sustainable sources for large-scale production of clinically useful cRBCs. Although the potential cost of one unit of cRBC is extrapolated to be around US$ 8000, it is a life-saving product for patients having rare blood groups and is a “ready to use” source of phenotype-matched, homogenous young red cells in emergency situations.

Blood Transfusion Increases Hospital Acquired Septicaemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3346-3346
Author(s):  
Stephen Kenneth O'Mara ◽  
John Ferguson ◽  
Michael Manolis
Keyword(s):  
Blood Transfusion ◽  
Odds Ratio ◽  
System Analysis ◽  
Conflicts Of Interest ◽  
Red Cells ◽  
University Teaching ◽  
Red Cell ◽  
Hospitalised Patients ◽  
Red Cell Transfusion ◽  
Hospital Acquired

Abstract Abstract 3346 The rate of septicaemia was measured following blood transfusion in patients admitted to hospital greater than 48 hours. The data was collected prospectively and analysed retrospectively. The aim of the analysis was to disprove that red cell transfusion increased the rate of hospital acquired septicaemia. Data was extracted on patients who had a transfusion and an episode of septicaemia between 1999 and 2008 at a university teaching hospital, John Hunter Hospital, Newcastle, Australia (JHH). The database contained information on 20161 transfusion events of 102,600 units of packed red cells. The septicaemia database contained 8375 patients with blood culture proven septicaemia. Blood was issued using a computerised cross matching system. Analysis of the issuing of blood found no bias in the age of blood issued based on age, sex, number of units or by year of issue. All patients having a septicaemic event recorded following transfusion were analysed and compared to septicaemia occurring in all admissions greater than 48hrs in which no transfusion occurred. All patients who were diagnosed with septicaemia in the 6 month prior to transfusion were excluded as were patients who received five or more units of packed red cells to exclude the bias from the sickest patients. All patients whose septicaemia occurred greater than 16 days after transfusion were excluded as were patients whose septicaemia occurred before the second day after transfusion. A total of 258 patients were reviewed to test the hypothesis. Table1. There was a statistically significant increase in septicaemia following red cell transfusion. The null hypothesis was rejected. The data base was examined by the age of red cells transfused and its effect on nosocomial septicaemia. There was a statistically significant effect of older blood on the rate of septicaemia. Packed red cells transfused less than 14 days of age had no effect on the septicaemia rate. Blood that was 14–28 days of age increased the rate of nosocomial septicaemia by 1.65. Red Cells that were between 29 and 35 days of age increased the rate of septicaemia by 2.5 times. Blood that was between 36 and 42 days of age increased the rate by approximately 4.4 times, with the absolute risk of developing septicaemia within 15 days being approximately 4%. Table2. The time course of septicaemia was examined for patients receiving at least one unit of 28 day old blood. It was found that the risk of sepsis lasted less than 15 days p<0.001. The conclusion of our analysis suggests that packed red cells older than 14 days increase the risk of septicaemia in hospitalised patients, this effect continues to rise until 42 days post collection. Table 1 N Odds Ratio (CI) Total cases of nosocomial septicaemia 1684     • Admissions greater than 48 hours 175,325 Prior to exclusions     • All transfusions     • No previous sepsis 790 10.2 (10.1–10.3) Study Group     • Less than 5 units Transfusion.     • No previous sepsis.     • Septicaemia >2 days <16days 258 2.02 (1.92–2.12) Table 2 Age of oldest red cell pack Less than 15 days of age 15 to 28 days of age 29–35 days of age 36–42 days of age Percentage transfused 24% 49% 14% 12% Number of transfusions < 5 units 3524 7161 2115 1802 Septicaemia observed 33 102 52 71 Odds ratio 0.98 1.54 2.55 4.4 Confidence Interval. 0.65–1.31 1.34–1.74 2.26–2.84 4.16–4.64 Disclosures: No relevant conflicts of interest to declare.

