Supply Of Red Blood Cells Research Articles

A systems pharmacology model for gene therapy in sickle cell disease.

We developed a mathematical model for autologous stem cell therapy to cure sickle cell disease (SCD). Experimental therapies using this approach seek to engraft stem cells containing a curative gene. These stem cells are expected to produce a lifelong supply of red blood cells (RBCs) containing an anti‐sickling hemoglobin. This complex, multistep treatment is expensive, and there is limited patient data available from early clinical trials. Our objective was to quantify the impact of treatment parameters, such as initial stem cell dose, efficiency of lentiviral transduction, and degree of bone marrow preconditioning on engraftment efficiency, peripheral RBC numbers, and anti‐sickling hemoglobin levels over time. We used ordinary differential equations to model RBC production from progenitor cells in the bone marrow, and hemoglobin assembly from its constituent globin monomers. The model recapitulates observed RBC and hemoglobin levels in healthy and SCD phenotypes. Treatment simulations predict dynamics of stem cell engraftment and RBC containing the therapeutic gene product. Post‐treatment dynamics show an early phase of reconstitution due to short lived stem cells, followed by a sustained RBC production from stable engraftment of long‐term stem cells. This biphasic behavior was previously reported in the literature. Sensitivity analysis of the model quantified relationships between treatment parameters and efficacy. The initial dose of transduced stem cells, and the intensity of myeloablative bone marrow preconditioning are predicted to most positively impact long‐term outcomes. The quantitative systems pharmacology approach used here demonstrates the value of model‐assisted therapeutic design for gene therapies in SCD.

Prediction Of Transfusion Based On Machine Learning

The capacity to anticipate transfusions during a hospital stay may allow for more efficient blood supply management, as well as increased patient safety by assuring a sufficient supply of red blood cells (RBCs) for a specific patient. As a result, we tested the accuracy of four machine learning–based prediction algorithms for predicting transfusion, large transfusion, and the number of transfusions in hospitalized patients. Between January 2008 and June 2017, researchers conducted a retrospective observational study at three adult tertiary care institutions in Western Australia. The area under the curve for the receiver operating characteristics curve, the F1 score, and the average precision of the four machine learning algorithms used: artificial neural networks (NNs), logistic regression (LR), random forests (RFs), and gradient boosting (GB) trees were the primary outcome measures for the classification tasks. Transfusion of at least 1 unit of RBCs could be predicted quite correctly using our four prediction models (sensitivity for NN, LR, RF, and GB: 0.898, 0.894, 0.584, and 0.872, respectively; specificity: 0.958, 0.966, 0.964, 0.965). The four approaches were less successful in predicting large transfusion (sensitivity: 0.780, 0.721, 0.002, and 0.797 for ANN, LR, RF, and GB, respectively; specificity: 0.994, 0.995, 0.993, 0.995). As a result, the total number of packed RBCs transfused was likewise very inaccurately predicted. This study shows that the need for intra-hospital transfusion can be predicted with reasonable accuracy, but the number of RBC units transfused throughout a hospital stay is more difficult to predict.

Capillary Oxygen Triggers Conducted Vasodilation via Connexin 40 Independent of Extracellular K + and Endothelial K IR 2.1

Mechanisms that fine-tune red blood cell (RBC) and O2 delivery to capillaries are critical to tissue function. Coupling RBC supply to demand is an intricate process requiring O2 sensing, generation of a capillary stimulus and triggering of a transduction process that alters the diameter of upstream arterioles. The sensor's identity, the stimulus it generates and the mechanisms that enable it are actively debated. Studies have implied the endothelial KIR2.1 channels drive O2 sensing whereas gap junctions, comprised of connexin 40, enable electrical signals to conduct upstream. This idea was tested in KIR2.1-/- and connexin 40-/- mice where the local O2 environment of skeletal muscle was precisely controlled. The extensor digitorum longus muscle was prepared for intravital microscopy; a custom stage insert with a gas chamber allowed for O2 control at the tissue surface. Second-by-second capillary RBC flow responses were recorded as O2 was: 1) reduced from 53 mmHg to 15 mmHg or 0 mmHg for 3 min; or 2) oscillated between 91 mmHg and 15 mmHg (1 cycle/min). Chamber O2 at 15 mmHg induces microvascular responses without diminishing mitochondrial function while 0 mmHg initiates additional metabolic responses. Dropping PO2 on the muscle surface (53 mmHg to 15 mmHg or 0 mmHg) significantly increased RBC supply rate in capillaries of control animals while elevated chamber O2 decreased capillary RBC supply rate in a graded manner. The RBC flow responses in control mice were rapid and tightly coupled to O2 levels as the chamber O2 was oscillated in a sinusoidal fashion. In stark contrast, this blood flow response failed to occur in connexin 40-/- mice. As this response was absent, capillary RBC O2 saturation was lower under resting conditions in the connexin 40-/- animals. Endothelial KIR2.1-/- mice, on the other hand, had normal resting RBC O2 saturation. Furthermore, KIR2.1-/- mice reacted normally to O2 changes, albeit an oscillation or a sustained O2 decrease (to 15 mmHg), with a rise in RBC supply rate. Likewise, the microcirculatory response to 0 mmHg O2 was similar between floxed control and endothelial KIR2.1-/- mice. We show that microvascular O2 responses depend on coordinated electrical signaling via gap junctions comprised of connexin 40 and that endothelial KIR2.1channels do not drive the initiating electrical event. These findings reform our understanding of blood flow regulation and how O2 initiates this process independent of metabolite production.

