Blood Red Blood Cell Research Articles

Ozone Improves Oxygenation and Offers Organ Protection after Autologous Blood Transfusion in a Simulated Carbon Dioxide Pneumoperitoneal Environment in a Rabbit Hemorrhagic Shock Model

Objectives: Autologous blood transfusion techniques are well applied in surgery, but the red blood cells (RBCs) collected during laparoscopic surgery may forfeit their ability to oxygenate. O3 is a potent oxidation gas. This study investigates whether O3 could improve the oxygen-carrying capacity of RBCs, reduce inflammatory reactions, and offer organ protection. Methods: We established a hemorrhagic shock model in rabbits, and simulated CO2 pneumoperitoneum and O3 were applied before autologous blood transfusion. Perioperative mean arterial pressure and arterial blood gas were recorded, blood gas and RBC morphology of collected blood were analyzed, plasma IL-6, ALT, AST, CRE, and lung histopathology POD0 and POD3 were tested, as well as postoperative survival quality. Results: Autologous blood that underwent simulated CO2 pneumoperitoneum had a lower pH and SaO2 and a higher PaCO2 than the control group. After O3 treatment, PaO2 and SaO2 increased significantly, with unchanged pH values and PaCO2. RBCs in autologous blood were drastically deformed after CO2 conditioning and then reversed to normal by O3 treatment. Rabbits that received CO2-conditioned autologous blood had a compromised survival quality after surgery, higher plasma IL-6 levels, higher lung injury scores on POD0, higher ALT and AST levels on POD3, and O3 treatment alleviated these adverse outcomes. Conclusion: O3 can restore RBC function, significantly improve blood oxygenation under simulated CO2 pneumoperitoneum, offer organ protection, and improve the postoperative survival quality in the rabbit hemorrhage shock model.

Effectiveness of hemocoagulase, tranexamic acid, and their combination for reducing blood loss in bimaxillary orthognathic surgery: a retrospective study.

Orthognathic surgery is a corrective intervention for maxillofacial deformities. Bleeding is a major concern for oral and maxillofacial surgeons. Various agents, such as hemocoagulase, tranexamic acid, and aprotinin have been developed to reduce intraoperative bleeding and transfusion requirements. Therefore, in this study we aimed to investigate the effects of hemocoagulase and tranexamic acid, as well as their simultaneous use, to reduce bleeding during orthognathic surgery. Patients and. This retrospective study included patients who had undergone simultaneous orthognathic surgery of the maxilla and mandible between January 2013 and September 2022 and were classified into three groups based on drugs administered: hemocoagulase (Botropase), tranexamic acid, and a combination of both drugs. We recorded patient age, sex, weight, blood loss, and duration of surgery. Red blood cell (RBC), hemoglobin, hematocrit, and platelet levels were measured before, immediately after, and one day after surgery. No statistically significant differences were found in blood loss, RBC, hemoglobin, hematocrit, or platelet levels between any of the groups. There were no differences in the drug effects between Le Fort I and bilateral mandibular sagittal split osteotomies, with or without double genioplasty. However, there were significant reductions in RBC, hemoglobin, hematocrit, and platelet levels during genioplasty. Tranexamic acid, hemocoagulase, and their combination had similar efficacy in patients who underwent Le Fort Ⅰ and bilateral mandibular sagittal split osteotomies with and without genioplasty.

Computational analysis of peripheral blood smears detects disease-associated cytomorphologies

Many hematological diseases are characterized by altered abundance and morphology of blood cells and their progenitors. Myelodysplastic syndromes (MDS), for example, are a group of blood cancers characterised by cytopenias, dysplasia of hematopoietic cells and blast expansion. Examination of peripheral blood slides (PBS) in MDS often reveals changes such as abnormal granulocyte lobulation or granularity and altered red blood cell (RBC) morphology; however, some of these features are shared with conditions such as haematinic deficiency anemias. Definitive diagnosis of MDS requires expert cytomorphology analysis of bone marrow smears and complementary information such as blood counts, karyotype and molecular genetics testing. Here, we present Haemorasis, a computational method that detects and characterizes white blood cells (WBC) and RBC in PBS. Applied to over 300 individuals with different conditions (SF3B1-mutant and SF3B1-wildtype MDS, megaloblastic anemia, and iron deficiency anemia), Haemorasis detected over half a million WBC and millions of RBC and characterized their morphology. These large sets of cell morphologies can be used in diagnosis and disease subtyping, while identifying novel associations between computational morphotypes and disease. We find that hypolobulated neutrophils and large RBC are characteristic of SF3B1-mutant MDS. Additionally, while prevalent in both iron deficiency and megaloblastic anemia, hyperlobulated neutrophils are larger in the latter. By integrating cytomorphological features using machine learning, Haemorasis was able to distinguish SF3B1-mutant MDS from other MDS using cytomorphology and blood counts alone, with high predictive performance. We validate our findings externally, showing that they generalize to other centers and scanners. Collectively, our work reveals the potential for the large-scale incorporation of automated cytomorphology into routine diagnostic workflows.