scholarly journals Ex vivo Engineering of Red Blood Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-5-SCI-5
Author(s):  
Jan Frayne ◽  
Deborah E Daniels ◽  
Jan Frayne ◽  
Timothy J Satchwell ◽  
Joseph Hawksworth ◽  
...  
Keyword(s):  
Blood Group ◽  
Gene Editing ◽  
Ex Vivo ◽  
Red Cells ◽  
Erythroid Cell ◽  
Blood Group Antigens ◽  
Erythroid Cells ◽  
Culture Systems ◽  
Cd34 Cells ◽  
Cell Source

Culture systems for human in vitro erythropoiesis are now well established. Using our 3-stage feeder-free erythroid culture system we can efficiently differentiate erythroid cells from adult and cord blood (CB) CD34+ cells with >105 fold expansion, enucleation rates of up to 95% and producing packed reticulocyte yields of >12ml post leukofiltration1. The final preparations for a first in man clinical trial of adult cultured reticulocytes produced under good manufacturing practice (RESTORE) are underway. Although we have shown that it is possible to modify the CD34+ derived cells using lentivirus for reengineering or additions to the medium, the finite proliferative capacity of CD34+ cells in culture currently limits yield. We therefore took the alternative approach of immortalising early erythroid cells differentiated from adult bone marrow (BM) CD34+ cells, creating the BEL-A (Bristol Erythroid Line Adult) line2, a sustainable erythroid cell source that recapitulates normal adult erythropoiesis, terminally differentiating to generate enucleated reticulocytes that express normal levels of adult globin. We have created a further 13 lines from BM, adult peripheral blood, CB and iPSC CD34+ cells, demonstrating reproducibility of the approach and its application to create lines from more accessible stem cell sources. Analysis of surface marker and globin profiles demonstrate the lines follow a similar differentiation profile to their respective primary cell source, with comparative proteomics confirming cell source representation of the lines. Lines always established at the pro-erythroblast/early basophilic stage, even when later stage erythroid cells were present in populations. As well as proof of principal as an alternative transfusion product and improved tools for studying erythropoiesis, such lines have far reaching additional applications, a number of which we are now exploring: Diagnostic and 'Universal' transfusion products: Presently serological testing by blood group reference laboratories relies on donated blood, which represent a finite resource and for some blood group phenotypes can be difficult to source. We used CRISPR-Cas9 gene editing to remove blood group antigens in order to generate a sustainable bank of cell lines with useful blood group phenotypes for diagnostic purposes3. Building on this, with the aim of developing a more compatible "universal" transfusion product to meet the needs of chronically transfused patients and those with rare blood group phenotypes, we used combinatorial gene targeting to create sublines deficient in multiple antigens responsible for the most common transfusion incompatibilities (ABO [Bombay], Rh [Rhnull], Kell [K0], Duffy [Fynull], GPB [S-s-U-]). Individual and multiple blood group knockout lines retained the ability to undergo terminal differentiation and enucleation, also illustrating the capacity for coexistence of multiple rare blood group phenotypes within viable reticulocytes3. Cytokine independent lines Cytokines represent a substantial cost contribution to erythroid culture systems. We therefore exploited activating mutations found in patient c-Kit and EPOR that cause hypersensitivity to ligand, to create cytokine independent lines thus increasing economic viability of cultured red cells. Bi-allelic EPOR or c-kit edits were introduced into BEL-A with confirmation and exploration of mechanism on differentiation in the absence, or with substantially reduced levels of cytokines. Model disease systems In addition to potential therapeutic applications we are also creating lines as model cellular disease systems for studying molecular mechanisms and as drug screening platforms, via CRISPR-Cas9 gene editing of BEL-A and by directly immortalising patient stem cells. To date we have made b-thalassemia major, HbE thalassemia and lines with KLF1 mutations. Furthermore, we have shown BEL-A reticulocytes support invasion and growth of Plasmodium falciparum and are utilising the line to study mechanisms of malaria parasite invasion4. Kupzig S, Parsons SF, Curnow E, Anstee DJ, Blair A. Superior survival of ex vivo cultured human reticulocytes following transfusion into mice. 2016;102:476-483Trakarnsanga K, Griffiths RE, Wilson MC, et al. An immortalized adult human erythroid line facilitates sustainable and scalable generation of functional red cells. Nat. Commun. 2017;8:14750Hawksworth J, Satchwell TJ, Meinders M, et al. Enhancement of red blood cell transfusion compatibility using CRISPR-mediated erythroblast gene editing. EMBO Mol. Med. 2018; 10:e8454Satchwell TJ, Wright K, Haydn-Smith K, et al. Genetic manipulation of cell line derived reticulocytes enables dissection of host malaria invasion requirements. Nat. Comm. 2019;10:3806 Disclosures No relevant conflicts of interest to declare.