Characterization of the Skeletal Muscle Microvascular Blood Flow Response to Tissue CO 2 Perturbations using a Gas Exchange Chamber

Objective To characterize the skeletal muscle microvascular blood flow response to changing tissue carbon dioxide (CO2) concentrations directly imposed using a microfluidic gas exchange chamber. Methods Eight male Sprague-Dawley rats (145-187g) were anaesthetized using sodium pentobarbital via an intraperitoneal injection. Cannulas were introduced into the left common carotid artery and right jugular vein for blood pressure and heart rate monitoring, administration of maintenance anaesthetic, and fluid resuscitation. The animal was then tracheotomized and mechanically ventilated. The extensor digitorum longus muscle of the right hindlimb was isolated, externalized, and reflected over a microfluidic gas exchange chamber set into the stage of an inverted Olympus IX73 microscope for in vivo visualization of blood flow. The muscle was allowed to equilibrate for 30 minutes before blood sampling and exposure to various gas levels. Using computer-controlled mass flow meters, the muscle was equilibrated with incremental levels of CO2 (0-10%) while maintaining a constant O2 state. One-minute intravital video microscopy sequences of the microcirculation were recorded at a range of CO2 levels (0-10%) while maintaining a constant O2 level. Analysis of hemodynamic variables, red blood cell (RBC) velocity, RBC supply rate (SR), RBC oxygen saturation (SO2), and hematocrit, was conducted offline using custom MATLAB software. Results Both the mean RBC velocity and SR increased incrementally as the CO2 increased. The graded increase of both parameters was significant between the low CO2 levels (0-2%) and moderate-high levels (4-10%); however, there was no significant difference between a physiologically relevant CO2 (5%) and elevated CO2 levels (6-10%) (See Figures 1A & 1B). The graded increase in CO2 had no significant impact on capillary SO2 or hematocrit (See Figures 1C & 1D). Conclusion The preliminary findings of the present study demonstrate the local CO2-sensitivity of the skeletal muscle microvascular system using a novel experimental technique. An expected graded increase in RBC velocity and SR were observed as CO2 increased; however, the lack of significant change following exposure to elevated CO2 suggests a higher relative sensitivity of the microvascular system to changes at low tissue CO2 concentrations. The observed invariance of SO2 at varying CO2 gas concentrations also suggests that local CO2-mediated blood flow regulation is not a result of RBC O2 saturation dependent ATP release. Direct effects of CO2 and the resulting change in pH are known to modulate microvascular blood flow, though the underlying mechanisms of CO2-mediated regulation remain to be defined.

Development and validation of a novel microfluidic device for the manipulation of skeletal muscle microvascular blood flow in vivo.

To develop and validate a novel liquid microfluidic approach to deliver drugs to microscale regions of tissue while simultaneously allowing for visualization and quantification of microvascular blood flow. Microfluidic devices were fabricated using soft lithographic techniques, molded in polydimethylsiloxane, and bound to a coverslip with a 600×300μm micro-outlet. Sprague-Dawley rats, anesthetized with pentobarbital, were instrumented to monitor systemic parameters. The extensor digitorum longus muscle was dissected, externalized, and reflected across the device mounted on the stage of an inverted microscope. Doses (10-8 to 10-3 M) of adenosine triphosphate (ATP), acetylcholine, and phenylephrine (PE) were administered to the muscle via perfusion through the device. Microvascular blood flow directly overlying the micro-outlet was recorded at multiple focal depths. Red blood cell (RBC) velocity, supply rate, and hematocrit were measured from recordings. ATP significantly increased RBC velocity and supply rate. Increasing concentrations of PE caused a decrease in RBC velocity and supply rate. Perfusion changes were restricted to areas directly overlying the micro-outlet and within 500μm. This novel microfluidic device allows for a controlled delivery of dissolved substances to constrained regions of microvasculature while simultaneously allowing for visualization and measurement of blood flow within discrete vessels and networks.

Transfusion support during childbirth for a woman with anti-U and the RHD*weak D type 4.0 allele.

Abstract D– red blood cells (RBCs), always in short supply, and Rh immune globulin (RhIG) are not needed for patient care if D+ RBCs can safely be transfused. According to a recent work group recommendation, patients with the RHD*weak D type 4.0 allele can be considered D+. We report an African American woman who presented for delivery at the end of the third trimester, at which time anti-U and a serologic weak D phenotype were recognized, requiring U–, D– RBC units. We obtained 3 U– RBC units, including 1 D– unit. Later, the RHD*weak D type 4.0 allele was determined by RHD genotyping, only 6 days before delivery. The patient had an uneventful vaginal delivery of a D+ baby. No transfusion was needed for mother or baby. In this case, a pregnant woman with the RHD*weak D type 4.0 allele can safely be managed as D+, relaxing the unnecessary D– restriction for the limited U– RBC supply. The procured U–, D– RBC unit was frozen with 14 days of shelf-life remaining. To conserve D– RBC units, not limited to U–, for patients with a definite need, we recommend molecular analysis of a serologic weak D phenotype before a transfusion becomes imminent. The best time to resolve a serologic weak D phenotype with RHD genotyping is early in a pregnancy. Immunohematology 2021;37:1–4 .