Early surgery within 48h was associated with reduced perioperative blood loss and red blood cell transfusion requirements in older patients with hip fracture: a retrospective study.

The aim of this study was to analyze the relationship between the timing of surgery and perioperative blood loss, red blood cell (RBC) transfusion rate, and RBC transfusion volume in older patients with hip fracture. From January 2020 to August 2022, this retrospective study enrolled older patients with hip fracture who underwent surgery in our hospital. The demographics, fracture type, type of surgery, time from injury to hospital, timing of surgery, medical history (hypertension, diabetes), duration of surgery, intraoperative blood loss, laboratory tests, and preoperative, postoperative and perioperative RBC transfusion requirements were recorded and analyzed. According to the surgical treatment within 48h or after 48h after admission, the patients were divided into early surgery group (ES) and delayed surgery group (DS). A total of 243 older patients with hip fracture were finally included in the study. Among these, 96 patients (39.51%) underwent surgery within 48h of admission and 147 (60.49%) underwent surgery after this time. Total blood loss (TBL) in the ES group was lower than that in the DS group (576.03 ± 265.57ml vs 699.26 ± 380.58ml, P = 0.003). Preoperative RBC transfusion rate, and preoperative and perioperative RBC transfusion volume in the ES group were significantly lower than those in the DS group (15.63% vs 26.53%, P = 0.046; 50.00 ± 128.15ml vs 117.01 ± 225.85ml, P = 0.004; 80.21 ± 196.63ml vs 144.90 ± 253.52ml, P = 0.027). Timing of surgery within 48h of admission for older patients with hip fracture was associated with reduced the total blood loss and RBC transfusion requirements during the perioperative period.

Haematological alterations of mullet Planiliza abu (Heckel, 1843) naturally infected by Ergasilus mosulensis Rahemo, 1982 (Copepoda: Ergaslidae) in a private fish farm from Basrah Province, Iraq

Generally, there is no previous information about haematological parameters of parasitized fish maintained in fish farms in Al- Mashab area, Basrah, Iraq. In this study, the effects of parasitism by the copepod Ergasilus mosulensis. on haemoglobin (Hb), haematocrit (HCT), red blood cells (RBCS), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and leucocytes were analysed in infected Planiliza abu. The high level of parasitism found in infected fishes was responsible for alterations in blood picture Hb, HCT, RBCS, MCV, MCH, MCHC, and leucocytes (P > 0.05). This is the first report regarding hematology of cultivated freshwater fish which are infected with parasitic copepods in Basrah Province.

Red blood cells of Labeo rohita express Toll-like receptors, NOD- like receptors, interleukins, and interferon-I in response to Gram-negative bacterial infections and lipopolysaccharide stimulations.

Red blood cells (RBCs) are the most abundant cell types in the circulatory system of vertebrates. In fish, RBCs retain their nuclei throughout their lifetime and remain transcriptionally and translationally active. While their primary function is typically associated with gas exchange, recent reports indicate that nucleated red blood cells can play a significant role in regulating the body's innate immune response. The current article describes the innate immune role of red blood cells in rohu (Labeo rohita), a freshwater fish species that holds significant commercial importance in India and South-East Asian nations. From the whole blood and mucosal surface RBCs have been isolated through density gradient centrifugation with HiSep™LSM 1077 (density 1.007 ± 0.0010) and their purity has been confirmed by the Giemsa staining followed by microscopical observations. Toll-like receptors (TLR2, 3, 4, 5) and nucleotide oligomerization domain (NOD)-like receptors (NOD1 and NOD2) in RBCs of rohu fingerlings were observed to be significantly activated (P < 0.05) on infection with Aeromonas hydrophila and Edwardsiella tarda. This activation resulted in increased expression of interleukins (IL-8, IL-1β) and interferon (IFN)-I genes. The activation of TLR4, NOD1 and NOD2, as well as the expression of interleukins and IFN-I genes have been observed in both in vivo and in vitro stimulation of rohu RBCs with lipopolysaccharides. These findings highlight the importance of fish RBCs in enhancing innate immunity against various pathogenic invasions in rohu.

