Senescent Red Blood Cells Research Articles

Significance of the age of transfused blood for prognosis after transcatheter aortic valve implantation.

INTRODUCTION Blood transfusion after transcatheter aortic valve implantation (TAVI) is frequently required owing to the high vulnerability of this patient group and procedure-related bleeding. OBJECTIVES We assessed the impact of postprocedural blood transfusion and the age of transfused red blood cell (RBC) units on prognosis after TAVI. PATIENTS AND METHODS This was a single-center, observational analysis conducted between the years 2009 and 2014. The adopted endpoints were early and long-term mortality after TAVI. The risk factors for mortality included in-hospital bleeding and vascular complications, the number of transfused RBC units, transfusion of at least 2 RBC units, the age of transfused RBCs, and standard deviation of the age of RBCs. RESULTS The study included 178 patients (mean [SD] age, 80.07 [7.47] years; range, 55-91 years). The follow-up ranged between 1 month and 5.8 years (mean [SD], 20.1 [15.2] months) after discharge; 14 early deaths (7.8%) and 27 late deaths (16.5%) were noted. In-hospital bleeding and vascular complications increased the risk of early deaths (hazard ratio [HR], 2.113; 95% CI, 1.011-4.418; P = 0.046 and HR, 2.265; 95% CI, 1.270-4.039; P = 0.005). Transfusion of younger RBCs (HR, 1.044; 95% CI, 1.004-1.085; P = 0.028) and a greater discrepancy in the age of transfused RBCs (HR, 1.153; 95% CI, 1.042-1.275; P = 0.006) were positively correlated with the risk of late deaths only in a univariate analysis. A higher number of transfused RBC units was the only independent predictor of long-term mortality (HR, 1.149; 95% CI, 1.024-1.291; P = 0.018). CONCLUSIONS The higher number of RBC units transfused early after TAVI worsens long-term prognosis. Shorter-storage RBCs and a greater discrepancy in RBC age in multitransfused elderly patients after TAVI might have a deleterious effect on life expectancy.

Abstract 266: Extracellular Hemoglobin From Stored Rbcs Binds Ago2 And Delivers Functional Microrna to Endothelial Cells

Red blood cell (RBC) transfusion is among the most common clinical therapies. Prior to transfusion, packed RBCs (PRBCs) may be stored for up to 42 days. During storage, RBCs undergo many changes, including hemolysis and the release of extracellular hemoglobin (Hb). Prior work has shown that endothelial cells exposed to free hemoglobin demonstrate upregulation of inflammatory and anti-inflammatory pathways including elevated ICAM-1, VCAM-1 and E selectin, as well as elevated Hemeoxygenase-1 and bilirubin levels. Our work tests the hypothesis that extracellular Hb from stored RBCs can transfer functional microRNA to endothelial cells. Stored RBCs (age 30-42 days) were obtained from the Emory University Hospital Blood Bank. Extracellular Hb was obtained from lysed RBCs. Binding of Argonaute 2 (Ago2) to Hb was assessed by immunoprecipitation (IP) of Hb-alpha followed by Western blot for Ago2. In addition, microRNA was extracted from the Hb immunoprecipitate. Levels of miR-451, -16, -584, and -92 were assessed by RT-qPCR analysis. Lastly, human aortic endothelial cells (HAECs) were treated with Hb from RBC lysates. After treatment, Qiazol was used to extract RNA from washed cells, and levels of miR-451 and target genes were measured by RT-qPCR and Western analysis. We found that Ago2, a crucial component of the RISC complex that executes the regulatory function of microRNA, co-immunopreciptated with Hb from RBC lysates. We also observed that miR-451, a microRNA known to be abundant in RBCs, was highly abundant in Hb immunoprecipates. Furthermore, miR-16, -584 and -92 could also be amplified from Hb immunoprecipitates. Endothelial cells treated with extracellular Hb from RBC lysates for 36 hours had a dramatic increase in levels of miR-451 and a significant decrease in mRNA abundance of genes known to be targets of miR-451, including ATF2 (p-value=0.03), CAV1 (p-value=0.02), and MIF (p-value=0.005). HAECs treated with extracellular Hb for 72 hours had decreased protein levels of ATF2 (p-value=0.01) and CAV1 (p-value=0.003). In conclusion, previous work has shown that extracellular Hb can have direct effects on endothelial cells; our data show, for the first time, that extracellular Hb also serves as a vehicle for the cell-to-cell delivery of microRNA.

