Fresh Red Blood Cells Research Articles

Large-scale production of Plasmodium falciparum gametocytes for malaria drug discovery

The tightly controlled induction of Plasmodium falciparum gametocytes in large-scale culture is a fundamental requirement for malaria drug discovery applications including, but not limited to, high-throughput screening. This protocol uses magnetic separation for isolation of hemozoin-containing parasites in order to (i) increase parasitemia, (ii) decrease hematocrit and (iii) introduce higher levels of young red blood cells in a culture simultaneously within 2-4 h. These parameters, along with red blood cell lysis products that are generated through schizont rupture, are highly relevant for enabling optimum induction of gametocytogenesis in vitro. No other previously published protocols have applied this particular approach for parasite isolation and maximization of fresh red blood cells before inducing gametocytogenesis, which is essential for obtaining highly synchronous gametocyte classical stages on a large scale. In summary, 500-1,000 million stage IV gametocytes can be obtained within 16 d from an initial 10 ml of asexual blood-stage culture.

Clearance of stored red blood cells is not increased compared with fresh red blood cells in a human endotoxemia model.

It is thought that the clearance of transfused red blood cells (RBCs) is related both to the storage time of the transfusion product and to the inflammatory status of the recipient. We investigated these effects in a randomized, "two-hit," healthy volunteer transfusion model, comparing autologous RBCs that were stored for 35 days with those that were stored for 2 days. Healthy male volunteers donated 1 unit of autologous RBCs either 2 days (2D) or 35 days (35D) before the study date. The experiment was started by infusion of 2 ng/kg lipopolysaccharide ("first hit"). Two hours later, the stored RBCs ("second hit") were reinfused, followed by the labeling of RBCs with biotin. Clearance of biotin-labeled RBCs (BioRBCs) was measured during the 5-hour posttransfusion endotoxemia period along with measurements of phosphatidylserine (PS) exposure, lactadherin binding, and expression of CD47 (cluster of differentiation 47; a transmembrane protein encoded by the CD47 gene). In the 2D stored RBCs group, 1.5% ± 3.4% of infused BioRBCs were cleared from the circulation 5 hours posttransfusion versus 4.8% ± 4.0% in the 35D stored RBCs group (p = 0.1). There were no differences in PS exposure, lactadherin binding, or CD47 expression between fresh and stored RBCs or between pretransfusion and posttransfusion measurements. Our study shows a low clearance of RBCs even during endotoxemia. Furthermore, short-term clearance of BioRBCs during endotoxemia was not related to storage duration. Consistent with these observations, PS exposure, lactadherin binding, and CD47 expression did not differ between 2D and 35D stored cells before or after transfusion. We conclude that, in the presence of endotoxemia, clearance of 35D stored autologous RBCs is not increased compared with 2D stored fresh RBCs.

2,3-Diphosphoglycerate Concentrations in Autologous Salvaged Versus Stored Red Blood Cells and in Surgical Patients After Transfusion

Stored red blood cells (RBCs) are deficient in 2,3-diphosphoglycerate (2,3-DPG), but it is unclear how autologous salvaged blood (ASB) compares with stored blood and how rapidly 2,3-DPG levels return to normal after transfusion. Therefore, we compared levels of 2,3-DPG in stored versus ASB RBCs and in patients' blood after transfusion. Twenty-four patients undergoing multilevel spine fusion surgery were enrolled. We measured 2,3-DPG and the oxyhemoglobin dissociation curve (P50) in samples taken from the ASB and stored blood bags before transfusion and in blood samples drawn from patients before and after transfusion. The mean storage duration for stored RBCs was 24 ± 8 days. Compared with fresh RBCs, stored RBCs had decreased 2,3-DPG levels (by approximately 90%; P < 0.0001) and a decreased P50 (by approximately 30%; P < 0.0001). However, ASB RBCs did not exhibit these changes. The mean 2,3-DPG concentration decreased by approximately 20% (P < 0.05) in postoperative blood sampled from patients who received 1 to 3 stored RBC units and by approximately 30% (P < 0.01) in those who received ≥4 stored RBC units. 2,3-DPG was unchanged in patients who received no stored blood or ASB alone. After surgery, 2,3-DPG levels recovered gradually over 3 postoperative days in patients who received stored RBCs. Stored RBCs, but not ASB RBCs, have decreased levels of 2,3-DPG and a left-shift in the oxyhemoglobin dissociation curve. Postoperatively, 2,3-DPG levels remain below preoperative baseline levels for up to 3 postoperative days in patients who receive stored RBCs but are unchanged in those who receive only ASB RBCs.