Reduction in blood pressure following acute dietary nitrate ingestion is correlated with increased red blood cell S-nitrosothiol concentrations

Dietary nitrate (NO3−) supplementation can enhance nitric oxide (NO) bioavailability and lower blood pressure (BP) in humans. The nitrite concentration ([NO2−]) in the plasma is the most commonly used biomarker of increased NO availability. However, it is unknown to what extent changes in other NO congeners, such as S-nitrosothiols (RSNOs), and in other blood components, such as red blood cells (RBC), also contribute to the BP lowering effects of dietary NO3−. We investigated the correlations between changes in NO biomarkers in different blood compartments and changes in BP variables following acute NO3− ingestion. Resting BP was measured and blood samples were collected at baseline, and at 1, 2, 3, 4 and 24 h following acute beetroot juice (∼12.8 mmol NO3−, ∼11 mg NO3−/kg) ingestion in 20 healthy volunteers. Spearman rank correlation coefficients were determined between the peak individual increases in NO biomarkers (NO3−, NO2−, RSNOs) in plasma, RBC and whole blood, and corresponding decreases in resting BP variables. No significant correlation was observed between increased plasma [NO2−] and reduced BP, but increased RBC [NO2−] was correlated with decreased systolic BP (rs = −0.50, P = 0.03). Notably, increased RBC [RSNOs] was significantly correlated with decreases in systolic (rs = −0.68, P = 0.001), diastolic (rs = −0.59, P = 0.008) and mean arterial pressure (rs = −0.64, P = 0.003). Fisher's z transformation indicated no difference in the strength of the correlations between increases in RBC [NO2−] or [RSNOs] and decreased systolic blood pressure. In conclusion, increased RBC [RSNOs] may be an important mediator of the reduction in resting BP observed following dietary NO3− supplementation.

Tissue Oxygenation Around Capillaries: Effects of Hematocrit and Arteriole Oxygen Condition.

Oxygen transfer in the microvasculature is a complex phenomenon that involves multiple physical and chemical processes and multiple media. Hematocrit, the volume fraction of red blood cells (RBCs) in blood, has direct influences on the blood flow as well as the oxygen supply in the microcirculation. On the one hand, a higher hematocrit means that more RBCs present in capillaries, and thus, more oxygen is available at the source end. On the other hand, the flow resistance increases with hematocrit, and therefore, the RBC motion becomes slower, which in turn reduces the influx of oxygen-rich RBCs entering capillaries. Such double roles of hematocrit have not been investigated adequately. Moreover, the oxygen-hemoglobin dissociation rate depends on the oxygen tension and hemoglobin saturation of the cytoplasm inside RBCs, and the dissociation kinetics exhibits a nonlinear fashion at different oxygen tensions. To understand how these factors and mechanisms interplay in the oxygen transport process, computational modeling and simulations are favorite since we have a good control of the system parameters and also we can access to the detailed information during the transport process. In this study, we conduct numerical simulations for the blood flow and RBC deformation along a capillary and the oxygen transfer from RBCs to the surrounding tissue. Different values for the hematocrit, arteriole oxygen tension, tissue metabolism rate and hemoglobin concentration and affinity are considered, and the simulated spatial and temporal variations of oxygen concentration are analyzed in conjunction with the nonlinear oxygen-hemoglobin reaction kinetics. Our results show that there are two competing mechanisms for the tissue oxygenation response to a hematocrit increases: the favorite effect of the higher RBC density and the negative effect of the slower RBC motion. Moreover, in the low oxygen situations with RBC oxygen tension less than 50mmHg at capillary inlet, the reduced RBC velocity effect dominates, resulting in a decrease in tissue oxygenation at higher hematocrit. On the opposite, for RBC oxygen tension higher than 50mmHg when entering the capillary, a higher hematocrit is beneficial to the tissue oxygenation. More interestingly, the pivoting arteriole oxygen tension at which the two competing mechanisms switch dominance on tissue oxygenation becomes lower for higher oxygen-hemoglobin affinity and lower hemoglobin concentration. This observation has also been analyzed based on the oxygen supply from RBCs and the oxygen-hemoglobin reaction kinetics. The results and discussions presented in this article could be helpful for a better understanding of oxygen transport in microcirculation.

Quantification of Blood Viscoelasticity under Microcapillary Blood Flow.