Haematological Responses to Detraining Following the Boston Marathon

PURPOSE: While vigorous exercise is well known to stimulate erythropoiesis, the hematological response to exercise detraining remains incompletely understood. We sought to characterize red blood cell (RBC) mediated determinants of oxygen carry capacity, including RBC population dynamics, during a period of detraining. METHODS: Recreational marathon runners participated in a structured 18-week training program (~7-8 h/w) then completed the 2016 Boston Marathon. Participants then reduced total exercise exposure to <2 h/w (no single session >1 hour) for 8 weeks. Exercise testing, carbon monoxide rebreathing tests and venous blood draws were performed 10-14 days before, and at 4 and 8 weeks after the marathon. Mixed linear modeling adjusting for age and marathon finish time was used to compare data across time points. RESULTS: Twenty-two runners (age = 34.5 ± 7.5 y, 50% men) completed the study protocol. Detraining was confirmed by serial reductions in time to exhaustion during treadmill testing (p<0.01, Figure 1). Plasma volume significantly declined by 4 weeks. In contrast, total hemoglobin mass (tHbmass) and serum ferritin remained stable. By 4 weeks, glycated hemoglobin was significantly elevated while RBC mean corpuscular volume was significantly reduced, indicating an increase in mean RBC age. By 8 weeks, there was a significant decrease in the RBC clearance threshold (Vc). CONCLUSION: tHbmass, a primary determinant of oxygen carrying capacity, appears to be stable during 8 weeks of exercise detraining. We speculate that this phenomenon is mediated by a subtle decrease in RBC production rate, and that an extended Vc after 8 weeks occurs to defend tHbmass in the absence of a sufficient erythropoietic stimulus.Figure 1: Changes in exercise capacity and hematologic parameters in response to 8 weeks of exercise detraining following completion of the Boston Marathon.

Age-structured population model of cell survival.

Age-structured cell population model was introduced to describe cell survival. The impact of the environment on the cell population is represented by drug plasma concentration. A key model variable is the hazard of cell removal that is a subject to the environment effect. The model is capable of describing cohort and random labeling cell survival data. In addition, it accounts for cell loss due to labeling of cell sample, but it lacks ability to describe the effect of label elution on the survival data. The model was applied to red blood cell (RBC) survival data in two groups of Wistar rats obtained by two techniques: cohort labeling using 14C-glycine (N=4) and random labeling using biotin (N=8). The Weibull probability density function was selected for the RBC lifespan distribution. The data were simultaneously fitted by the mixed effects model implemented in Monolix 4.3.3. The estimated typical values of RBC lifespan and age were 53.7 and 27.8days, respectively. A noticeable effect of biotinylation on RBC survival was observed that resulted in a significant difference between the means of individual RBC lifespan for two groups. The model provides a mechanistic framework flexible enough to account for various experimental designs to generate the cell survival data. Despite model qualification using animal data, the model has the same potential to be applied to cell survival data analysis in humans.

Abstract 141: Increasing Storage Age of Transfused Red Blood Cell Units Is Not Associated with Cardiac Surgical Outcomes

Introduction: First-in, first-out institutional blood management practices are based on evidence associating increased risk of sequelae from exposure to older red blood cell (RBC) transfusions. Nonetheless, research to date within the setting of cardiac surgery has been limited by previously unmeasured confounding (i.e., use of leukoreduction, timing and volume transfused). Hypothesis: After accounting for exposure to leukoreduction and the timing and volume of RBC units, older RBC units will not increase a patient’s odds of developing post-operative sequelae following cardiac surgery. Methods: We reviewed 659 adult patients undergoing CABG and/or valve operations between January 1, 2008-December 31, 2015 at a tertiary center. During this time period, all RBCs given to cardiac surgery patients were leukoreduced. To address confounding by timing and volume of RBCs, we analyzed patients receiving 1 or 2 units of RBCs intraoperatively. Logistic regression was used to model the maximum storage age of RBCs against a composite endpoint (prolonged mechanical ventilation, pneumonia, renal failure, operative mortality). We further explored any differences when modeling age of RBCs as quintiles and dichotomously (≥14 vs. &lt;14 days). We present our results using odds ratios and 95% confidence intervals. Results: The average maximum storage age of RBCs ranged from 5 to 42 days (mean 20.8, sd: 7.0). The primary endpoint occurred among 122 (18.5%) subjects. There was neither a significant association between increasing age of RBCs and our composite endpoint (OR 1.0; 95% CI 0.98, 1.04), nor evidence of confounding in univariate analysis. These findings were qualitatively unchanged when modeling age of RBCs as quintiles (Figure; ptrend =0.50) or dichotomously (OR 1.6; 95% CI 0.88, 2.79). Conclusion: In this single center study, we did not detect a significant adverse effect of increasing age of RBCs contrary to prior conclusions. Our findings suggest that current institutional RBC storage policies that prioritize distribution of older age RBC units may not adversely impact patient outcomes following cardiac surgery.

From the Cradle to the Grave: The Role of Macrophages in Erythropoiesis and Erythrophagocytosis.