Transfusion of recently donated (fresh) red blood cells (RBCs) does not improve survival in comparison with current practice, while safety of the oldest stored units is yet to be established: a meta-analysis.

Preclinical studies generated the hypothesis that older stored red blood cells (RBCs) can increase transfusion risks. To examine the most updated and complete clinical evidence and compare results between two trial designs, we assessed both observational studies and randomized controlled trials (RCTs) studying the effect of RBC storage age on mortality. Five databases were searched through December 2014 for studies comparing mortality using transfused RBCs having longer and shorter storage times. Analysis of six RCTs found no significant differences in survival comparing current practice (average storage age of 2 to 3weeks) to transfusion of 1- to 10-day-old RBCs (OR 0·91, 95% CI 0·77-1·07). RBC storage age was lower in RCTs vs. observational studies (P=0·01). The 31 observational studies found an increased risk of death (OR 1·13, 95% CI 1·03-1·24) (P=0·01) with increasing age of RBCs, a different mortality effect than RCTs (P=0·02). RCTs established that transfusion of 1- to 10-day-old stored RBCs is not superior to current practice. The apparent discrepancy in mortality between analyses of RCTs and observational studies may in part relate to differences in hypotheses tested and ages of stored RBCs studied. Further trials investigating 1- to 10-day-old stored RBC benefits would seem of lower priority than studies to determine whether 4- to 6-week stored units have safety and efficacy equivalent to the 2- to 3-week-old stored RBCs commonly transfused today.

Biopreservation of RBCs for invitro Plasmodium falciparum culture.

Biopreservation of RBCs for invitro Plasmodium falciparum culture.

Identified metabolic signature for assessing red blood cell unit quality is associated with endothelial damage markers and clinical outcomes.

There has been interest in determining whether older red blood cell (RBC) units have negative clinical effects. Numerous observational studies have shown that older RBC units are an independent factor for patient mortality. However, recently published randomized clinical trials have shown no difference of clinical outcome for patients receiving old or fresh RBCs. An overlooked but essential issue in assessing RBC unit quality and ultimately designing the necessary clinical trials is a metric for what constitutes an old or fresh RBC unit. Twenty RBC units were profiled using quantitative metabolomics over 42 days of storage in SAGM with 3- to 4-day time intervals. Metabolic pathway usage during storage was assessed using systems biology methods. The detected time intervals of the metabolic states were compared to clinical outcomes. Using multivariate statistics, we identified a nonlinear decay process exhibiting three distinct metabolic states (Days 0-10, 10-17, and 17-42). Hematologic variables traditionally measured in the transfusion setting (e.g., pH, hemolysis, RBC indices) did not distinguish these three states. Systemic changes in pathway usage occurred between the three states, with key pathways changing in both magnitude and direction. Finally, an association was found between the time periods of the metabolic states with the clinical outcomes of more than 280,000 patients in the country of Denmark transfused over the past 15 years and endothelial damage markers in healthy volunteers undergoing autologous transfusions. The state of RBC metabolism may be a better indicator of cellular quality than traditional hematologic variables.

Oxidative stress and rheologic properties of stored red blood cells before and after transfusion to surgical patients.

The loss of structural and functional integrity of red blood cells (RBCs) during storage, collectively referred to as "storage lesion," has been implicated in reduced oxygen delivery after transfusion. RBCs are highly susceptible to oxidative damage from generation of reactive oxygen species by autoxidation of hemoglobin. Therefore, we examined whether increased oxidative stress (OS) in stored RBCs is associated with impaired cell membrane deformability before or after transfusion. Thirty-four patients undergoing multilevel spine fusion surgery were enrolled. OS in RBCs was assessed by the presence of fluorescent heme degradation products and methemoglobin, which were measured with fluorimetric and spectrophotometric methods, respectively. Deformability and aggregation were determined by ektacytometry in stored RBCs, autologous salvaged RBCs, and posttransfusion blood samples. OS in stored RBCs was significantly increased with longer storage (R = 0.54, p = 0.032) and significantly higher than that in fresh RBCs (9.1 ± 1.3 fluorescent arbitrary units vs. 7.7 ± 0.9 fluorescent arbitrary units, p < 0.001). Deformability decreased (R = -0.60, p = 0.009) with increasing storage duration. OS was elevated (p < 0.05) and deformability was decreased (p < 0.05) in postoperative blood from patients who had undergone moderate (≥4 RBC units) but not minimal or no transfusion. Neither the decrease in deformability of RBCs nor the aggregation changes were correlated with OS. Although stored RBCs show signs of increased OS and loss of cell membrane deformability, these changes were not directly correlated and were only evident after moderate but not lower dose transfusion in postoperative surgical patients. These findings suggest that factors other than OS may contribute to impaired rheology with stored RBCs in the clinical setting.

