Human red blood cells with normal or improved oxygen transport function prepared and frozen in the primary polyvinyl chloride plastic blood collection container.

This is a report a a new system for freezing human red blood cells in the same polyvinyl chloride plastic container in which the blood is collected and separated into components. This polyvinyl chloride plastic collection bag with integrally attached transfer packs for blood collection, component separation, red blood cell biochemical modification, freezing, storage, and post-thaw dilution before washing, represents a major advancement in the freeze-preservation process. The label with the donor's blood type and identification number affixed to the bag at the time of collection remains in place throughout the freezing and thawing process. The transfused red blood cells are of superior quality, and the processing cost is less than with other methods of freeze-preservation. There is a lower risk of contamination with these red blood cells because manipulation of the product is kept at a minimum. "Rejuvenation", a bioengineering process by which outdated red blood cells can be salvaged, can be incorporated into the preservation process using one of the attached transfer packs of the primary collection bag. This process has been introduced as a possible means of alleviating the dramatic blood shortages which occur periodically. Red blood cells may also be "rejuvenated" after storage in the liquid state to increase their 2,3 DPG and ATP levels to 150 to 200% of normal, and these red blood cells with improved oxygen transport function have been administered to anemic patients with and without cardiopulmonary insufficiency, patients undergoing cardiopulmonary bypass and treatment with hypothermia during cardiac surgery, and in instances where nonhemolytic transfusion reactions might be expected.     

Combined action of nicotinamide and adenine in preservation of packed red cells

An alkaline resuspension solution "Erythronaph" with adenine and nicotinamide has been developed. It allows to preserve red blood cells for 35 days. Removing leukocytes and platelets up to 90% by brief warning of 24-hour blood prior to centrifugation combined with return of 10% of plasma provide further prolongation of storage of red blood cells in "Erythronaph".     

Erythrocyte storage increases rates of NO and nitrite scavenging: implications for transfusion-related toxicity

Storage of erythrocytes in blood banks is associated with biochemical and morphological changes to RBCs (red blood cells). It has been suggested that these changes have potential negative clinical effects characterized by inflammation and microcirculatory dysfunction which add to other transfusion-related toxicities. However, the mechanisms linking RBC storage and toxicity remain unclear. In the present study we tested the hypothesis that storage of leucodepleted RBCs results in cells that inhibit NO (nitric oxide) signalling more so than younger cells. Using competition kinetic analyses and protocols that minimized contributions from haemolysis or microparticles, our data indicate that the consumption rates of NO increased ~40-fold and NO-dependent vasodilation was inhibited 2-4-fold comparing 42-day-old with 0-day-old RBCs. These results are probably due to the formation of smaller RBCs with increased surface area: volume as a consequence of membrane loss during storage. The potential for older RBCs to affect NO formation via deoxygenated RBC-mediated nitrite reduction was also tested. RBC storage did not affect deoxygenated RBC-dependent stimulation of nitrite-induced vasodilation. However, stored RBCs did increase the rates of nitrite oxidation to nitrate in vitro. Significant loss of whole-blood nitrite was also observed in stable trauma patients after transfusion with 1 RBC unit, with the decrease in nitrite occurring after transfusion with RBCs stored for >25?days, but not with younger RBCs. Collectively, these data suggest that increased rates of reactions between intact RBCs and NO and nitrite may contribute to mechanisms that lead to storage-lesion-related transfusion risk.     

Pyridoxalated hemoglobin polyoxyethylene: a nitric oxide scavenger with antioxidant activity for the treatment of nitric oxide-induced shock

Hemoglobins modified for therapeutic use as either hemoglobin-based oxygen carriers or scavengers of nitric oxide are currently being evaluated in clinical trials. One such product, pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), is a human-derived and chemically modified hemoglobin that has yielded promising results in Phase II clinical trials, and is entering a pivotal Phase III clinical trial for the treatment of shock associated with systemic inflammatory response syndrome (SIRS). Shock associated with SIRS is a NO-induced shock. PHP, a new mechanism-based therapy, has been demonstrated in clinical trials to have the expected hemodynamic activity of raising blood pressure and reducing catecholamine use, consistent with its mechanism of action as a NO scavenger. PHP is conjugated with polyoxyethylene, which results in a surface-decorated molecule with enhanced circulation time and stability as well as in attachment of soluble red blood cell enzymes, including catalase and superoxide dismutase. PHP thus contains an antioxidant profile similar to the intact red blood cell and is therefore resistant to both initial oxidative modification by oxidants such as hydrogen peroxide and subsequent ferrylhemoglobin formation. These studies suggest both that the redox activity of modified hemoglobins can be attenuated and that modified hemoglobins containing endogenous antioxidants, such as PHP, may have reduced pro-oxidant potential. These antioxidant properties, in addition to the NO-scavenging properties, may allow the use of PHP in other indications in which excess NO, superoxide, or hydrogen peroxide is involved, including ischemia-reperfusion injury and hemorrhagic shock.     