scholarly journals Reduction in massive postpartum haemorrhage and red blood cell transfusion during a national quality improvement project, Obstetric Bleeding Strategy for Wales, OBS Cymru: an observational study

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sarah F. Bell ◽  
Rachel E. Collis ◽  
Philip Pallmann ◽  
Christopher Bailey ◽  
Kathryn James ◽  
...  
Keyword(s):  
Risk Assessment ◽  
Quality Improvement ◽  
Blood Loss ◽  
Postpartum Haemorrhage ◽  
Red Cells ◽  
National Database ◽  
Care Bundle ◽  
Red Cell ◽  
National Quality ◽  
Red Cell Transfusion

Abstract Background Postpartum haemorrhage (PPH) is a major cause of maternal morbidity and mortality and its incidence is increasing in many countries despite management guidelines. A national quality improvement programme called the Obstetric Bleeding Strategy for Wales (OBS Cymru) was introduced in all obstetric units in Wales. The aim was to reduce moderate PPH (1000 mL) progressing to massive PPH (> 2500 mL) and the need for red cell transfusion. Methods A PPH care bundle was introduced into all 12 obstetric units in Wales included all women giving birth in 2017 and 2018 (n = 61,094). The care bundle prompted: universal risk assessment, quantitative measurement of blood loss after all deliveries (as opposed to visual estimation), structured escalation to senior clinicians and point-of-care viscoelastometric-guided early fibrinogen replacement. Data were submitted by each obstetric unit to a national database. Outcome measures were incidence of massive PPH (> 2500 mL) and red cell transfusion. Analysis was performed using linear regression of the all Wales monthly data. Results Uptake of the intervention was good: quantitative blood loss measurement and risk assessment increased to 98.1 and 64.5% of all PPH > 1000 mL, whilst ROTEM use for PPH > 1500 mL increased to 68.2%. Massive PPH decreased by 1.10 (95% CI 0.28 to 1.92) per 1000 maternities per year (P = 0.011). Fewer women progressed from moderate to massive PPH in the last 6 months, 74/1490 (5.0%), than in the first 6 months, 97/1386 (7.0%), (P = 0.021). Units of red cells transfused decreased by 7.4 (95% CI 1.6 to 13.2) per 1000 maternities per year (P = 0.015). Red cells were transfused to 350/15204 (2.3%) and 268/15150 (1.8%) (P = 0.001) in the first and last 6 months, respectively. There was no increase in the number of women with lowest haemoglobin below 80 g/L during this time period. Infusions of fresh frozen plasma fell and there was no increase in the number of women with haemostatic impairment. Conclusions The OBS Cymru care bundle was feasible to implement and associated with progressive, clinically significant improvements in outcomes for PPH across Wales. It is applicable across obstetric units of widely varying size, complexity and staff mixes.