Capillary Oxygen Triggers Conducted Hyperpolarization and Dilation Independent of Extracellular K+ and Endothelial KIR2.1

The matching of red blood cell (RBC) supply to oxygen demand is an intricate process which requires generation of a capillary stimulus and triggering of a transduction process that dilates upstream arterioles. The sensor’s identity, the stimulus it generates and the mechanisms that enables it are openly debated and a source of active inquiry. This study tested whether extracellular K+ is the stimulus and if capillary KIR2.1 channels enable upstream arteriolar dilation via conduction. Experiments were performed in vivo, using a live microcirculatory preparation (extensor digitorum longus); the local oxygen environment was tightly controlled (custom stage insert) and capillary RBC kinetics monitored in mice lacking endothelial KIR2.1 or connexin 40. Dropping PO2 on the muscle surface (53 mmHg to 15 mmHg or 0 mmHg (3 min)) induced a stark microvascular response in control animals (RBC supply rate, velocity and hematocrit increased by 56%, 28% an 18%, respectively) without diminishing mitochondrial respiration. This blood flow response failed to materialize in connexin 40−/− mice. As this response was absent, capillary RBC O2 saturation was lower under normal conditions in the connexin 40−/− animals. In contrast, endothelial KIR2.1−/− mice reacted normally to O2 challenges, with RBC supply rate, velocity and hematocrit rising by 43%, 28%, and 11%, respectively. These findings show that coupling O2 demand‐to‐O2 supply requires coordinated electrical signaling among cells connected by gap junctions comprised of connexin 40. They also show that endothelial KIR2.1 channels are not responsible for initiating the hyperpolarization needed to drive conduction. These findings begin the process of restructuring our understanding of blood flow regulation and how O2 initiates this process independent of metabolite production.Support or Funding InformationThis work was funded by NSERC Discovery grants to CGE and to DGW.

Hyperinsulinemic Microvascular Blood Flow Response in Skeletal Muscle is Oxygen Mediated

ObjectiveTo determine if the blood flow response to insulin is partially mediated by local metabolic demand reflecting increased oxygen consumption required to fix glucose.MethodsTwo separate protocols were used to address the objective. 8 Sprague‐Dawley (171–204g) rats, 4 per protocol, were anesthetized with sodium pentobarbital via intraperitoneal injection, and mechanically ventilated. Arterial and venous catheters were introduced to maintain and monitor the animals’ cardiovascular state. The extensor digitorum longus (EDL) muscle was isolated and reflected onto the stage of an Olympus IX73 inverted microscope. Intravital video microscopy sequences were recorded during baseline and at hyperinsulinemic euglycemia in both protocols. Euglycemia was achieved through intravenous infusion of 2U insulin/kg/hr and variable rates of 50% D‐glucose were titrated against repeated blood glucose samples until stable normoglycemic levels were reached. In protocol 1, the EDL was reflected over a glass stage insert and isolated from room air with polyvinylidene film and a glass coverslip. In protocol 2, the muscle was instead reflected over a 100μm thick semi‐permeable membrane interfacing the EDL with a gas exchange chamber and similarly isolated from room air. An oxygen (O2) challenge was imposed in protocol 2 where O2 levels were oscillated at one minute intervals from 7%‐12%‐2%‐7%, with a stable 5% carbon dioxide concentration and the balance of gas composed of nitrogen. For both protocols, offline analysis of microvascular flow was conducted using custom MATLAB software yielding frame‐by‐frame measurements of capillary red blood cell (RBC) velocity, hematocrit, RBC supply rate (SR), and oxygen saturation (SO2). Animal protocols were approved by Memorial University’s Institutional Animal Care Committee.ResultsNo significant changes were found between glucose infusion rates (GIR) at euglycemia between both protocols, as well as blood glucose levels at both baseline and euglycemic conditions between experiments. In protocol 1, SR increased to 11.2 cells/s following euglycemic clamp, compared to 8.0 cells/s at baseline (p = 0.0255) consistent with flow increases reported in the literature. As expected, the imposed O2 challenges in protocol 2 caused significant changes in SO2 at 12% and 2% concentrations, which were found to be the same under both baseline and euglycemic conditions. In protocol 2, SR decreased at 12% O2 and increased at 2% O2 during the oscillation, compared to 7% O2, under both baseline and euglycemic conditions. SR was the same under euglycemia compared to baseline in the last 15 seconds of each step of the O2 oscillation from 7%‐12%‐2%‐7%, with p values >0.9999, >0.9999, 0.9984, and 0.9963 respectively.ConclusionsGas exchange chamber was effective at manipulating tissue oxygen levels, observed through profound SO2 changes during chamber oscillations at both baseline and euglycemic conditions. Our results suggest that the increase in muscle blood flow observed in response to insulin, as seen in experiment 1, is eliminated if the tissue oxygen environment is fixed at a given oxygen concentration as demonstrated in experiment 2.Support or Funding InformationProject funded through a CIHR project grant awarded to GM Fraser (PJT‐162119)Time series showing the change in capillary red blood cell oxygen saturation over the imposed oxygen oscillations under baseline and hyperinsulinemic euglycemic conditions (N=4 animals, 52 capillaries).Figure 1Time series of capillary red blood cell supply rate response to imposed oxygen oscillations under baseline and hyperinsulinemic euglycemic conditions (N=4 animals, 135 capillaries).Figure 2

High-fat diet pre-conditioning improves microvascular remodelling during regeneration of ischaemic mouse skeletal muscle.