Erythropoiesis is a highly regulated process where sequential events ensure the proper differentiation of hematopoietic stem cells into, ultimately, red blood cells (RBCs). Macrophages in the bone marrow play an important role in hematopoiesis by providing signals that induce differentiation and proliferation of the earliest committed erythroid progenitors. Subsequent differentiation toward the erythroblast stage is accompanied by the formation of so-called erythroblastic islands where a central macrophage provides further cues to induce erythroblast differentiation, expansion, and hemoglobinization. Finally, erythroblasts extrude their nuclei that are phagocytosed by macrophages whereas the reticulocytes are released into the circulation. While in circulation, RBCs slowly accumulate damage that is repaired by macrophages of the spleen. Finally, after 120 days of circulation, senescent RBCs are removed from the circulation by splenic and liver macrophages. Macrophages are thus important for RBCs throughout their lifespan. Finally, in a range of diseases, the delicate interplay between macrophages and both developing and mature RBCs is disturbed. Here, we review the current knowledge on the contribution of macrophages to erythropoiesis and erythrophagocytosis in health and disease.

A national review of the clinical use of group O D- red blood cell units.

There has been an international decline in the demand for red blood cell (RBC) units. In Australia, there has been a 21% reduction in demand between 2012 and 2015. In contrast, the demand for the "universal" group O D- RBC units is in fact proportionally increasing. The clinical use of the entire O D- RBC distribution for a 5-week period throughout Australia was reviewed. Fate data on each unit issued (n = 9733) were collected that included the indication and urgency of transfusion, reason for discard, component age, and patient demographics. A total of 74% of audit forms were returned (n = 7143). The national discard rate of issued units was 7.9%. A total of 6387 units were transfused into an estimated total of 3008 patients (55% males) with median patient age of 67 years and median RBC age of 21 days. Forty-seven percent were transfused to group O D- patients. A total of 17.4% were chosen for specific phenotype requirements, 24.5% of units were transfused close to expiry, and 24.5% were transfused into patients of other ABO groups. The data appear broadly representative of the current transfusion and inventory management practices surrounding the use of group O D- RBC units. Strategies to reduce O D RBC demand include reevaluation of inventory holdings particularly at smaller centers, increasing the panel of phenotyped RBC units across all ABO groups, more regular rotation of units between hospitals to minimize time expiry, and continuing education for promoting transfusion of ABO-identical RBC units.

Abstract A32: A randomized, controlled trial of fish oil supplementation on eicosanoid production in patients at risk for colorectal cancer

Abstract Long-chain n-3 polyunsaturated fatty acids (LCPUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have anti-inflammatory properties and may be a potential chemopreventive agent for colorectal cancer. Dietary n-3 LCPUFAs decreases tissue content of arachidonic acid, inhibits prostaglandin-endoperoxide synthase, and reduces prostaglandin E2 (PGE2) production. Eleven alpha-hydroxy-9, 15-dioxo-2, 3, 4, 5-tetranorprostane-1, 20-dioic acid (PGE-M) is a urinary metabolite of prostaglandin E2 that is associated with an increased risk for colorectal adenomas and cancer. Limited data exists investigating the impact of n-3 LCPUFAs on urinary PGE-M production. We conducted a 6-month, randomized, placebo-controlled trial of 1395 mg/day of EPA plus 1125 mg/day of DHA (3 capsules of Lovaza®) versus 3 grams/day oleic acid as a placebo in patients at increased risk for colorectal cancer (NCT01661764). Participants were ages 40 to 80 years and had a past medical history of one or more adenomatous polyps. Exclusion criteria included a known hereditary cancer syndrome, a prior history of any cancer, current use of fish oil supplements, a known fish allergy, and a personal history of inflammatory bowel disease or cardiovascular disease. Red blood cells and urine were collected at baseline, 12-weeks, and 24-weeks while rectal mucosal biopsies were collected at baseline and 24-weeks. The primary outcome of the trial was change in markers of rectal epithelial cell proliferation and apoptosis and these pathological evaluations are ongoing. In this abstract we present results concerning our measurements of urinary PGE-M, a secondary outcome of the study. We determined red blood cell phospholipid membrane fatty acid content using gas chromatography. We measured urinary PGE2 metabolite (PGE-M) using liquid chromatography/tandem-mass spectrometry. A total of 141 subjects were randomized. For this analysis, we included 125 participants (88.7%) who had urine collected at all three time points. Urinary PGE-M results were significantly skewed and we present geometric means. We calculated P-values using repeated measures ANOVA with log-transformed urinary PGE-M values. Regression models were adjusted for age, sex, tobacco use, aspirin use, and baseline red blood cell phospholipid membrane n-3 LCPUFA percentages. All analyses were conducted as intention-to-treat. The median age of participants was 58 years (interquartile range [IQR] 54, 64), with 49.6% female (n = 62), 9.6% reporting tobacco use (n = 12) and 31.2% reporting regular aspirin use (n = 39). Over the 24-week study, red blood cell phospholipid membrane total n-3 LCPUFA concentrations (EPA + DHA) increased by 105% from a median of 3.57 (IQR 2.95, 5.19) to 7.32 (IQR 5.42, 9.07) in participants allocated to fish oil supplementation compared to a 5% increase in the placebo group (median at baseline 3.97 (IQR 2.91, 4.87) to 4.15 (IQR 3.28, 5.09). This difference was statistically significant (P =&amp;lt; 0.0001) and indicated good compliance with the study medication. Individuals allocated to fish oil supplements (n = 61) had a urinary PGE-M measurement of 8.45 ± 9.01 [geometric mean and standard deviation] at baseline, 5.85 ± 4.49 at 12-weeks and 7.21 ± 6.26 at 24-weeks. Participants allocated to the placebo arm (n = 64) had a baseline urinary PGE-M of 10.6 ± 11.0, a 12-week measurement of 10.4 ± 11.5, and a 24-week measurement of 9.89 ± 11.0. Supplemental n-3 LCPUFAs were associated with a statistically significant decrease in urinary PGE-M measurements (P = 0.05). In conclusion, n-3 LCPUFA supplementation appears to reduce urinary PGE-M levels compared to placebo; however, this effect seemed to be more pronounced at 12-weeks than 24-weeks. The reduction of urinary PGE-M seen with fish oil supplementation suggests these agents might have use as a colorectal cancer chemopreventive agent. Citation Format: Harvey J. Murff, Martha J. Shrubsole, Qiuyin Cai, Timothy Su, Stephanie M. Martin, Sandra Motley, Ginger Milne, Wei Zheng, Qi Dai. A randomized, controlled trial of fish oil supplementation on eicosanoid production in patients at risk for colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr A32.