Blood elasticity is quantified using a single compliance model by analyzing pulsatile blood flow. However, one compliance coefficient is influenced substantially by the microfluidic system (i.e., soft microfluidic channels and flexible tubing). The novelty of the present method comes from the assessment of two distinct compliance coefficients, one for the sample and one for the microfluidic system. With two compliance coefficients, the viscoelasticity measurement can be disentangled from the influence of the measurement device. In this study, a coflowing microfluidic channel was used to estimate blood viscoelasticity. Two compliance coefficients were suggested to denote the effects of the polydimethylsiloxane (PDMS) channel and flexible tubing (C1), as well as those of the RBC (red blood cell) elasticity (C2), in a microfluidic system. On the basis of the fluidic circuit modeling technique, a governing equation for the interface in the coflowing was derived, and its analytical solution was obtained by solving the second-order differential equation. Using the analytic solution, two compliance coefficients were obtained via a nonlinear curve fitting technique. According to the experimental results, C2/C1 is estimated to be approximately 10.9-20.4 with respect to channel depth (h = 4, 10, and 20 µm). The PDMS channel depth contributed simultaneously to the increase in the two compliance coefficients, whereas the outlet tubing caused a decrease in C1. The two compliance coefficients and blood viscosity varied substantially with respect to homogeneous hardened RBCs or heterogeneous hardened RBCs. In conclusion, the proposed method can be used to effectively detect changes in blood or microfluidic systems. In future studies, the present method can contribute to the detection of subpopulations of RBCs in the patient's blood.

5613165 INVESTIGATING VARIANTS ASSOCIATED WITH QUANTITATIVE RED BLOOD CELL PHENOTYPES FROM TOPMED PREDICTIONS IN AFRICAN SICKLE CELL DISEASE POPULATIONS.

Background: Sickle cell disease (SCD) is caused by a single point mutation (c.20A>T, p.Glu6Val), which results in the formation of mutant haemoglobin S (HbS) instead of haemoglobin A (HbA). It is a life-long inherited recessive disorder that predominantly affects people of African, Indian, and Mediterranean descent. The polymerisation of HbS within red blood cells (RBCs) is the primary pathophysiological alteration in SCD, which results in the sickle-shape of RBCs. Sickle-shaped RBCs are more prone to haemolysis and blocking blood flow, resulting in anaemia and painful episodes. Haematological indices including haematocrit (HCT), haemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), red blood cell width (RDW) and RBC count indicate the size, development, and haemoglobin content of RBCs. Haematological indices are routinely measured as a means of monitoring SCD. A study recently found associations between 14 novel variants and haematological indices when using whole genome sequencing (WGS) data. The association between these variants and haematological indices have not yet been investigated in African SCD patients. This study aims to determine the associations between these variants and haematological indices in SCD patients from Cameroon and Senegal compared to non-SCD individuals. Aim: Investigating the association of 13 novel variants previously found in the Trans-Omics for Precision Medicine (TOPMed) project to haematological indices in African SCD patients. Methods: The 14 variants from the TOPMed study will be genotyped using a TaqMan Assay platform. Sanger sequencing will then be performed on 10% of the cohort to validate the TaqMan Assay result. The application R will be used for the descriptive statistics using sociodemographic, clinical, and haematological indices to evaluate the cases and controls. The Hardy-Weinberg Equilibrium will be used to ensure quality control. A linear regression analysis will be performed in R to determine the association between the variants and RBC phenotypes. Expected results: We are expecting to find an association between the SNPs and haematologocial indices. Conclusion: The variants of focus in this study were prioritized using WGS data, therefore capturing variants that are usually not included in GWAS arrays or imputation reference panels. The associations of the 13 novel variants with haematological indices have not yet been tested in African SCD populations. The results of this investigation could positively impact future studies into predictive modelling using haematological indices.

Risk factors of intraoperative blood loss and transfusion for pediatric patients undergoing brain tumor removal: a retrospective cohort study.

Intraoperative blood loss is a major challenge in pediatric brain tumor removal. Several clinical and surgical factors may influence the occurrence of intraoperative blood loss and blood transfusion. This study aimed to identify the risk factors of intraoperative blood loss and intraoperative red blood cell (RBC) transfusion for pediatric patients undergoing brain tumor removal. A total of 297 pediatric patients undergoing brain tumor removal were selected in this retrospective, singlecenter study. Demographic data, laboratory data, imaging data, and surgical records were collected, and then independent risk factors for intraoperative blood loss and transfusion were identified using multivariate stepwise regression analysis. The median intraoperative blood loss in our cohort was 23.1 ml/kg (IQR 10.0-60.0 ml/kg). In total, 284 (95.6%) patients received intraoperative RBC transfusion, with a median amount of 0.2 U/kg (IQR 0.0-2.6 U/kg). Age (β = -0.189; 95% CI [-1.359, -0.473]; p < 0.001); preoperative hemoglobin level (β = -0.141; 95% CI [-1.528, -0.332]; p = 0.003); anesthesia time (β = 0.189; 95% CI [0.098, 0.302]; p < 0.001); unclear tumor boundary (β = 0.100; 95% CI [2.067, 41.053]; p = 0.031); tumor size (β = 0.390; 95% CI [14.706, 24.342]; p < 0.001); and intraoperative continuous infusion of vasopressor (β = 0.155; 95% CI [13.364, 52.400]; p = 0.001) were independent predictors of intraoperative blood loss. Independent predictors of the need for RBC transfusion included age (β = -0.268; 95% CI [-0.007, -0.004]; p < 0.001); preoperative hemoglobin level (β = -0.117; 95% CI [-0.005, -0.001]; p = 0.003); anesthesia time (β = 0.221; 95% CI [0.001, 0.001]; p < 0.001); unclear tumor boundary (β = 0.110; 95% CI [0.024, 0.167]; p = 0.010); tumor size (β = 0.370; 95% CI [0.056, 0.092]; p < 0.001); intraoperative continuous infusion of vasopressor (β = 0.157; 95% CI [0.062, 0.205]; p < 0.001); and tumor grade (β = 0.107; 95% CI [0.007, 0.062]; p = 0.014). Overall, age, preoperative hemoglobin, tumor size, anesthesia time, continuous infusion of vasopressors, and unclear tumor boundary were the main determinants for intraoperative blood loss and RBC transfusion in pediatric patients undergoing brain tumor removal. Clinical trial registration no.: ChiCTR1900024803 (ChiCTR.org).