Critical limb ischaemia (CLI) is characterized by inadequate angiogenesis, arteriolar remodelling and chronic myopathy, which are most severe in type 2 diabetic patients. Hypertriglyceridaemia, commonly observed in these patients, compromises macrovascular function. However, the effects of high-fat diet-induced increases in circulating lipids on microvascular remodelling are not established. Here, we investigated if high-fat diet would mimic the detrimental effect of type 2 diabetes on post-ischaemia vascular remodelling and muscle regeneration, using a mouse model of hindlimb ischaemia. Male C57Bl6/J mice were fed with normal or high-fat diets for 8weeks prior to unilateral femoral artery ligation. Laser doppler imaging was used to assess limb perfusion recovery. Vascular recovery, inflammation, myofibre regeneration and fibrosis were assessedat 4 or 14days post-ligation by histology and RNA analyses. Capillary-level haemodynamics were assessed by intravital microscopy of control and regenerating muscles 14days post-ligation. High-fat diet increased muscle succinate dehydrogenase activity and capillary-level oxygen supply. At 4days post-ligation, no diet differences were detected in muscle damage, inflammatory infiltration or capillary activation. At 14days post-ligation, high fat-fed mice displayed accelerated limb blood flow recovery, elevated capillary and arteriole densities as well as greater red blood cell supply rates and capillary-level oxygen supply. Regenerating muscles from high fat-fed mice displayed lower interstitial fat and collagen deposition. The muscle-level adaptations to high-fat diet improved multiple aspects of muscle recovery in response to ischaemia and did not recapitulate the worse outcomes seen in diabetic CLI patients.

A Reproducible and Simple Method to Generate Red Blood Cells from Human Pluripotent Stem Cells

Erythroid cells generated from human pluripotent stem cells (hPSCs) can potentially offer an unlimited and safe supply of red blood cells (RBCs) for transfusion. Human PSC-derived erythroid cells at various stages of differentiation can also be used to model blood diseases, test new drug candidates, and develop cellular and genetic therapies. Although several protocols for deriving RBCs from hPSCs have been described, these are typically complex, involving multiple culture steps that may include co-culture with feeder cells, and exhibit large variability in erythroid cell yields between hPSC lines and replicate experiments. We have developed a straightforward, serum-free and feeder-free culture method to generate erythroid cells from hPSCs with high yields and high purity. The method has been validated on multiple human embryonic stem (ES) cell lines (H1, H7, H9) and induced pluripotent stem (iPS) cell lines (WLS-1C, STiPS-F016, STiPS-B004). The protocol involves two steps: hematopoietic specification of hPSCs, followed by differentiation of hPSC-derived hematopoietic stem and progenitor cells (HSPCs) into erythroid cells. Lineage specification and differentiation is driven by only three supplements that combine cytokines and other factors to support optimal differentiation efficiency and cell yield across cell lines. In the first step, small hPSC aggregates routinely maintained in feeder-free maintenance medium, are plated onto matrigel-coated microwells, and specification to mesoderm and subsequent hematoendothelial differentiation is induced by addition of successive expansion supplements. This phase promotes extensive hematopoietic progenitor cell generation, with a single hPSC producing on average 142 HSPCs (range: 50 - 360, n = 3 experiments) by day 10 across all six ES and iPS cell lines tested. The average frequency of cells expressing CD43, an embryonic pan-hematopoietic marker, is 92% (range: 85 - 95%), and the frequency of CD34+ cells ranges between 24-55%. In the second, erythroid differentiation step, hPSC-derived HSPCs expand on average 300-fold (range: 80 - 1000) within 10 - 14 days, and the average frequency of GlyA+ cells is 75% (range: 70 - 85%). Cumulatively, this results in the generation of on average 30,000 GlyA+ cells (range: 10,000 - 80,000) per initial hPSC after 20 - 24 days. Further maturation in 7-day cultures containing EPO and human serum resulted in a > 90% pure population of GlyA+ erythroid cells. Notably, no cell loss was observed during the maturation phase, resulting in an average yield of 50,000 GlyA+ cells (range: 10,000 - 220,000) per single initial hPSC on day 31. Differentiated cells were characterized by orthochromatic normoblast morphology and decreased CD71 expression, consistent with erythroid maturation. Erythroid cells generated in this differentiation culture system expressed a mix of 'primitive' and 'definitive' hemoglobin types, but with adult and fetal hemoglobin being expressed at higher levels than embryonic hemoglobin. The observed enucleation rates of hPSC-derived erythroid cells are consistent with current reports and are subject to further optimization. In summary, we have developed a two-step, serum- and feeder-free erythroid differentiation method to generate large numbers of erythroid cells from multiple hPSC lines. This culture system provides a simple, standardized and reproducible platform to generate RBCs from hPSCs with high yields and efficiency for basic and translational research. Disclosures Walasek: STEMCELL Technologies, Inc: Employment. Chau:STEMCELL Technologies, Inc: Employment. Barborini:STEMCELL Technologies, Inc: Employment. Richardson:STEMCELL Technologies, Inc: Employment. Szilvassy:STEMCELL Technologies, Inc: Employment. Louis:STEMCELL Technologies, Inc: Employment. Thomas:STEMCELL Technologies, Inc: Employment. Eaves:STEMCELL Technologies, Inc: Employment. Wognum:STEMCELL Technologies, Inc: Employment.