Replication Study: The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors.

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Chroscinski et al., 2015) that described how we intended to replicate selected experiments from the paper "The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors "(Willingham et al., 2012). Here we report the results of those experiments. We found that treatment of immune competent mice bearing orthotopic breast tumors with anti-mouse CD47 antibodies resulted in short-term anemia compared to controls, consistent with the previously described function of CD47 in normal phagocytosis of aging red blood cells and results reported in the original study (Table S4; Willingham et al., 2012). The weight of tumors after 30 days administration of anti-CD47 antibodies or IgG isotype control were not found to be statistically different, whereas the original study reported inhibition of tumor growth with anti-CD47 treatment (Figure 6A,B; Willingham et al., 2012). However, our efforts to replicate this experiment were confounded because spontaneous regression of tumors occurred in several of the mice. Additionally, the excised tumors were scored for inflammatory cell infiltrates. We found IgG and anti-CD47 treated tumors resulted in minimal to moderate lymphocytic infiltrate, while the original study observed sparse lymphocytic infiltrate in IgG-treated tumors and increased inflammatory cell infiltrates in anti-CD47 treated tumors (Figure 6C; Willingham et al., 2012). Furthermore, we observed neutrophilic infiltration was slightly increased in anti-CD47 treated tumors compared to IgG control. Finally, we report a meta-analysis of the result.

Biophysical and Biochemical Markers of Red Blood Cell Fragility

Background: Red blood cells (RBCs) undergo a natural aging process occurring in the blood circulation throughout the RBC lifespan or during routine cold storage in the blood bank. The aging of RBCs is associated with the elevation of mechanical fragility (MF) or osmotic fragility (OF) of RBCs, which can lead to cell lysis. The present study was undertaken to identify RBC properties that characterize their susceptibility to destruction under osmotic/mechanical stress. Methods: RBCs were isolated from freshly donated blood or units of packed RBCs (PRBCs) and suspended in albumin-supplemented phosphate-buffered saline (PBS). In addition, PRBCs were separated by filtration through a microsphere column into two fractions: enriched with rigid (R-fraction) and deformable (D-fraction) cells. The RBCs were subjected to determination of deformability, MF and OF, moreover, the level of cell surface phosphatidylserine (PS) and the stomatin level in isolated RBC membranes were measured. Results: In the RBC population, the cells that were susceptible to mechanical and osmotic stress were characterized by low deformability and increased level of surface PS. The OF/MF was higher in the R-fraction than in the D-fraction. Stomatin was depleted in destroyed cells and in the R-fraction. Conclusion: RBC deformability, the levels of surface PS, and membrane stomatin can be used as markers of RBC fragility.