Hematological Parameters in Hair Goats During and out of Breeding Season Hair Goats Seasonal Hematological Parameters

Background: The hair goat of Anatolia is a breed that has adapted to rugged and mountainous areas and is known to be very resistant to diseases. As the world faces the problem of climate change, we believe that knowing the hematological characteristics of hair goats will provide great benefits for goat breeding. Objectives: This study was performed to determine possible differences in the blood parameters of adult (male and female) hair goats during and out of the breeding season. Methods: For this purpose, blood samples were taken from clinically healthy adult male and female hair goats (n=36, 3-4 years old) in the spring and autumn seasons. Regarding the whole blood, white blood cell (WBC), red blood cell (RBC), hemoglobin (Hb), hematocrit (Hct), mean red blood cell volume (MCV), mean red blood cell hemoglobin (MCH), average red blood cell hemoglobin concentration (MCHC) and red blood cell distribution width (RDW) values were determined. Results: In the study, MCV values in males were not significantly (P≥0.05) different between spring and autumn, while all other parameters showed significant differences (P≤0.05). In females, WBC, RBC, Hb, Hct, and RDW blood values were higher in the autumn (estrus period) than in the spring (anestrus period), and the differences were found to be significant (P≤0.05). Comparing males and females between the estrus and anestrus seasons, WBC and MCHC were found to be significantly different (P≤0.05) between males and females in May. In contrast, the difference between other parameters was not significant. In October (estrus season), the MCV value was not significantly different between males and females; however, all other parameters were significantly different (P≤0.05). Conclusion: As a result, there were significant differences in hematological parameters of hair goats based on gender and the breeding season.

Red blood cell lingering modulates hematocrit distribution in the microcirculation

The distribution of red blood cells (RBCs) in the microcirculation determines the oxygen delivery and solute transport to tissues. This process relies on the partitioning of RBCs at successive bifurcations throughout the microvascular network, and it has been known since the last century that RBCs partition disproportionately to the fractional blood flow rate, therefore leading to heterogeneity of the hematocrit (i.e., volume fraction of RBCs in blood) in microvessels. Usually, downstream of a microvascular bifurcation, the vessel branch with a higher fraction of blood flow receives an even higher fraction of RBC flux. However, both temporal and time-average deviations from this phase-separation law have been observed in recent studies. Here, we quantify how the microscopic behavior of RBC lingering (i.e., RBCs temporarily residing near the bifurcation apex with diminished velocity) influences their partitioning, through combined in vivo experiments and in silico simulations. We developed an approach to quantify the cell lingering at highly confined capillary-level bifurcations and demonstrate that it correlates with deviations of the phase-separation process from established empirical predictions by Pries et al. Furthermore, we shed light on how the bifurcation geometry and cell membrane rigidity can affect the lingering behavior of RBCs; e.g., rigid cells tend to linger less than softer ones. Taken together, RBC lingering is an important mechanism that should be considered when studying how abnormal RBC rigidity in diseases such as malaria and sickle-cell disease could hinder the microcirculatory blood flow or how the vascular networks are altered under pathological conditions (e.g., thrombosis, tumors, aneurysm).

Effects of a Maximal Exercise Followed by a Submaximal Exercise Performed in Normobaric Hypoxia (2500 m), on Blood Rheology, Red Blood Cell Senescence, and Coagulation in Well-Trained Cyclists.