Static Laser Light Scattering Studies from Red Blood Cells

A homemade Static Light scattering studies has been used to determine angle resolved scattered intensity for different polarization states of the incident laser light. Classical light scattering set ups are being used to study morphological aspects of scatterers using simple set ups using low power lasers. Red blood cells form rather interesting as well as a challenging system for scattering experiments. The scattering spectrometer consists of a scattering arm, a scattering turn table and collimating arm. Along with polarizers integrated in the collimating arm as well as scattering arms ensures collection of scattered flux with the required polarization state. This technique is being developed for its in vitro studies using fresh red blood cells. A brief review of the theoretical models used for scattering from Red Blood Cells (RBC) has been discussed in the paper. Scattering pattern (scattering plots) as well as polar plots of scattered flux have been determined for different polarization state of the incident light. Insight into the orientation of major axis of particles can be inferred from the polar plots.

An exploratory, hypothesis-generating, meta-analytic study of damage control resuscitation in acute hemorrhagic shock: Examining the behavior of patient morbidity and mortality in the context of plasma-to-packed red blood cell ratios

Both traumatic and nontraumatic hemorrhagic shock continues to be associated with unacceptably high mortality and morbidity. Although significant progress has been made within the transfusion science in terms of research and subsequent implementation of life-saving massive transfusion protocols, controversies persist regarding the optimal fresh frozen plasma-to-packed red blood cell (FFP-to-PRBC) ratios in the setting of hemorrhagic shock resuscitation, especially in the context of postresuscitation sequelae. To further compound the problem, there continues to be a paucity of prospective and high-quality retrospective data in this important clinical area. The goal of this hypothesis-generating, meta-analytic study was to combine data from all available high-quality literature sources in order to enhance our understanding of the relationship between FFP-to-PRBC ratios and associated morbidity/mortality across the entire reported spectrum of transfusion component combinations. Major findings of this analysis include the significant association between increasing FFP-to-PRBC ratios and decreasing mortality, as well as the concurrent increase in morbidity among survivors. More specifically, mortality odds were significantly lower with “higher” versus “lower” FFP-to-PRBC ratios (odds ratio [OR] 0.569; 95% confidence interval [CI] 0.463–0.700) in a combined cohort of 10,610 patients. At the same time, multi-organ failure was more likely to occur in the “higher” FFP-to-PRBC ratio group (OR 1.417, 95% CI 1.243–1.616). Formal studies that focus on risk-benefit aspects of higher FFP-to-PRBC ratios are needed. Research efforts should be directed at continued mortality reduction following massive transfusion while focusing on strategies designed to minimize the incidence and severity of complications among survivors of hemorrhagic shock. The current study provides a potentially useful platform for planning and implementation of future research efforts in the area of damage control resuscitation. The following core competencies are addressed in this article: Patient care, practice-based learning and improvement, systems based practice, medical knowledge

Improving Oxygen Binding of Desiccated Human Red Blood Cells

Desiccating human red blood cells (RBCs) to increase their storage life has been the subject of intense research for a number of years. However, drying RBCs invariably compromises their integrity and has detrimental effects on hemoglobin function due to autoxidation. We have previously demonstrated an RBC desiccation and rehydration process that preserves RBC antigenic epitopes better than frozen RBCs. This study expands on those observations by examining what effects this desiccation process has on RBC hemoglobin function with respect to oxygen binding properties. In this paper, we examined RBCs from normal donors which were desiccated to 25% moisture content and stored dry for 2 weeks at room temperature prior to rehydration with plasma followed by structural and functional studies. Our data showed that approximately 98% of the RBCs were intact upon rehydration based on hemolysis assays. Oxygen dissociation curves for the desiccated/rehydrated RBCs showed a left shift compared to fresh RBCs (pO2 = 17 mmHg vs. 26 mmHg, respectively). The desiccated/rehydrated RBCs also showed an increase in methemoglobin compared to fresh RBCs (4.5% vs 0.9%, respectively). 2,3-Diphosphoglycerate concentration of the desiccated/rehydrated RBCs was reduced by 20%. In conclusion, although this RBC dehydration process preserves RBC integrity and hemoglobin oxygen binding properties better than most other dehydration techniques described so far, further optimization and long-term studies are needed to make this procedure acceptable for human transfusion.