Blood supply chain management: robust optimization, disruption risk, and blood group compatibility (a real-life case)

Based on the uncertain conditions such as uncertainty in blood demand and facility disruptions, and also, due to the uncertain nature of blood products such as perishable lifetime, distinct blood groups, and ABO-Rh(D) compatibility and priority rules among these groups, this paper aims to contribute blood supply chains under uncertainty. In this respect, this paper develops a bi-objective two-stage stochastic programming model for managing a red blood cells supply chain that observes above-mentioned issues. This model determines the optimum location-allocation and inventory management decisions and aims to minimize the total cost of the supply chain includes fixed costs, operating costs, inventory holding costs, wastage costs, and transportation costs along with minimizing the substitution levels to provide safer blood transfusion services. To handle the uncertainty of the blood supply chain environment, a robust optimization approach is devised to tackle the uncertainty of parameters, and the TH method is utilized to make the bi-objective model solvable. Then, a real case study of Mashhad city, in Iran, is implemented to demonstrate the model practicality as well as its solution approaches, and finally, the computational results are presented and discussed. Further, the impacts of the different parameters on the results are analyzed which help the decision makers to select the value of the parameters more accurately.

Application of a Novel Microfluidic Device for Studying Microvascular Blood Flow Regulation In Vivo

ObjectiveValidate the efficacy of a novel liquid microfluidic device (LMFD) to manipulate microvascular blood flow by maintaining a fixed solute concentration, temperature, and physiologic gas conditions in a microscale region of tissue while simultaneously allowing for visualization and quantification of vascular geometry and hemodynamics.MethodsLMFD flow channels were moulded in polydimethylsiloxane (PDMS) using soft lithography techniques. A diffusive exchange window was created by patterning a 600×300 μm laser cut micro‐outlet into a glass coverslip. Cured PDMS flow channels were plasma bound to both the coverslip containing the micro‐outlet, and a glass slide to produce a complete LMFD.Ten Sprague‐Dawley rats (149–197g) were anesthetized with sodium pentobarbital; indwelling cannulas were placed into the left common carotid and right jugular vein for systemic monitoring, anaesthetic administration and fluid resuscitation; animals were then tracheotomized and mechanically ventilated. The extensor digitorum longus muscle of the lower hind‐limb was carefully dissected, externalized, and reflected across the LMFD secured in the stage of an inverted Olympus IX73 microscope. The muscle was allowed to equilibrate for 30 minutes while the LMFD was perfused with buffered Krebs solution at 37□.Logarithmic doses (10−8 M to 10−3 M) of adenosine triphosphate (ATP), acetylcholine (Ach), and phenylephrine (PE) were sequentially administered to the muscle via perfusion through the LMFD. The LMFD was perfused with each drug concentration for 5 minutes prior to data collection. Video of microvascular blood flow was recorded from multiple focal planes within the volume directly overlying the micro‐outlet. Recordings were analyzed offline to measure velocity, hematocrit, and supply rate. All animal protocols were approved by Memorial University's Institutional Animal Care Committee.ResultsATP significantly increased red blood cell supply rate (RBC SR) to 36.1 and 36.5 cell/s at concentrations of 10−4 and 10−3 M respectively (p < 0.01) compared to the baseline level of 14.4 cell/s. Increasing concentrations of PE caused a graded change in RBC SR with significant decreases to 9.1, 3.5, and 0.8 cells/s at 10−5, 10−4, 10−3 M respectively (p < 0.02). Changes in perfusion were observed only in areas directly overlying the micro‐outlet and within 200–300 μm of the window. Functional images generated at the highest drug concentrations showed that microvascular perfusion was unaffected in regions at greater distances from the micro‐outlet. Evan's Blue dye was used to confirm there were no leaks around the exchange window and that only tissue overlying the micro‐outlet was directly exposed to solutions perfusing the LMFD.ConclusionsOur novel LMFD allows for a controlled, continuous delivery of dissolved substances to highly constrained regions of microvasculature while simultaneously allowing visualization and measurement of blood flow within discrete vessels and networks. Blood flow changes can be directly observed in the region of interest while the surrounding vasculature is unaffected thus preserving integrated regulation to the tissue as a whole.Support or Funding InformationProject funded through a NSERC Discovery grant to GM Fraser.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Measurement and interpretation of hemoglobin concentration in clinical and field settings: a narrative review.

Anemia affects over 800 million women and children globally. Defined as a limited or insufficient functional red blood cell supply in peripheral blood, anemia causes a reduced oxygen supply to tissues and can have serious health consequences for women and children. Hemoglobin (Hb) concentration is most commonly measured for anemia diagnosis. Methods to measure Hb are usually invasive (requiring a blood sample); however, advances in diagnostic and clinical chemistry over the past decade have led to the development of new noninvasive methods. Accurate diagnosis at the individual level is important to identify individuals who require treatment. At the population level, anemia prevalence estimates are often the impetus for national nutrition policies or programs. Thus, it is essential that methods for Hb measurement are sensitive, specific, accurate, and reproducible. The objective of our narrative review is to describe the basic principles, advantages, limitations, and quality control issues related to methods of Hb measurement in clinical and field settings. We also discuss other biomarkers and tests that can help to determine the severity and underlying causes of anemia. In conclusion, there are many established and emerging methods to measure Hb concentration, each with their own advantages, limitations, and factors to consider before use.