Acute normoxic exercise impacts the rheological properties of red blood cells (RBC) and their senescence state; however, there is a lack of data on the effects of exercise performed in hypoxia on RBC properties. This crossover study compared the effects of acute hypoxia vs. normoxia on blood rheology, RBC senescence, and coagulation during exercise. Nine trained male cyclists completed both a session in normoxia (FiO2 = 21%) and hypoxia (FiO2 = 15.3% ≈ 2500 m). The two sessions were randomly performed, separated by one week, and consisted of an incremental and maximal exercise followed by a 20 min exercise at the first ventilatory threshold (VT1) on a home-trainer. Blood samples were taken before and after exercise to analyze hematological parameters, blood rheology (hematocrit, blood viscosity, RBC deformability and aggregation), RBC senescence markers (phosphatidylserine (PS) and CD47 exposure, intraerythrocyte reactive oxygen species (ROS), and calcium content), and blood clot viscoelastic properties. Hemoglobin oxygen saturation (SpO2) and blood lactate were also measured. In both conditions, exercise induced an increase in blood viscosity, hematocrit, intraerythrocyte calcium and ROS content, and blood lactate concentration. We also observed an increase in blood clot amplitude, and a significant drop in SpO2 during exercise in the two conditions. RBC aggregation and CD47 exposure were not modified. Exercise in hypoxia induced a slight decrease in RBC deformability which could be related to the slight increase in mean corpuscular hemoglobin concentration (MCHC). However, the values of RBC deformability and MCHC after the exercise performed in hypoxia remained in the normal range of values. In conclusion, acute hypoxia does not amplify the RBC and coagulation changes induced by an exercise bout.

Manipulating omega-3 and omega-6 fatty acids in sheep: depletion of long-chain omega-3 polyunsaturated fatty acids by feeding different grain types

Context Plasma fatty acid profiles provide evidence of short-term feeding, while red blood cells (RBC) indicate longer-term feeding effects. Information on the rate of change in fatty acid profiles of sheep blood over time after alterations in diet is lacking. This study monitored the short- and longer-term effects of feeding grain-based diets to lambs on changes to omega-3 polyunsaturated fatty acids (PUFA) in blood fractions over 21 days. The data provide a guide to managing omega-3 PUFA availability for adequate tissue accretion of these fatty acids. Aims To determine the rate of depletion over 21 days of omega-3 PUFA in the blood fractions of sheep fed on grain-based diets. Methods Blood samples were taken from ewes taken off pasture and fed grain-based diets for 21 days. Fatty acid profiles in the blood plasma and RBC were used to monitor the rate of depletion of omega-3 PUFA. Key results Immediate changes in plasma omega-3 PUFA concentrations occurred from the first day of grain feeding. The omega-3 PUFA content declined continuously on all five grains, but with substantial differences among them. RBC omega-3 PUFA contents displayed great variation across individual sheep but depletion was not significant. Conclusions Plasma omega-3 PUFA depletion immediately on grain feeding indicated a lower availability for tissue accretion in growing sheep following short-term changes in diet. The lack of change in RBC omega-3 PUFA concentrations over 21 days indicated a differential accumulation of these fatty acids in this lipid depot compared with plasma, and changes in other tissues should also be examined. Implications The effect of these short- and long-term changes on LCn-3PUFA accumulation in meat and reproductive tissues warrants further investigation.

GBT021601 Prevents Microvascular Dysfunction and Improves Hypoxia-Reoxygenation-Induced Vaso-Occlusion in a Murine Model of Sickle Cell Disease