Acetylcholinesterase in Human Erythroid Cells

1973 ◽  
Vol 12 (3) ◽  
pp. 911-923
Author(s):  
R. J. SKAER
Keyword(s):  
Endoplasmic Reticulum ◽  
Nervous System ◽  
Golgi Apparatus ◽  
Red Cells ◽  
The Other ◽  
Red Cell ◽  
Erythroid Cells ◽  
Cell Replacement ◽  
Kinetics Of ◽  
Human Red Cells

Acetylcholinesterase is present in human red cells but cannot be demonstrated by the copper thiocholine test. The enzyme is revealed, however, in the perinuclear cisterna, endoplasmic reticulum and Golgi apparatus of red cell precursors. It is suggested that 2 forms of the enzyme are present, one of which can be demonstrated by the copper thiocholine test, the other cannot; one form may be the precursor of the other. These observations may cast light on the kinetics of red cell replacement and on the interpretation of the results from the copper thiocholine test on other tissues such as the nervous system.

Developing a National Registry for Hemochromatosis

2016 ◽  
pp. 154-164
Author(s):  
Indu Singh ◽  
Janelle Guerrero ◽  
Michael J. Simmonds
Keyword(s):  
Blood Transfusion ◽  
Hereditary Hemochromatosis ◽  
National Registry ◽  
Red Cross ◽  
Blood Collection ◽  
Healthy Population ◽  
Red Cell ◽  
Blood Products ◽  
Patient Health ◽  
Red Cell Transfusion

Hereditary Hemochromatosis (HH) is a disorder where iron and ferritin concentrations in a patient's blood are much higher than normal healthy levels. The main therapeutic intervention for individuals with HH is removing 300-500 mL of blood every few months to maintain ferritin concentration within acceptable ranges. The blood collected during these venesections is usually discarded as there is a belief that blood with high levels of ferritin are not suitable for blood transfusion purposes. Australian Red Cross Blood Services voluntarily collects blood from donors for subsequent use in blood transfusion. Annually more than 700 thousand units are transfused within Australia and there is a constant need for new donors given the significant imbalance between supply and demand of blood products. Besides red cell transfusions, the Red Cross also issues donor blood for development of many other blood products essential for patient health care. The HH blood can currently be used for other blood products if not for red cell transfusion. However, there is evidence to suggest that there is no significant difference between the red cells of the normal healthy population compared to those from HH patients. Australian Red Cross has developed a mobile computer application (High Ferritin “app”) as they have started collecting blood from HH patients. Though there is little or no awareness about the existence and use of this High Ferritin app in general HH population, their doctors and nurses collecting their blood for therapeutic purposes. This chapter describes possibility of saving and utilizing the blood collected from hemochromatosis patients for therapeutic purposes. A national hemochromatosis patients registry, in collaboration with High Ferritin app (HFa) developed by Australian Red Cross Blood Services, accessible to the patients, their doctors and Red Cross Blood Collection Sservices 24 hours a day anywhere in the country can allow the patients to donate the blood collected for therapeutic purposes at any affiliated blood collection center in the country after they automatically get a message either by email or text message after their blood results have been reviewed by their doctor and they are required to go for venesection.

scholarly journals The Effect of Anemic Anoxia on the Cellular Development of Nucleated Red Cells

Blood ◽  
1959 ◽  
Vol 14 (4) ◽  
pp. 386-398 ◽  
Author(s):  
ALLAN J. ERSLEV ◽  
Elva Ruiz
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Nitrogen Mustard ◽  
Mitotic Division ◽  
Red Cells ◽  
Red Cell ◽  
Cell Production ◽  
Cellular Development

Abstract The action of an anoxic stimulus on red cell production was studied in rabbits bled 20 ml./Kg., kept anemic for 20 hours and then reinfused with the previously removed blood. This 20-hour period of anemic anoxia was followed by a characteristic reticulocyte response, a response which was modified by nitrogen mustard or colchicine administered immediately after the 20-hour period of anemia, but was not influenced by anoxia or hyperoxia in the postanemic period. When mitotic division was arrested by colchicine during the 20-hour period of anemic anoxia, the onset of the reticulocyte response, though delayed by 1 to 2 days, was otherwise of characteristic magnitude. These observations indicate that (1) the anoxic stimulus operates in the bone marrow by accelerating the differentiation of stem cells into pronormoblasts, and that thereafter (2) the maturation and multiplication of differentiated nucleated red cells proceed at fixed rates independent of the anoxic stimulus.