Bodybuilders pneumonia.

Bodybuilders pneumonia.

Extracellular Vesicles from Red Blood Cell Products Induce a Strong Pro-Inflammatory Host Response, Dependent on Both Numbers and Storage Duration

Background: Transfusion of red blood cells (RBCs) is associated with adverse outcome, but the causative factor is unknown. Extracellular vesicles (EVs) have pro-inflammatory properties. We hypothesized that EVs released from both fresh and stored RBC products can induce a host inflammatory response in a dose-dependent manner. Methods: Whole blood was incubated with supernatant from RBC units stored for different time periods, either containing (different numbers of) EVs or depleted from EVs. Results: Incubation with both fresh and stored supernatant containing EVs induced a strong host response with production of TNF, IL-6 and IL-8. In supernatant depleted from EVs, this host response was completely abrogated. IL-10 levels were not affected. EV-induced host response was both dependent on the number of EVs as well as on storage time. Conclusions: EVs from both fresh and stored RBC units illicit a strong inflammatory host response in recipients and may therefore contribute to adverse outcome of RBC transfusion.

Transfusion of fresh versus non-fresh (older) red blood cell in neonates

Transfusion of fresh versus non-fresh (older) red blood cell in neonates

Validating the measurement of red blood cell diameter in fresh capillary blood by darkfield microscopy: A pilot study

ObjectivesAnalysis of fresh capillary blood using darkfield microscopy involves measuring red blood cell (RBC) diameter to assess for microcytosis or macrocytosis. The reference range for normal RBC diameter currently used in fresh capillary blood analysis by darkfield microscopy (FCB-DM) is the same as that used in haematology, i.e. 6.2–8.2μm. However, the haematology reference range for RBC diameter refers to stained and dried blood specimens. Evidence from early research suggests that fresh RBCs are larger than stained and dried RBCs. The following article presents methodology and findings for the objective measurement of RBC diameter using the FCB-DM technique. MethodsFCB-DM screenings of 14 consenting participants were photographed to permit a quantitative analysis of RBC size and morphology. Participants also provided a venous blood sample for pathology testing, which included markers for RBC size. The researcher was blinded to the pathology results until all FCB-DM data analysis was complete. ResultsAll participants had a normal reference range for all pathology markers tested. The mean RBC diameter, determined by FCB-DM, was 8.51μm (s=0.161, s2=0.026, range=±0.656) and the mode was 8.55μm. ConclusionThese findings support evidence from early studies of RBC diameter, that fresh blood cells observed with darkfield microscopy are approximately 1.3μm larger than dried and stained RBCs. Further research using a larger sample size is recommended.

The interaction of sterically stabilized magnetic nanoparticles with fresh human red blood cells.

Sterically stabilized superparamagnetic iron oxide nanoparticles (SPIONs) were incubated with fresh human erythrocytes (red blood cells [RBCs]) to explore their potential application as magnetic resonance imaging contrast agents. The chemical shift and linewidth of 133Cs+ resonances from inside and outside the RBCs in 133Cs nuclear magnetic resonance spectra were monitored as a function of time. Thus, we investigated whether SPIONs of two different core sizes and with three different types of polymeric stabilizers entered metabolically active RBCs, consuming glucose at 37°C. The SPIONs broadened the extracellular 133Cs+ nuclear magnetic resonance, and brought about a small change in its chemical shift to a higher frequency; while the intracellular resonance remained unchanged in both amplitude and chemical shift. This situation pertained over incubation times of up to 90 minutes. If the SPIONs had entered the RBCs, the intracellular resonance would have become broader and possibly even shifted. Therefore, we concluded that our SPIONs did not enter the RBCs. In addition, the T2 relaxivity of the small and large particles was 368 and 953 mM−1 s−1, respectively (three and nine times that of the most effective commercially available samples). This suggests that these new SPIONs will provide a superior performance to any others reported thus far as magnetic resonance imaging contrast agents.

Accelerated clearance of human red blood cells in a rat transfusion model.

BackgroundAnimal models are valuable in transfusion research. Use of human red blood cells (RBCs) in animal models facilitates extrapolation of the impact of storage conditions to the human condition but may be hampered by the use of cross species.MethodsInvestigation of clearance and posttransfusion recovery in a rat model using fresh and stored human RBCs.ResultsDirectly following transfusion, human RBCs could be detected in the circulation of all recipients, with higher recovery rates for stored RBCs than for fresh RBCs. After 24 h following transfusion, no donor RBCs could be detected in the circulation, but donor RBCs could be detected in all organs of all recipients.ConclusionThe use of human donor RBCs in a rat transfusion model resulted in clearance from cells from the circulation. Donor cells were found in different organs of the recipients. Rat transfusion models are thus not appropriate to study the efficacy of human RBC transfusion.