Background: In sickle cell disease (SCD), a single nucleotide change in the β-globin gene coding sequence results in the production of hemoglobin S (HbS), which polymerizes when deoxygenated and leads to red blood cell (RBC) sickling, hemolysis, anemia, diminished microvascular blood flow, vascular endothelial cell activation, vaso-occlusion, and ischemia. Pharmacological increase of the oxygenated HbS concentration in RBCs has been shown to be a well-tolerated and effective approach to inhibiting HbS polymerization, reducing RBC sickling, and improving oxygen (O2) delivery by reducing anemia and improving blood rheology. GBT021601, a potent second-generation small-molecule HbS polymerization inhibitor, is currently under investigation and has previously been shown to increase hemoglobin (Hb), improve hemolysis, and improve RBC health in a murine model. However, little is known regarding its impact on vaso-occlusion and microvascular dysfunction. Objective: To examine the effects of GBT021601 on SCD-induced microvascular dysfunction, capillary perfusion and density, Hb-O2 oxygen affinity, and Hb and hematocrit levels in a murine model of SCD. Methods: Capillary perfusion was measured by functional capillary density (FCD, a marker of capillary perfusion defined as the number of capillaries with RBCs in transit) in a murine model of SCD with an implanted dorsal window chamber (Figure). HbSS Townes mice were treated with GBT021601 (75 mg/kg) by oral gavage daily for 14 days and were compared with untreated HbSS and HbAA Townes mice. Results: Chronic treatment of HbSS mice with GBT021601 led to increased Hb-O2 affinity, evidenced by a reduction in blood p50 (partial O2 pressure at which Hb is 50% saturated with O2) from 32 mm Hg to 14 mm Hg. GBT021601 significantly reduced anemia by increasing total Hb and hematocrit from 8.3 g/dL and 26% in untreated HbSS mice to 11.2 g/dL and 37% in GBT021601-treated HbSS mice, respectively (P<0.01). Moreover, GBT021601 treatment prevented microvascular dysfunction (reduced RBC velocities and blood flow) in the arterioles and venules of HbSS mice. Increased capillary density is indicative of greater oxygen transport. Challenging HbSS mice with hypoxia (8% O2 for 1 hour) followed by reoxygenation (21% O2) induced vaso-occlusion, causing reduced microvascular blood flow and FCD in untreated mice. However, in GBT021601-treated HbSS mice, microvascular blood flow and FCD were significantly higher than those in untreated HbSS mice. Unlike in untreated HbSS mice, microvascular blood flow and FCD returned to baseline values after 72 hours of reoxygenation in treated HbSS mice. These results suggest that by increasing HbS-O2 affinity and thereby inhibiting HbS polymerization, GBT021601 may attenuate hypoxia-induced vaso-occlusion and impaired capillary perfusion. Furthermore, GBT021601 treatment in HbSS mice reduced both circulating adhesion markers and leukocyte-endothelium interactions in venules after hypoxia and reoxygenation. Conclusions: GBT021601 increased microvascular perfusion, reduced vaso-occlusion, and improved the hypoxia-induced reduction in capillary perfusion in HbSS mice, suggesting that chronic GBT021601 treatment prevents SCD-induced microvascular dysfunction and mitigates the rebound vaso-occlusion induced by hypoxia and reoxygenation in a murine model of SCD. Funding: Global Blood Therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular Determinants of Human Red Blood Cell Survival in Murine Circulation

Introduction: Transfusion of human red blood cells (RBCs) is an irreplaceable life-saving medical intervention. However, as the expression of surface proteins on donor RBCs can regulate their circulatory lifespan, these proteins can affect the efficiency of transfusion. A murine preclinical transfusion model was used to better understand the impact of these proteins on human RBC survival. Methods: Human peripheral blood RBCs were obtained from qualified blood donors as aliquots of leukoreduced, packed CP2D/AS1 RBCs, or separately collected from healthy research donors. RBCs were biotinylated at densities ≤18 mg/ml following established protocols. Unlabeled or biotinylated human RBCs were transfused into immunodeficient NCG mice treated with chlodronate and cobra venom factor before and after transfusion. Human RBCs in murine circulation were identified with FITC-conjugated anti-human CD235a antibodies and/or PE-conjugated streptavidin. Aliquots of transfused RBCs were analyzed by flow cytometry for expression levels of the surface molecules CD35, CD36, CD38, CD44, CD47, CD55, CD59, CD147, CD233 and CD235a before transfusion, and also in peripheral blood samples collected from mice after transfusion. In some studies, human RBCs were treated with antibodies against selected surface molecules before transfusion. Results: Samples collected from transfused mice at serial timepoints showed significantly decreased expressions over time of several human RBC surface molecules: CD47, CD55, CD59, CD147, CD233 and CD235a. In contrast, there were no significant changes in RBC expression of CD35, CD36, CD38 and CD44 in the samples. Among the surface molecules showing lower levels over time, there was a significant positive correlation between CD47 or CD59 expression levels on human RBCs and circulatory survival of those RBCs in the mice. When mixtures of RBCs from two donors were transfused simultaneously, those RBCs with higher CD47 or CD59 expression demonstrated significantly longer survival in murine peripheral blood in comparison with the co-transfused RBCs. Furthermore, pre-incubation of human RBCs with antibodies against CD47 or CD59 prior to transfusion markedly reduced their survival in murine circulation. In contrast, antibodies against CD55, CD147, CD233 or CD235a had no effect on human RBC circulation. Conclusions: Our data show that CD47 and CD59 expression on human RBCs increases their survival after transfusion, and suggest that pre-transfusion profiling of CD47 and CD59 molecules could help predict the efficiency of human RBC transfusion.

Red blood cell transfusion is associated with an increased risk of splanchnic vein thrombosis in patients with cirrhosis.