Comparative Blood Group Profiling of Human Erythroid Cells (EBs) Generated from Adult Blood (AB), Cord Blood (CB), Human Embryonic Stem Cells (hESC) and Induced Pluripotent Stem Cells (iPS)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1027-1027 ◽  
Author(s):  
Barbara Ghinassi ◽  
Maria Themeli ◽  
Kai-Hsin Chang ◽  
Gregory Halverson ◽  
Ghazala Hashmi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Blood Group ◽  
Ex Vivo ◽  
Antigen Expression ◽  
Band 3 ◽  
Blood Group Antigens ◽  
Dna Genotyping

Abstract Abstract 1027 Red blood cells (RBC) survive shear forces in the microvasculature because trans-membrane complexes embedded in the lipid bilayer attach their membrane to the cytoskeleton assuring its flexibility. The expression of clinically relevant red blood cell antigens present on these complexes is determined by genetic polymorphisms and their developmental regulation. Therefore, flow cytometry studies of blood group antigens may provide insights both on potential immunogenicity and on membrane structure of ex-vivo generated EBs. Blood group antigen profiles of EBs expanded ex vivo from one AB (three experiments), three CB, the H1 hESC line and one iPS line derived from mononuclear cells from a healthy donor were compared by flow cytometry using commercially available antibodies recognizing antigens present on proteins in the 4.1R [Duffy (Fya and Fy3), Kell (Kell prot, K/k, Kpa/Kpb, Jsb) and glycophorin C (GPC, Ge2)] and ankyrin R [glycophorin A (GPA, CD235a, M and EnaFS) RhAG and band 3 (Wrb)] complexes and on other important membrane proteins [glycophorin B (GPB, s and U), urea transporter (Kidd, Jk3), the complement receptor (CD35) and inhibitors of complement-mediated lysis (CD55 and CD59)]. Controls included DNA genotyping (CB, AB and H1-hESC) (HEA-Bead Chip, Immunocor, Norcross, GA) and immunophenotyping of blood red cells from the same AB and CB. Antigen expression similar to that observed on in vivo generated RBC was considered normal. EBs were generated from AB and CB at day 10 in HEMAser cultures whereas EBs from hESC and iPS were derived using previously optimized protocols. The maturation state was determined by morphological analyses and CD36/CD235a profiles. Irrespective of the stem cell source, the immunophenotype of ex-vivo expanded EBs was consistent with that predicted by genotyping. However, source specific differences in the magnitude of antigen expression and in the changes with maturation were observed (see Figure). Immature EBs from AB expressed normal levels of the antigens present on both the 4.1R (Duffy, Kell, GPC) and ankyrin R (GPA, M/N, EnaFS, RhAG and band 3) complexes. With maturation, expression of 4.1R-associated antigens remained normal while that of ankyrin R associated antigens varied (M decreased and RhAG increased). EBs from CB expressed normal levels of antigens present on the ankyrin R complex and of some of those present on the 4.1R complex (Duffy, Kell protein and GPA). However, expression of epitopes on Kell protein varied with some antigens expressed at normal levels (k and Jsb) and others (Kpa/Kpb) at levels 2x greater than normal. With maturation, CB-derived EBs maintained normal levels of ankyrin R associated antigens while those associated with complex 4.1R became barely detectable. EB from hESC expressed unbalanced levels of proteins associated with both ankyrin R (2x levels of GPA and barely detectable levels of RhAG) and 4.1R [3x levels of Duffy and 2x levels of Jsb (Kell) with normal levels of k and Kpb (Kell) antigens] complexes. The variegation in expression of different epitopes on the same protein observed with CB- and hESC-derived EBs likely reflect altered structural conformation of the complexes rather than differences in protein concentration on the membrane. EBs from iPS, as those from AB, expressed normal levels of antigens present on Ankyrin R and 4.1R complexes which increased with maturation. Irrespective of stem cell sources, EBs expressed normal levels of GPB and Kidd. EBs from AB expressed normal levels of the complement regulatory proteins tested which in the case of CD59 CD59 decreased with maturation. EBs from CB expressed normal levels of CD35 and CD59 but 2x levels of CD55 with expression of CD35 and CD55 decreasing with maturation. EBs from iPS expressed 2x levels of CD35 and CD55 and expression of these antigens was not affected by maturation. The observation that blood group antigenic profiles of ex-vivo generated EBs are consistent with those predicted by DNA-genotyping suggests that these cells are unlikely to be immunogenic for known epitopes. However, the antigen profiles of ankyrin R and 4.1R complexes were normal only for AB and iPS-derived EBs raising the possibility that antigenic deviations seen in EBs derived from CB and hESC may have immunologic or functional consequences in vivo. Disclosures: No relevant conflicts of interest to declare.