Membrane Remodelling and Vesicle Formation During Ageing of Human Red Blood Cells

Background/Aims: A high surface-to-volume ratio and a spectrin membrane-skeleton (MS) confer to the mammalian red blood cells (RBCs) their characteristic deformability, mechanical strength and structural stability. During their 120 days of circulatory life in humans, RBCs decrease in size, while remaining biconcave disks, owing to a coordinated decrease in membrane surface area and cell water. It is generally believed that part of the membrane is lost with the shedding of spectrin-free vesicles of the same type that can be obtained in vitro by different treatments. If this were true, an excess of MS would arise in old RBCs, with respect to the lipid bilayer. Aim of this paper was to investigate this aspect. Methods: Quantification of spectrin by electrophoretic methods was carried out in RBCs of different age. Results: Spectrin decreases, on a per cell basis, with RBC ageing. On the other hand, the membrane raft protein marker flotillin-2, while decreasing in the membrane of old cells, was found to be strongly depleted in the membrane of in vitro-induced vesicles. Conclusion: Part of the membrane-skeleton is probably lost together with part of the lipid bilayer in a balanced way. These findings point to a mechanism for the in vivo release of membrane that is different from that which is known to occur in vitro.

Spectrin and Other Membrane-Skeletal Components in Human Red Blood Cells of Different Age

Background: Old human red blood cells (RBCs) have a reduced surface area with respect to young RBCs. If this decrease occurred through the release of vesicles similar to the spectrin-free vesicles that are shed in vitro under different experimental conditions or during storage, there would be no decrease of membrane-skeleton, but only of lipid bilayer surface area, during RBC ageing in vivo. However, we observed a decrease in spectrin and other membrane-skeletal proteins in old RBCs. Because RBCs contain components of the ubiquitin-proteasome system and other hydrolytic systems for protein degradation, we asked whether increased membrane-skeleton fragments could be detected in older RBCs. Methods: Four different anti-spectrin antibodies and an antibody anti-ubiquitin conjugates were used to analyse, by Western blotting, fragments of spectrin and other proteins in RBCs of different age separated in density gradients and characterized for their protein 4.1a/4.1b ratio as a cell age parameter. Results: spectrin fragments do exist in RBCs of all ages, they represent a minute fraction of all spectrin, are membrane-bound and not cytoplasmic and do not increase with cell age. Besides spectrin, other membrane-skeletal components decrease with cell age. Conclusion: Observed results challenge the commonly accepted view that decrease in cell membrane throughout RBC life in vivo occurs via the release of spectrin-free vesicles.

Red Blood Cells: A Source of Extracellular Vesicles.

During their lifetime, like all other cell types, red blood cells (RBCs) release both exosomes and plasma membrane derived EVs (ectosomes). RBC exosomes are formed only during the development of RBCs in bone marrow, and are released following the fusion of microvesicular bodies (MVB) with the plasma membrane. On the other hand, RBC EVs are generated during normal aging of RBCs in circulation by budding of the plasma membrane due to complement -mediated calcium influx, followed by vesicle shedding. This makes red blood cells and stored red cells a reliable source of EVs for basic and clinical research.

Evidencing the Role of Erythrocytic Apoptosis in Malarial Anemia.

In the last decade it has become clear that, similarly to nucleated cells, enucleated red blood cells (RBCs) are susceptible to programmed apoptotic cell death. Erythrocytic apoptosis seems to play a role in physiological clearance of aged RBCs, but it may also be implicated in anemia of different etiological sources including drug therapy and infectious diseases. In malaria, severe anemia is a common complication leading to death of children and pregnant women living in malaria-endemic regions of Africa. The pathogenesis of malarial anemia is multifactorial and involves both ineffective production of RBCs by the bone marrow and premature elimination of non-parasitized RBCs, phenomena potentially associated with apoptosis. In the present overview, we discuss evidences associating erythrocytic apoptosis with the pathogenesis of severe malarial anemia, as well as with regulation of parasite clearance in malaria. Efforts to understand the role of erythrocytic apoptosis in malarial anemia can help to identify potential targets for therapeutic intervention based on apoptotic pathways and consequently, mitigate the harmful impact of malaria in global public health.