Haemorrhage and coagulation disorders are common complications in cirrhotic patients, which cause blood products transfusion, and mounting evidence suggested that red blood cells (RBCs) were associated with pathologic thrombosis and RBC transfusion increased the risk of venous thromboembolism (VTE). The aim of the study was to investigate the association of RBC transfusion with splanchnic vein thrombosis (SVT) in cirrhotic patients. We retrospectively reviewed patients with cirrhosis admitted in the Hunan Provincial People's Hospital between January 2010 and September 2020. Demographic data, the development of SVT, blood transfusion product type and RBC transfusion dose were collected. Multivariate logistic regression analyses and propensity matching analysis (PSM) were performed to identify the association between RBC transfusion and development of SVT. A total of 4479 patients with cirrhosis were enrolled in the study. SVT occurred in 48 (12.4%) cirrhotic patients in RBC transfusion group, and 233 (5.7%) cirrhotic patients in non-RBC transfusion group. RBC transfusion was significantly associated with an increased risk of SVT (unadjusted odds ratio [OR] 2.345, 95% confidence interval [CI] 1.686-3.262, p < 0.001). Notably, this association remained robust after PSM, and the volume of RBC transfusion was associated with SVT in a dose-dependent manner. This study suggested that RBC transfusion was associated with an increased risk of SVT in cirrhotic patients. High quality clinical study will be needed to further validate the association between RBC transfusion and SVT.

Differential Cytokine Levels during Normothermic Kidney Perfusion with Whole Blood- or Red Blood Cell-Based Perfusates-Results of a Scoping Review and Experimental Study.

The ideal composition of the perfusate for normothermic kidney perfusion is unknown, though the perfusate commonly used to perfuse human kidneys contains leukocyte-depleted packed red blood cells (RBC), as this is believed to prevent excessive inflammation. We performed a systematic search identifying 19 articles reporting on cytokine levels during normothermic pig or human kidney perfusion. Cytokine levels varied widely across the reported studies. No direct comparisons of perfusate cytokines during perfusion with RBC or whole blood were performed, and no data on how these levels are influenced by ischemia are available. Therefore, we compared perfusate IL-6, IL-1β, TNF-α, TGF-β, IL-10, IL-8, and CCL2 levels during 4 h normothermic pig kidney perfusion with a whole blood- or RBC-based perfusate. Kidneys were exposed to either 1 h of warm or 22 h of cold ischemia. We found no evidence of different perfusate cytokine or gene expression levels in whole blood or RBC perfusions. There was no clear evidence to suggest that cytokine concentrations differ between ischemically injured kidneys and controls. In conclusion, pro-inflammatory and anti-inflammatory cytokines and chemokines are detectable in the perfusate and urine of kidneys undergoing normothermic perfusion. It is unclear how cytokine levels change in different ischemic conditions and whether the use of a leukocyte filter plays a role.

Role of oral lactoferrin as a source of iron supplementation in correction of anemia in pediatric patients with chronic kidney disease stages 2–4

Background Children with chronic kidney disease (CKD) have multiple risk factors for anemia such as primary erythropoietin deficiency, blood loss, decreased red blood cell (RBC) survival, bone marrow suppression, iron deficiency, inflammation and infection, malnutrition, hyperparathyroidism, vitamin B12 and folate deficiency, aluminum toxicity, and carnitine deficiency. This study was performed to evaluate the effect of oral bovine lactoferrin on patients with iron deficiency with CKD stages 2–4. Patients and methods This follow-up cohort clinical study was conducted on children with CKD in the conservative clinic, Pediatric Nephrology Unit, Children's Hospital, Ain Shams University. It included 45 pediatric patients with CKD stages from 2 to 4 for 6 months without a control group. This is a follow-up case study in which all the included patients were on erythropoietin therapy ranging from 150 to 300 IU/kg once per week. The patients were subjected to history and laboratory evaluation, including hemoglobin (Hb), serum iron, serum ferritin, and total iron-binding capacity (TIBC), which were done for the patients at baseline and 6 months after treatment with bovine lactoferrin for 6 months. Results Blood Hb and RBC volume were significantly increased beginning from first month after oral lactoferrin therapy, serum iron and serum ferritin were significantly increased 6 months after intervention, and serum TIBC was significantly decreased after intervention. The current study had shown no significant difference between males and females regarding laboratory changes 6 months after intervention. Laboratory improvements were significantly lowest among cases with stage 4, followed by stage 3, and the highest among cases with stage 2. There was a decrease in all anemia clinical manifestations after 6 months of lactoferrin administration; the differences were significant only in easy fatigability, constipation, and gastrointestinal upset, which were the most frequent adverse effects. Conclusion Oral lactoferrin was found to be effective in treating iron-deficiency anemia regarding blood Hb, blood RBCs, serum iron, serum ferritin, and TIBC in association with erythropoietin therapy. The effect declines with the progression of CKD.