Efficient Erythropoiesis From Human Embryonic Stem Cells Through Dimerization of Intracellular MPL.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2291-2291
Author(s):  
William Sang Kim ◽  
Gautam G. Dravid ◽  
Yuhua Zhu ◽  
Chintan Parekh ◽  
Qiming Deng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Total Yield ◽  
Embryonic Stem ◽  
Erythroid Differentiation ◽  
Akt Signaling ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Enucleated Rbc ◽  
Negative Controls

Abstract Abstract 2291 Objectives: Unlimited self renewal capacity and the ability to differentiate into any cell type make human pluripotent stem cells (PSC) a potential source for the ex vivo manufacture of red blood cells (RBC) for safe transfusion. Current methods of RBC differentiation from PSC suffer from low yields of RBCs, most of which contain embryonic rather than adult or fetal hemoglobins. Therefore, efficient clinical translation of this strategy is critically dependent on the development of novel methods to enhance the generation of functional RBCs from PSC. We have previously shown that dimerization of the intracellular component of MPL (the thrombopoietin receptor), induces expansion of myelo-erythroid progenitors (MEP) from human cord blood as well as their terminal differentiation into enucleated RBC through unique, EPO-independent mechanisms (Parekh et al, 2012). Our goal was to investigate the potential of intracellular MPL dimerization to induce erythropoiesis from human PSC and to identify the signaling pathways activated by this strategy. Methods: Human embryonic stem cell (hESC) lines H1 and HES3 were transduced with a lentiviral vector to express the fusion protein F36V-MPL (containing the ligand binding domain F36V and the intracytoplasmic portion of MPL). Dimerization of F36V-MPL was accomplished by addition of the synthetic ligand AP20187 (aka CID) during culture (with or without erythropoietin) on OP9 stroma in the absence of other cytokines. F36V-MPL transduced-hESC that did not receive CID and F36V-transduced hESC cultured with CID served as negative controls. Flow cytometry and Colony Forming Unit (CFU) assays were used to analyze erythroid differentiation. Phosflow and Western Blot were used to analyze cell signaling. MEP generated during hESC differentiation were defined as cells co-expressing GlyA and CD41a/CD42a. Results: F36V-MPL dimerization induced significantly more Glycophorin A+ cells (P=0.0001; n=5) and 10-fold higher number of erythroid CFU (P=0.0007; n=15) as compared to negative controls. The effect was consistent across different hESC cell lines. The increased yield of erythroid cells was not due to an overall increase in cell proliferation as the total yield of cells was not statistically different between treated and untreated cultures. This effect was seen in the absence of any hematopoietic cytokines, including erythropoietin (EPO), a critical cytokine for erythropoiesis and an integral component of all ex vivo PSC erythroid differentiation protocols, indicating that MPL dimerization alone is sufficient to induce erythropoiesis from hESCs. Erythroid output was further enhanced in an additive manner in the presence of EPO (P=0.0058; n=5). In order to identify the point at which MPL dimerization affects erythropoiesis, CID was added during differentiation directly from hESC or to isolated MEP generated from hESC. CID and EPO increased the number of MEP compared to untreated controls, demonstrating that MPL dimerization induces the generation of early erythroid progenitors. In addition, CID drove erythroid differentiation from MEP more efficiently than EPO, demonstrated by a significantly higher frequency of total erythroid cells (P=0.02; n=3), and 4-fold increase in yield of enucleated RBC. This indicates that CID has a greater effect on terminal erythroid differentiation than EPO. We then investigated the signaling mechanism activated by F36V-MPL dimerization and found that, unlike the full-length MPL receptor, which activates both STAT5/JAK2 and AKT pathways, F36V-MPL dimerization activated AKT but not STAT5 or JAK2 phosphorylation. PI3K/AKT inhibitors (LY294002 and AKT inhibitor IV) effectively inhibited erythroid differentiation of transduced hESC cultured in the presence of CID (P=0.0442; n=2) indicating that MPL dimerization induced erythropoiesis is dependent on AKT signaling. Conclusion: F36V-MPL dimerization during hESC-derived hematopoiesis induces EPO-independent erythroid differentiation through AKT signaling, by both generating erythroid progenitors and promoting maturation of RBC. MPL dimerization also is more potent than EPO in inducing erythropoiesis from hESC and has an additive effect when combined with EPO, making this a potential strategy for the generation of therapeutically relevant levels of functional enucleated RBCs from PSC. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Packed red cell blood transfusion practices review in medical oncology unit in a tertiary cancer center, South India