Establishing performance management objectives and measurements of red blood cell inventory planning in a large tertiary care hospital in British Columbia, Canada

BackgroundTraditional key performance indicators (KPIs) for red blood cell (RBC) inventory management such as blood shortage rate (BSR) and outdate rate (ODR) alone are observed to be insensitive for large hospitals, often due to fluctuating demands and rapid turnover. We hypothesized that improvement in complementary KPIs for RBC supply chains, including age of blood as a surrogate for the pre‐hospital and in‐hospital supply chains, may further improve efficiency.MethodsRed blood cell supply, inventory and disposition data from a large tertiary care hospital blood bank were retrospectively assessed from June 2014 to 2015 as the baseline period. From June 2015 to 2016, (1) collaboration with the blood supplier to improve logistics and (2) a ‘demand‐driven inventory planning policy’ (DDIP) to determine better inventory levels aided by discrete‐event simulation modelling were instituted. Age of blood transfused (ABT) was chosen as the main KPI for the efficiency of the entire RBC supply chain.ResultsImprovements in age of blood received (ABR) led to the greatest efficiency gains. Reduction in ABT (28.7 ± 8.8 days vs. 22.1 ± 9.5 days, P < 0.01), ABR (19.4 ± 8.8 days vs. 13.4 ± 7.0 days, P < 0.01), inventory‐to‐transfusion ratio (P < 0.01), O‐negative RBC utilization (7.4 vs. 6.4 units/day, P < 0.01) and ODR (P < 0.01) compared to the baseline period was observed without a significant increase in BSR.ConclusionCollaboration with the blood supplier to improve logistics, implementation of DDIP to determine better inventory levels and use of KPIs other than BSR and ODR led to inventory efficiency gains in a large tertiary care hospital blood bank.

Effectiveness of intervention due to feedback on errors arising from inappropriate transportation and storage of blood bags in hospitals: a quasi-experimental study.

BackgroundThe limited supply of red blood cells (RBCs) for transfusion has clinical implications, and can lead to the introduction of different approaches in decreasing blood wastage and the safe and effective delivery of blood products.ObjectiveTo identify the influences of feedback-based intervention on inappropriate temperatures during transportation and storage of blood bags.MethodsThis was a quasi-experimental pretest-posttest design with a non-equivalent control group that was carried out on 200 RBC. The study was conducted in a teaching hospital in Mashhad, Iran, and in two periods during the same season: winter of 2015 (pre-intervention period) and winter of 2016 (post-intervention period). Staff of the blood bank department, as the intervention group, received the intervention including feedback regarding the inappropriate temperature in the blood bank during the pre-intervention period. The control group included personnel and nurses from the OR, CSICU, and transportation department, who did not receive any feedback. The effect of the intervention on the RBCs’ temperature was evaluated by comparing the percentage of inappropriate temperature of RBC bags before and after the implementation of the intervention.ResultsInappropriate temperature in the blood bank decreased from 30% to 12% after the intervention was implemented (relative reduction of 60%). Meanwhile, there was even an increase in inappropriate temperature in the control group including OR (from 6.5% to 20.5%), CSICU (from 1% to 2.5%) and transportation (from 0.6% to 16.6%).ConclusionThe implementation of a feedback-based intervention to increase the awareness of the staff of critical conditions can improve conditions of blood storage and transport. The use of various interventions along with this type of intervention seems necessary to increase effectiveness.FundingThis research was part of the first author’s MSc thesis which was supported by a grant from Mashhad University of Medical Science Research Council (Ref. no.: 950116; Date: September 7, 2016).

Analysis on distribution of leukocyte-free suspended red blood cells clinical supply of different ABO blood groups and seasons in Binzhou City of Shandong Province from 2007 to 2016