Design of a Cryogenic Videocamera-Recorder and Image Processing System and Calculation of Volumes of Red Cells

Since Rowe reported that the storage technique for red cells at very low temperatures had been realized successfully (4, 5), many experts who work in the fields of cryobiology and medicine have turned their attention to this storage technique for tissues and organs (3). Since the first quantitative cryomicroscope was made successfully about 20 years ago (1), it has been possible to observe changes in shape and phase. Particularly the image processing technique has laid the foundation for quantitative analysis of the relationship between changes in shape and damage from freezing of cells and cooling rate and storage temperature. In this work, we constructed a system consisting of a cryomicroscope, a videocamera-recorder, and an image processor. We carried out many experiments with red cells in cold storage and have established a model for calculating the volumes of red cells. In our experiments we also dynamically traced the shape changes of cells with various experimental parameters.     

Rapid removal of glycerol from frozen‐thawed red blood cells

The storage of red blood cells (RBCs) in a refrigerated state allows a shelf life of a few weeks, whereas RBCs frozen in 40% glycerol have a shelf life of 10 years. Despite the clear logistical advantages of frozen blood, it is not widely used in transfusion medicine. One of the main reasons is that existing post‐thaw washing methods to remove glycerol are prohibitively time consuming, requiring about an hour to remove glycerol from a single unit of blood. In this study, we have investigated the potential for more rapid removal of glycerol. Using published biophysical data for human RBCs, we mathematically optimized a three‐step deglycerolization process, yielding a procedure that was less than 32 s long. This procedure was found to yield 70% hemolysis, a value that was much higher than expected. Consequently, we systematically evaluated three‐step deglycerolization procedures, varying the solution composition and equilibration time in each step. Our best results consisted of less than 20% hemolysis for a deglycerolization time of 3 min, and it is expected that even further improvements could be made with a more thorough optimization and more reliable biophysical data. Our results demonstrate the potential for significantly reducing the deglycerolization time compared with existing methods. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:609–620, 2013     

Impact of blood manufacturing and donor characteristics on membrane water permeability and in vitro quality parameters during hypothermic storage of red blood cells

Several factors have been proposed to influence the red blood cell storage lesion including storage duration, blood component manufacturing methodology, and donor characteristics [1,18]. The objectives of this study were to determine the impact of manufacturing method and donor characteristics on water permeability and membrane quality parameters.Red blood cell units were obtained from volunteer blood donors and grouped according to the manufacturing method and donor characteristics of sex and age. Membrane water permeability and membrane quality parameters, including deformability, hemolysis, osmotic fragility, hematologic indices, supernatant potassium, and supernatant sodium, were determined on day 5 ± 2, day 21, and day 42. Regression analysis was applied to evaluate the contribution of storage duration, manufacturing method, and donor characteristics on storage lesion.This study found that units processed using a whole blood filtration manufacturing method exhibited significantly higher membrane water permeability throughout storage compared to units manufactured using red cell filtration. Additionally, significant differences in hemolysis, supernatant potassium, and supernatant sodium were seen between manufacturing methods, however there were no significance differences between donor age and sex groups.Findings of this study suggest that the membrane-related storage lesion is initiated prior to the first day of storage with contributions by both blood manufacturing process and donor variability. The findings of this work highlight the importance of characterizing membrane water permeability during storage as it can be a predictor of the biophysical and chemical changes that affect the quality of stored red blood cells during hypothermic storage.     

Specific binding of red blood cells to endothelial cells is regulated by nonadsorbing macromolecules

Abnormal adhesion of red blood cells to the endothelium has been linked to the pathophysiology of several diseases associated with vascular disorders. Various biochemical changes, including phosphatidylserine exposure on the outer membrane of red blood cells as well as plasma protein levels, have been identified as being likely to play a key role, but the detailed interplay between plasma factors and cellular factors remains unknown. It has been proposed that the adhesion-promoting effect of plasma proteins originates from ligand interaction, but evidence substantiating this assumption is often missing. In this work, we identified an alternative pathway by demonstrating that nonadsorbing macromolecules can also have a marked impact on the adhesion efficiency of red blood cells with enhanced phosphatidylserine exposure to endothelial cells. It is concluded that this adhesion-promoting effect originates from macromolecular depletion interaction and thereby presents an alternative mechanism by which plasma proteins could regulate cell-cell interactions. These findings should thus be of potential value for a detailed understanding of the pathophysiology of diseases associated with vascular complications and might be applicable to a wide range of cell-cell interactions in plasma or plasma-like media.     