2019 ◽  
Vol 7 (12) ◽  
pp. 4433
Author(s):  
Veerendra Angadi ◽  
Manjunath Nandennavar ◽  
Shashidhar V. Karpurmath ◽  
Roshan Jacob ◽  
Yamini Donekal
Keyword(s):  
Blood Transfusion ◽  
Cancer Patients ◽  
Intravenous Iron ◽  
Red Blood Cell Transfusion ◽  
Cancer Center ◽  
Red Cell ◽  
Retrospective Case ◽  
Case Record ◽  
Red Cell Transfusion ◽  
Oncology Unit

Background: Anaemia is a very common complication in cancer patients. Up to 60% of solid tumor patients and 70-90% of patients receiving myelosuppressive chemotherapy have anaemia. Pathophysiology of anaemia in cancer patients is multifactorial. The treatments for cancer related anaemia include Erythropoietin Stimulating Agents (ESAs), iron supplementing therapies (intravenous iron, oral iron) and blood transfusion. There are various safety concerns regarding usage of ESAs; also, their usage is less in India due to cost factor. There is scant literature regarding blood transfusion practices in patients undergoing chemotherapy.Methods: Patients diagnosed with cancer and patients receiving chemotherapy were included in the study. Retrospective case record review of cancer patients who received chemotherapy between January to March 2019 was done. Type of malignancy, presence of symptoms related to anemia and trigger for packed red cell transfusion were recorded.Results: Among 342 patients received total of 1365 cycles of chemotherapy in this time period. Mean age of patients was 46 years. 46 of the 342 patients received blood transfusion. Only 13% of the patients had symptoms of anemia like weakness and fatigue the average hemoglobin level at which transfusion was given was 6 gm/dL.Conclusions: Packed Red blood cell transfusion was usually administered at Hb <7 gm/dL. Very few patients reported anaemia related symptoms prior to transfusion. No patient received erythropoietin. Further data is needed from other tertiary cancer centres to understand the blood transfusion practices in Indian cancer patients undergoing chemotherapy.

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