Objective To investigate different ABO blood groups and seasonal distribution of leukocyte-free suspended red blood cells clinical supply in Binzhou City of Shandong Province from 2007 to 2016, in order to provide scientific basis for the development of blood collection and blood supply plans for Binzhou Blood Center, and to facilitate the effective and rational use of blood resources. Methods From 2007 to 2016, a total of 578 848.5 U leukocyte-free suspended red blood cells which supplied to all medical institutions in Binzhou City from Binzhou Blood Center were selected as research subjects. The clinical supply of leukocyte-free suspended red blood cells from 2007 to 2016 was collected using the Qi′ao Blood Station Information Management System. The composition ratios of leukocyte-free suspended red blood cells clinical supply in different ABO blood groups and quarters from 2007 to 2016 were calculated, respectively. Trend graphs of leukocyte-free suspended red blood cells clinical supply in the different ABO blood groups and quarters, as well as composition ratio trend graph of leukocyte-free suspended red blood cells clinical supply in different quarters were drew by Microsoft EXCEL 2003 software. The composition ratios of leukocyte-free suspended red blood cells clinical supply in different ABO blood groups and quarters were compared through χ2 test, respectively. Results ① From 2007 to 2016, leukocyte-free suspended red blood cells supply for all clinical institutions in Binzhou City were 41 249.0 U, 44 888.0 U, 52 608.0 U, 60 477.5 U, 63 663.5 U, 64 699.5 U, 59 698.5 U, 63 199.5 U, 61 631.5 U and 66 733.5 U in each year, respectively. From 2007 to 2010, the supply of leukocyte-free suspended red blood cells for all clinical institutions in Binzhou City showed a rapid upward trend. The rising trend from 2010 to 2012 slowed down and declined from 2013 to 2015. In 2016, it rebounded significantly, reaching the highest value during 10 years. ② From 2007 to 2016, leukocyte-free suspended red blood cells supply for all clinical institutions in Binzhou City in different ABO blood groups from high to low in turn were 197 278. U in B blood group, 164 624.5 U in O blood group, 152 728.5 U in A blood group and 64 217.5 U in AB blood group, which were with the composition ratios of 34.08%, 28.44%, 26.38% and 11.09%, respectively. From 2007 to 2016, the difference of composition ratios of leukocyte-free suspended red blood cells clinical supply in different ABO blood groups in Binzhou City was statistically significant (χ2=12 967.41, P<0.01). ③ From 2007 to 2016, leukocyte-free suspended red blood cells supply for all clinical institutions in Binzhou City in different quarters from high to low in turn were 147 501.0 U in the fourth quarter, 145 777.0 U in the second quarter, 143 993.5 U in the third quarter and 141 577.0 U in the first quarter, which were with the composition ratios of 25.48%, 25.18%, 24.88% and 24.46%, respectively. From 2007 to 2016, the difference of composition ratios of leukocyte-free suspended red blood cells clinical supply of different quarters in Binzhou City was statistically significant (χ2=43.75, P<0.01). Conclusions There is an ABO blood groups and seasonal disequilibrium of leukocyte-free suspended red blood cells clinical supply in Binzhou City. Blood collection and supply institutions should adjust the blood supply and collection plan in time according to their changing trends, in order to prevent seasonal blood shortage and storage deviation of ABO blood group. Key words: Blood donors; Blood component transfusion; Erythrocyte transfusion; ABO blood-group system; Blood group distribution; Seasonal

Inhibition of NOS Does Not Affect Oxygen Dependent Capillary Blood Flow Response In Vivo

ObjectiveTo determine if nitric oxide (NO) plays a significant role in regulating blood flow, in response to changing oxygen (O2) conditions within skeletal muscle capillaries.MethodsFive male Sprague‐Dawley rats, 140 – 190g rats were anaesthetized with pentobarbital, tracheotomized, ventilated, and maintained in a normotensive state. A laparotomy was performed and the left common iliac was catheterized with a cannula advanced retrograde to the distal abdominal aorta. The extensor digitorum longus muscle was blunt dissected and reflected onto a glass cover slip (dose response), or permeable membrane above a gas exchange chamber (O2 response) set within a microscope stage. The muscle was bathed in warm saline and isolated from the air via polyvinylidene chloride film and a glass coverslip. Dose response was determined in 3 animals by injecting a nitric oxide synthase inhibitor, L‐NMMA, in logarithmic increasing concentrations ranging from 10−5 to 1 mg/kg into the iliac artery. O2 dependent blood flow responses were determined in 2 animals. Gas concentrations (N2, CO2, and O2) within the exchange chamber were dynamically controlled using mass flow valves connected to a computer. The muscle was allowed to equilibrate for 30 minutes at 5% CO2, 5% O2 and 90% N2. O2 concentrations at the surface of the muscle were oscillated from 7%–12%‐2%–7% in a square‐wave with each level maintained for 60s. CO2 was held at a constant 5% with N2 composing the balance of gas within the exchange chamber. Video sequences of capillary blood flow at 10× magnification were recorded in multiple fields in each animal. Videos were analyzed offline using custom software written in MATLAB yielding frame‐by‐frame measurements of capillary velocity, hematocrit and red blood cell supply rate (SR).ResultsCapillary blood flow dose response for L‐NMMA was measured in 178 capillaries under baseline, and increasing concentrations of L‐NMMA. All L‐NMMA concentrations caused a significant decrease in SR compared to vehicle (Figure 1) with a 42% reduction at 10−2 mg/kg; higher doses did not result in a further decrease of SR. The time dependent O2 blood flow response was measured in 58 capillaries under baseline conditions and after infusion of 10−2 mg/kg L‐NMMA (Figure 1). An average phase lag of 20 s was observed in the SR response to O2 concentration changes in the chamber under both conditions. Phase lag was partially due to the intrinsic time delay of the gas delivery system. Average SR under 7% O2 concentration was 45% lower with L‐NMMA compared to control. High O2 caused a decrease in SR of 53% for control vs. 60% under L‐NMMA, normalized to the baseline mean SR at 7% O2. Low O2 caused an increase in SR of 14% and 47% with control and L‐NMMA respectively. Post oscillation, 7% O2 restored average SR to 93% and 100% of baseline in control and L‐NMMA respectively.ConclusionAlteration of tissue surface O2 causes an O2 dependent capillary blood flow response to both low and high O2 challenges that is preserved following the introduction of a nitric oxide synthase inhibitor. This suggests that NO does not play a significant role in regulating blood flow, in response to changing O2 conditions within skeletal muscle capillaries.

Trends in age and red blood cell donation habits among several racial/ethnic minority groups in the United States.