Proteomics and transfusion medicine: future perspectives

Limited number of important discoveries have greatly contributed to the progresses achieved in the blood transfusion; ABO histo-blood groups, citrate as anticoagulant, fractionation of plasma proteins, plastic bags and apheresis machines. Three major types of blood products are transfused to patients: red cell concentrates, platelet concentrates and fresh frozen plasma. Several parameters of these products change during storage process and they have been well studied over the years. However, several aspects have completely been ignored; in particular those related to peptide and protein changes. This review presents what has been done using proteomic tools and the potentials of proteomics for transfusion medicine.     

Studies of the deformability of preserved red blood cells using a simple filtration method]

The applied method to measure the filtrability of red blood cells is accurate enough for the study of the effects of the conditions of storage on their deformability. In this work the standard deviation of the method, the mean biological deviation of the deformability of human red blood cells and their deformability in the course of storage in the ACD-AG-medium have been investigated. Considerable loss of deformability occurs towards the end of storage time of the cells, which is not reversible be restitution of ATP alone. The applicability of this method as a simple test of the vitatity of preserved erythrocytes if to be tested.     

Stored red blood cells: A changing universe waiting for its map(s)

The availability of stored red blood cells (RBCs) for transfusion remains an important aspect of the treatment of polytrauma, acute anemia or major bleedings. RBCs are prepared by blood banks from whole blood donations and stored in the cold in additive solutions for typically six weeks. These far from physiological storage conditions result in the so-called red cell storage lesion that is of importance both to blood bankers and to clinical practitioners. Here we review the current state of knowledge about the red cell storage lesion from a proteomic perspective. In particular, we describe the current models accounting for RBC aging and response to lethal stresses, review the published proteomic studies carried out to uncover the molecular basis of the RBC storage lesion, and conclude by suggesting a few possible proteomic studies that would provide further knowledge of the molecular alterations carried by RBCs stored in the cold for six weeks.     

The RAINFOR database: monitoring forest biomass and dynamics

Problem: Data from over 100 permanent sample plots which have been studied for 10–20 years need a suitable system for storage which allows simple data manipulation and retrieval for analysis. Methods: A relational database linking tree records, taxonomic nomenclature and corresponding environmental data has been built in MS Access as part of the RAINFOR project. Conclusion: The database allows flexible and long‐term use of a large amount of data: more than 100 tree plots across Amazonia, incorporating over 80 000 records of individual trees and over 300 000 total records of tree diameter measurements from successive censuses. The database is designed to enable linkages to existing soil, floristic or plant‐trait databases. This database will be a useful tool for exploring the impact of environmental factors on forest structure and dynamics at local to continental scales, and long term changes in forest ecology. As an early example of its potential, we explore the impact of different methodological assumptions on estimates of tropical forest biomass and carbon storage.     

Optimal synthesis of chromatographic trains for downstream protein processing

Downstream bioprocessing and especially chromatographic steps, commonly used for the purification of multicomponent systems, are significant cost drivers in the production of therapeutic proteins. There has been an increased interest in the development of systematic methods for the design of such processes, and the appropriate selection of a series of chromatographic steps is still a major challenge to be addressed. Several models have been developed previously but have assumed that 100% recovery of the desired product is obtained at each chromatographic step. In this work, a mathematical framework is proposed, based on mixed integer optimisation techniques, that removes this assumption and allows full flexibility on the position of retention time cut-points, between which the desired product fraction is collected. The proposed model is demonstrated on three example protein mixtures, each containing up to 13 contaminants and selecting from a set of up to 21 candidate steps. The proposed model results in a reduction of one to three chromatographic steps over solutions that no losses are allowed.     

Dynamics of the measured oxidative potential of preserved blood]

An expression for oxidative potential (phi) of conservated blood is suggested proceeding from the assumption of the steady character of the system methemoglobin -- hemoglobin, oxihemoglobin included. This system is suggested to be responsible for the formation of phi in the blood. The equation qualitatively describes the experimental relation of phi on time at the contact of blood with air. Relationship between the changes of oxidative potential in the blood during its storage with the state of conservated blood hemoglobin is discussed. The experimental data show that measuring of the oxidative potential may be the basis for creating a control method for the state of conservated blood during its storage.     