Heme Oxygenase 1 Inhibition Reverses Anemia in β-Thalassemia Mice

Thalassemias are a heterogeneous group of red blood cell (RBC) disorders ranging from a clinically severe phenotype requiring lifesaving transfusions (thalassemia major) to a relatively moderate symptomatic disorder, sometimes requiring transfusions (thalassemia intermedia). Though considered a major cause of morbidity and mortality worldwide, there is still no universally available cure for thalassemia major. The reason for this is, at least in part, due to the lack of full understanding of pathophysiology of thalassemia. The underlying basis of thalassemia pathology is the premature apoptotic destruction of erythroblasts causing ineffective erythropoeisis. In β-thalassemia, β-globin synthesis is diminished causing α-globin accumulation. Unpaired globin chains that accumulate in thalassemic erythroblasts are bound to heme. Moreover, in β-thalassemia an erythroid-specific protease destroys excess α-globin chains, likely leading to the generation of a pool of "free" heme in erythroblasts.Physiologically, heme can be degraded only via heme oxygenases (HO). Circulating erythrocytes contain the majority of heme destined for catabolism; this process takes place primarily in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Heme oxygenase, in particular its heme-inducible isoform HO1, has been extensively studied in hepatocytes and many other non-erythroid cells. Recently, we have provided unequivocal evidence that this enzyme is present in erythroid progenitors as well as their differentiated progenies.1"Unshielded" heme is toxic, but this toxicity will likely be augmented, if HO1 releases iron from heme. We hypothesize that in β-thalassemic erythroblasts HO1-mediated release of iron from heme is the major culprit responsible for cellular damage. Additionally, it has been shown that prevention of heme-derived iron release from splenic and hepatic macrophages improves β-thalassemia phenotype2. Therefore, suppression of HO1-mediated heme catabolism from senescent RBC could be beneficial in reversing thalassemic phenotype.To test this hypothesis, we exploited the mouse model of β-thalassemia known as th3/th3; we obtained these mice from Dr. Stefano Rivella. Our data indicates that HO1 expression is increased in the liver of β-thalassemic mice as compared to wild type mice. Importantly, we observed that erythropoietin-mediated erythroid differentiation of fetal liver (FL) cells from β-thalassemic fetuses increased HO1 mRNA and protein levels to a higher degree than in their wild type counterparts. Ferritin levels were increased in β-thalassemic FL cells suggesting increased heme catabolism and iron release from the tetrapyrrole macrocycle. To investigate the contribution of HO1 to the pathology associated with β-thalassemia, wild type and thalassemic (th3/+) mice were injected intraperitoneally with 40 µmoles/kg/d of tin-protoporphyrin IX (SnPP, HO inhibitor) during a 4-weeks, 3-times a week. Our results show that β-thalassemic mice injected with SnPP have increased hemoglobin levels and red blood cell counts, and display a decrease in the spleen index, reticulocyte counts and liver iron content when compared to PBS-injected β-thalassemic mice. Furthermore, while hepcidin levels remain unchanged, liver ferroportin expression decreases in SnPP-injected β-thalassemic mice.Our results indicate that β-thalassemic erythroblasts have high levels of HO1, which would be expected to degrade any "free" heme. Further research is needed to determine whether iron liberated from heme by HO1 is directly responsible for the damage of β-thalassemic erythroblasts.1GarciaSantos D, et al. Heme oxygenase 1 is expressed in murine erythroid cells where it controls the level of regulatory heme. Blood 123 (14): 226977, 2014.2Nai A, et al. Deletion of TMPRSS6 attenuates the phenotype in a mouse model of β-thalassemia. Blood 119 (21): 5021, 2012. DisclosuresNo relevant conflicts of interest to declare.

Band 3 Phosphorylation Induces Irreversible Alteration of Stored Red Blood Cells

Introduction: Storage of red blood cells (RBCs) for transfusion purposes is accompanied by a number of morphological and biochemical changes (storage lesions) that reduce post-transfusion survival/efficacy and increase risk for adverse reactions in the recipients. The clearance of altered and older RBCs from circulation is triggered by the clustering of Band 3, an aggregate state that is recognized by a low-affinity naturally occurring IgG antibody (Nab). Considering the key role of Band 3 in the maintenance of RBC structure and survival, elucidation of functional and structural modifications of Band 3 during storage should lead to new approaches aiming to improve RBC storage and post-transfusion viability.Results: Immunoblot analysis of RBC membrane proteins using an anti-phosphotyrosine antibody showed a progressive increase in the phosphorylation status of Band 3 during RBC storage (Figure 1).In addition, using the quenching fluorescence of eosin-5-maleimide (EMA), we showed an increase of the mobile fraction of Band 3. These findings are consistent with previous demonstration that tyrosine phosphorylation of Band 3 reduces its affinity for ankyrin, leading to the release of the immobile fraction of Band 3 from the skeleton complex, and enhancement of the lateral mobility of Band 3 into the lipid bilayer. Immunoblot experiments using an antibody that specifically recognizes the clustered form of Band 3 revealed an increase of Band 3 cluster formation from the 28th day of storage. We also showed that the release of microparticles (MPs) that occurs during RBC aging increases from the 28th day of storage (Figure 2).Finally, stopped-flow-based functional studies showed a decrease of the anion exchanger activity of Band 3 from the 28th day of storage.Conclusion: Altogether, our results suggest that the 28th of storage represents a key moment for the molecular processes leading to irreversible lesions of RBCs and allow us to propose a new Band 3 phosphorylation/clustering-based mechanism of RBC aging. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Red Blood Cell Deformability, Age, Ethnicity and Susceptibility to Malaria in Africa