To meet the needs of a diverse patient population, an adequate supply of red blood cells (RBCs) from ethnic/racial minority donors is essential. We previously described the 10-year changes in minority blood donation in the United States. This study describes donation patterns by donor status, age, and race/ethnicity. Data on the age and the number of unique black/African American, Hispanic/Latino, Asian, and white RBC donors were obtained from eight US blood collectors for 2006, 2009, 2012, and 2015. Donors self-identified their race/ethnicity. First-time (FT) and repeat (R) donors were analyzed separately. Overall, for both FT and R donor groups, whites constituted the majority of unique donors (FT 66.7% and R 82.7%) and also donated the greatest proportion of RBC units (FT 66.6% and R 83.8%). Donors less than 20 years old comprised the greatest proportion of FT donors for all racial/ethnic groups (39.2%) and had the highest mean number of RBC donations per donor (1.12) among FT donors. Conversely, R donors less than 20 years old had some of the lowest mean number of RBC donations per donor (1.55) among R donors, whereas R donors at least 60 years old had the highest mean (1.88). Year by year, the percentage of FT donors who were less than 20 years old increased for all race/ethnicities. For R donors, whites were more frequently older, while Hispanics/Latinos and Asians were younger. Greater efforts to convert FT donors less than 20 years into R donors should be undertaken to ensure the continued diversity of the blood supply.

Group O RBCs: where is universal donor blood being used

There have been recurrent shortages of group O blood due to insufficient inventory and use of group O blood in ABO non-identical recipients. We performed a 12-year retrospective study to determine utilization of group O Rh-positive and Rh-negative red blood cells (RBCs) by recipient ABO group. Reasons for transfusing group O blood to ABO non-identical recipients were also assessed. Utilization data from all group O Rh-positive and Rh-negative RBCs transfused at three academic hospitals between April 2002 and March 2014 were included. Data were extracted from Transfusion Registry for Utilization Surveillance and Tracking, a comprehensive database with inventory information on all blood products received at the hospitals. Extracted data included product type, ABO and Rh, final disposition (transfused, wasted, outdated), and demographic and clinical data on all patients admitted to hospital. Descriptive statistics were performed using sas 9.3. There were 314 968 RBC transfusions: 151 645 (48·1%) were group O, of which 138 136 (91·1%) RBC units were transfused to group O individuals. ABO non-identical recipients received 13 509 group O RBCs (8·9%). The percentage of group O RBCs transfused to ABO non-identical recipients by fiscal year varied from 7·8% to 11·1% with a steady increase from 2011 to 2013. Reasons for this included: trauma, outdating, outpatient usage and shortages. The practice of transfusing O RBCs to non-O individuals has been increasing. Specific hospital and blood supplier policies could be targeted to change practice, leading to a more sustainable group O red blood cell supply.

Sepsis impairs microvascular autoregulation and delays capillary response within hypoxic capillaries

IntroductionThe microcirculation supplies oxygen (O2) and nutrients to all cells with the red blood cell (RBC) acting as both a deliverer and sensor of O2. In sepsis, a proinflammatory disease with microvascular complications, small blood vessel alterations are associated with multi-organ dysfunction and poor septic patient outcome. We hypothesized that microvascular autoregulation—existing at three levels: over the entire capillary network, within a capillary and within the erythrocyte—was impaired during onset of sepsis. This study had three objectives: 1) measure capillary response time within hypoxic capillaries, 2) test the null hypothesis that RBC O2-dependent adenosine triphosphate (ATP) efflux was not altered by sepsis and 3) develop a framework of a pathophysiological model.MethodsThis was an animal study, comparing sepsis with control, set in a university laboratory. Acute hypotensive sepsis was studied using cecal ligation and perforation (CLP) with a 6-hour end-point. Rat hindlimb skeletal muscle microcirculation was imaged, and capillary RBC supply rate (SR = RBC/s), RBC hemoglobin O2 saturation (SO2) and O2 supply rate (qO2 = pLO2/s) were quantified. Arterial NOx (nitrite + nitrate) and RBC O2-dependent ATP efflux were measured using a nitric oxide (NO) analyzer and gas exchanger, respectively.ResultsSepsis increased capillary stopped-flow (p = 0.001) and increased plasma lactate (p < 0.001). Increased plasma NOx (p < 0.001) was related to increased capillary RBC supply rate (p = 0.027). Analysis of 30-second SR–SO2–qO2 profiles revealed a shift towards decreased (p < 0.05) O2 supply rates in some capillaries. Moreover, we detected a three- to fourfold increase (p < 0.05) in capillary response time within hypoxic capillaries (capillary flow states where RBC SO2 < 20 %). Additionally, sepsis decreased the erythrocyte’s ability to respond to hypoxic environments, as normalized RBC O2-dependent ATP efflux decreased by 62.5 % (p < 0.001).ConclusionsSepsis impaired microvascular autoregulation at both the individual capillary and erythrocyte level, seemingly uncoupling the RBC acting as an “O2 sensor” from microvascular autoregulation. Impaired microvascular autoregulation was manifested by increased capillary stopped-flow, increased capillary response time within hypoxic capillaries, decreased capillary O2 supply rate and decreased RBC O2-dependent ATP efflux. This loss of local microvascular control was partially off-set by increased capillary RBC supply rate, which correlated with increased plasma NOx.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-015-1102-7) contains supplementary material, which is available to authorized users.