In vitro antiplasmodial activity of Kani herb Alstonia scholaris against Plasmodium falciparum

Background: The Alstonia scholaris plant grows throughout India, in deciduous and evergreen forests and also in plains. The plant is known to possess a lot of medicinal properties in folk medicine by the Kani tribals in Kanyakumari district. A. scholaris bark has been as one of the ingredient used in the marketed Ayurveda preparation Ayush-64, NRDC, India. The milky juice of the plant is applied on wounds, ulcers and rheumatic pains. Tincture of the bark and juice of the leaves act as powerful galactogogue in certain cases. The drug is also used in case of snake bite. Objectives: The present study has been made an attempt to find out the antiplasmodial activity of different plant parts (Leaf, root, bark, stem bark and fruit) of A. scholaris against the most prevalent Indian malarial Parasite P. falciparum. Methods: Different plant arts Viz., Leaf, stem, bark, root and fruit of A. scholaris plant was collected from SouthWest coast of India, Kanyakumari, Tamil Nadu during monsoon and non-monsoon months. The plant parts were cut into pieces and kept for shade drying. Moisture free samples were subjected for percolation by soaking it in ethanol: water mixture (3:1 ratio). Different concentrations of filter-sterilized crude extract from plants (100, 50, 25, 12.5, 6.25 and 3.125 µg. ml −1 ) was incorporated into 96-well tissue culture plate containing 200 μl of P. falciparum culture with fresh red blood cells diluted to 2% haematocrit. Results: Among the plant parts tested against P. falciparum, the bark and leaf extract of A. scholaris collected during non-monsoon season exhibited IC 50 <3.125 µg. ml -1 ; followed by the stem extract (IC 50 3.125 µg. ml -1 ). Conclusion: It can be concluded from the present study that the bark and leaf extracts of A. scholaris potentiate the antiplasmodial activity against P. falciparum.

Transfusion of Old RBCs Induces Neuroinflammation and Cognitive Impairment.

Transfusing RBCs stored for longer than 14 days (old RBC) in humans is common. This transfusion can injure organs, such as lungs and kidneys. We determined whether transfusion with old RBC injured brain. Prospective, controlled animal study. University research laboratory. Adult male Sprague-Dawley rats. Six-month-old Sprague-Dawley rats lost 20% total blood volume and then received RBC prepared from equal volume of the lost blood. RBC was stored for 1 day (fresh RBC) or 7 days (old RBC, storage lesions similar to those of human RBC stored for 28 d). Some rats received IV cell-free hemoglobin. These rats were not subjected to hemorrhage and RBC transfusion. Rats were subjected to Barnes maze and fear conditioning tests from 1 week after blood transfusion. Rats transfused with old RBC but not fresh RBC took a longer time to identify the target hole in the Barnes maze and had less context-related fear conditioning behavior than control rats. Old RBC significantly increased interleukin 6 and ionized calcium-binding adapter molecule 1 in the hippocampus at 24 hours after the transfusion. These effects were attenuated by sulforaphane and minocycline, an antibiotic with anti-inflammatory property. Old RBC solution had a higher concentration of cell-free hemoglobin. Sulforaphane increased haptoglobin, a chelator of cell-free hemoglobin. Rats that received cell-free hemoglobin had a pattern of neuroinflammation and impairment of learning and memory similar to that of rats that received old RBC. These results provide initial evidence to suggest that transfusion of old RBC induces neuroinflammation and impairment of learning and memory. These effects may be mediated by cell-free hemoglobin.

Transfusing Older Red Cells Does Not Affect Mortality

According to this study: * Compared with standard-issue red blood cells, fresh red blood cells do not improve mortality rates among critically ill adults. * Using standard-issue red cells (which may be stored for as long as 42 days) would eliminate the costs associated with the storage and handling of fresh cells.

ACP Journal Club. In critically ill adults, transfusion of fresh vs standard-issue red blood cells did not differ for 90-day mortality.

Source Citation Lacroix J, Hebert PC, Fergusson DA, et al; ABLE Investigators; Canadian Critical Care Trials Group. Age of transfused blood in critically ill adults. N Engl J Med. 2015;372:1410-8. ...