Introduction: malaria is one of the most frequent hematological diseases worldwide. Because the malaria parasite Plasmodium falciparum develops mainly in red blood cells (RBC), splenic retention of infected and uninfected RBC is likely a key player in the variable susceptibility of humans to malaria. Age and ethnicity are important determinants of the manifestations of malaria in Africa (Reyburn JAMA 2005, Dolo Am J Trop Med Hyg 2005; Greenwood Ann Trop Med Parasitol 1987; Torcia PNAS 2008), Asia (Price Am J Trop Med Hyg 2001), and in travelers (Seringe Emerg Infect Dis 2011). We had speculated that variations in the splenic sensing of RBC contribute to the innate protection/susceptibility of infants against distinct forms of severe malaria and to the pathogenesis of chronic malaria (Buffet Curr Opin Hematol 2009; Buffet Blood 2011). Here, we explore the deformability and morphology of circulating RBC in populations living in a malaria-endemic area.Materials and methods: experiments were embedded in an integrated study driven by Institut de Recherche pour le Développement, which aims at the identification of genetic, epidemiologic and anthropologic determinants of susceptibility to malaria. IRB approval was obtained from Institut des Sciences Biomédicales Appliquées, Benin. Clinical and biological data were collected at the beginning of the rainy season from 627 individuals, belonging to 4 different ethnic groups living in sympatry in Atakora, North Benin and included age, gender, ethnicity, body temperature, presence and grade of splenomegaly, rapid diagnostic test for malaria (RDT), thick film and rapid hemoglobin determination with HemoCue©. Venous blood was collected for determination of RBC morphology and deformability. Using microsphiltration, a RBC filtering method that uses microsphere layers to mimic the mechanical retention of RBC in the splenic red pulp (Deplaine Blood 2011) we quantified the ability of a mix of labeled and non-labeled RBCs to squeeze between calibrated slits, results being expressed as retention or enrichment rates (RER) of subject’s RBC compared to normal RBC (from a single French O-positive donor) stored in blood bank conditions. Microsphiltration has been adapted to high-throughput experimentation using microplates (Duez AAC 2015). Experiments were performed in a field laboratory established on site; microplates were prepared in Paris and brought to North Benin in luggage with constant care to avoid shocks during transportation. All RBC samples were filtered in triplicate less than 8 hours after blood collection. Up- and downstream samples were brought back to France at 4°C in sealed micro-well plates and analyzed for individual RER calculation in the next 2 weeks by flow cytometry.Results: over 10 days, 262 adults and 249 children were included, 31% Bariba, 17% Gando (genetically related to Bariba), 24% Otamari, 27% Peulhs. Prevalences of splenomegaly, positive RDT, and fever were 13%, 27%, and 2%, respectively. Of 629 blood samples collected, 511 could be analyzed. RER of controls remained stable with time and across 17 microfiltering plates, with a median (IQR) retention rate of 12% (5% - 21%). Ethnicity and age were the only two factors associated with statistically significant differences in RER (figures 1 and 2). Infants (less than 2 year-old) had a more important enrichment than older children and adults (median in 2 years old or less 287%; 3 to 5 years 103%; 6 to 10 years: 64%; more than ten years: 91%; p=0.0161). Peulhs and Otamari also had higher median enrichment rates than Bariba and Gando (RER: 122% and 118%, 75%, 64%, respectively; p=0.0246). Conversely, splenomegaly, gender, positivity of RDT or anemia at the time of sampling were not associated with RER.Discussion: higher averaged enrichment rates in specific ethnic subgroups, namely Peulhs and Otamari, likely result from a more stringent splenic retention, leaving more deformable RBC in circulation. An innate spleen-RBC interaction process was also observed in infants, which is consistent with the higher incidence of severe malarial anemia and splenomegaly observed in this population (Reyburn JAMA 2005; Price Am J Trop Med Hyg 2001). Our results show that innate factors (e.g. ethnicity and age) tend to influence the deformability of RBC, and therefore the phenotypic expression of malaria in Africa. Ongoing experiments aim at deciphering the mechanisms responsible for these differences. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Rejuvenation of Stored Red Blood Cells Increases Oxygen Release Capacity

Introduction:The goal of a red blood cell (RBC) transfusion is to treat anemia and improve oxygen delivery to tissues (Sharma 2011). RBC metabolic changes during liquid storage increases the affinity of hemoglobin for oxygen by depletion of 2,3-diphosphoglycerate (2,3-DPG). This change reduces the partial pressure of O2 where the oxygen tension of hemoglobin is 50% saturated (p50). Transfusion of stored RBCs manifests immediate deficits in patient 2,3-DPG concentration after surgery with incomplete in vivo restoration 72 hours post-surgery (Scott 2016). This change may bring into question the efficiency of peripheral oxygen unloading of liquid stored RBCs following transfusion.Ex-vivo rejuvenation of allogeneic RBCs increases the levels of ATP and 2,3-DPG and increases the p50 of stored RBCs by right-shifting the Oxyhemoglobin Dissociation Curve (ODC) (Dennis 1979). RBC Oxygen Release Capacity (ORC) is determined by the percent of oxygen removed from hemoglobin across the arterial (100 mmHg O2) - venous (40 mmHg O2) pressure gradient (Li 2016). The objective was to evaluate the changes in 2,3-DPG and p50 during routine blood bank storage for 35 days and the impact on ORC after RBC rejuvenation.Methods:Five (5) units of human whole blood were collected in CPD, processed into leukocyte reduced RBC units and stored in an additive solution (AS-1). Nearly fresh RBC were obtained from a local blood center after days 3 - 6 of storage at 1-6 °C and then stored up to 35 days at 1-6 °C. A ten (10) mL aliquot was withdrawn from each unit on the day of receipt, then on Days 7, 14, 21, 28, and 35. Each aliquot was split equally by volume into Control (untreated) and Rejuvenated Groups (n=5 per group). The Rejuvenated samples (5 mL) were incubated with 0.8 mL rejuvesol™ Solution (Zimmer Biomet) in a dry air blood warmer (Sarstedt SAHARA-III) for one hour at 37 °C. Complete blood counts (CELL-DYN 3700), ODC (TCS Scientific Corp Hemox-Analyzer), and 2,3-DPG (Roche) on perchloric acid extracts were collected. The ORC was calculated from the ODC as previously described (Li 2016).Results:Five (5) units of CPD/AS-1 RBC units were received less than one week post-donation (5.0 ± 1.2 Days). As expected in the Control Group aliquots (n = 5), 2,3-DPG concentration and the p50 value declined significantly (p < 0.001, ANOVA) from Day 7 through Day 35 (Figure 1). Rejuvenated Group aliquots exhibited a significant increase in 2,3-DPG concentration and improved p50 (p < 0.001, t-test) at each storage interval after incubation with rejuvesol Solution compared to untreated Control aliquots (Figure 1). RBC rejuvenation shifted the ODC to the right (Figure 2) and significantly increased the ORC compared to Control aliquots (Figure 3). The ORC of Rejuvenated aliquots did not decline significantly with storage duration (p = 0.11, ANOVA) while Control aliquots were significantly impacted with storage duration (p < 0.001, ANOVA).Conclusion:Reduction in ORC with storage duration of unrejuvenated RBCs suggests impaired oxygen tissue delivery occurs with stored RBCs to the tissue microenvironment. Transfusion practices designed to increase hemoglobin concentration may be less effective with increased RBC age because of reduced oxygen release capacity. These in vitro results confirm previous reports regarding 2,3-DPG changes during storage and treatment with rejuvenation (Valeri 2000). Additional research is proposed to confirm these observations on full RBC units, the clinical impact of reduced oxygen release capacity, and what impact RBCs with a superphysiological ORC have on the tissue microenvironment. [Display omitted] [Display omitted] [Display omitted] DisclosuresInglut:Zimmer Biomet: Employment. Kausch:Zimmer Biomet: Employment. Gray:Zimmer Biomet: Employment. Landrigan:Zimmer Biomet: Employment.

Guidelines on transfusion for fetuses, neonates and older children.

Guidelines on transfusion for fetuses, neonates and older children.

Red blood cell transfusion and skeletal muscle tissue oxygenation in anaemic haematologic outpatients.

BackgroundStored red blood cells (RBCs) accumulate biochemical and biophysical changes, known as storage lesion. The aim of this study was to re-challenge current data that anaemia in chronically anaemic haematology patients is not associated with low skeletal muscle tissue oxygen (StO2), and that RBC storage age does not influence the tissue response after ischaemic provocation, using near-infrared spectroscopy.Patients and methodsTwenty-four chronic anaemic haematology patients were included. Thenar skeletal muscle StO2 was measured at rest (basal StO2), with vascular occlusion testing (upslope StO2, maximum StO2) before and after transfusion.ResultsBasal StO2 was low (53% ± 7%). Average RBC storage time was 10.5 ± 3.9 days. Effects of RBC transfusions were as follows: basal StO2 and upslope StO2 did not change significantly; maximum StO2 increased compared to baseline (64 ± 14% vs. 59 ± 10%, p = 0.049). Change of basal StO2, upslope StO2 and maximum StO2 was negatively related to age of RBCs. The decrease of maximum StO2 was predicted (sensitivity 70%, specificity 100%), after receiving RBCs ≥ 10days old.DiscussionResting skeletal muscle StO2 in chronic anaemic patients is low. RBC storage time affects skeletal muscle StO2 in the resting period and after ischaemic provocation.