Using storage organelles for the accumulation and encapsulation of recombinant proteins

Plants have been used to produce many diverse and valuable recombinant proteins, including subunit vaccines, antibodies and antibody fragments, hormones, blood products, cytokines, and enzymes. Different plant species and platforms have been explored as production hosts, each with unique properties in terms of the gene transfer method, production time, environmental containment, scalability, downstream processing strategy, protein folding and accumulation, and overall costs. Seed-based systems have many advantages because they exploit the natural storage properties of seeds, which facilitate batch processing and distribution. Seeds possess specialized storage organelles that may be used to accumulate recombinant proteins, offering stability both in planta and after harvest in the final preparation/formulation. The post-harvest stabilizing effect of seeds allows recombinant subunit vaccines and antibodies to be delivered via the mucosal route because they are better able to withstand this harsh microenvironment when protected by the plant matrix. Native storage organelles such as starch granules and protein bodies offer this protective effect, but protein storage organelles can also be induced ectopically in vegetative tissues. In this paper, we discuss the technical capabilities of storage organelle-based expression platforms and their potential applications.     

Survival of the fittest?--survival of stored red blood cells after transfusion.

During the last 90 years many developments have taken place in the world of blood transfusion. Several anticoagulants and storage solutions have been developed. Also the blood processing has undergone many changes. At the moment, in The Netherlands, red blood cell (RBC) concentrates (prepared from a whole blood donation and leukocyte-depleted by filtration) are stored for a maximum of 35 days at 4 degrees C in saline adenine glucose mannitol (SAGM). Most relevant studies show that approximately 20% of the RBCs is lost in the first 24 hr after transfusion. Even more remarkable is that the average life span is 94 days after a storage period of 42-49 days. Such observations create the need for a parameter to measure the biological age of RBCs as a possible predictor of the fate of RBCs after transfusion. The binding of IgG to RBCs can lead to recognition and subsequent phagocytosis by macrophages. This occurs during the final stages of the RBC life span in vivo. We determined the quantity of cell-bound IgG during storage, and found considerable variation between RBCs, but no significant storage-related change in the quantity of cell-bound IgG. The significance of this finding for predicting the survival of transfused RBCs in vivo remains to be established. Hereto we developed a flow cytometric determination with a sensitivity of 0.1% for the measurement of survival in vivo based on antigenic differences. This technique has various advantages compared with the 'classical' 51Cr survival method.     

Osmotic hemolysis and fragility A new model based on membrane disruption,and a potential clinical test

Red cell osmotic hemolysis has traditionally been defined by the loss of hemoglobin, in response to reduced osmotic pressure, as measured spectroscopically. Previous work from this laboratory using resistive pulse spectroscopy (RPS) has shown that in a mixed population of hemolyzing cell, ghosts can be detected as being more deformable, and hence appearing distinctly smaller, than the remaining intact cells. Other researchers using similar methods have reported detection of ghosts as apparently smaller objects, resulting from their greater sensitivity to dielectric breakdown. We now confirm both of these results, and demonstrate by kinetic studies that changes which occur in the rheological and electrical properties of ghosts are independent phenomena. We include in our analysis the explicit calculation of ghost and intact spherocyte resistivity after dielectric breakdown. The two different characterizations for ghosts are integrated into a proposed model of osmotic hemolysis based on known red blood cell membrane and cytoplasmic properties. This work provides both a theoretical and a practical foundation for RPS-based measures of osmotic fragility, including a potential new clinical test, measures which provide very early detection of the ultimate fate of osmotically stressed red cells.     

Frozen-thawed red cells--standard article for hospital use?

The freezing and storing of glycerol protected red cells allows the present 20---40 day limitation of red cell storage to be indefinitely lengthened. Although frozen thawed cells are more expensive to prepare than ordinary packed cells, the longer shelf-life of the red cells, the reduced risk of hepatitis transmission, and the reduction of immunization against leuko- and thrombocytes, are all advantages which out-weighed this additional cost. To effect an increase in their use, frozen thawed cells could be distributed from central blood banks to hospitals were they would be stored until needed for transfusion. Thawing and washing would necessarily be done by the hospital blood bank personnel.     

Optical tweezers as a new biomedical tool to measure zeta potential of stored red blood cells

During storage, red blood cells (RBCs) for transfusion purposes suffer progressive deterioration. Sialylated glycoproteins of the RBC membrane are responsible for a negatively charged surface which creates a repulsive electrical zeta potential. These charges help prevent the interaction between RBCs and other cells, and especially among each RBCs. Reports in the literature have stated that RBCs sialylated glycoproteins can be sensitive to enzymes released by leukocyte degranulation. Thus, the aim of this study was, by using an optical tweezers as a biomedical tool, to measure the zeta potential in standard RBCs units and in leukocyte reduced RBC units (collected in CPD-SAGM) during storage. Optical tweezers is a sensitive tool that uses light for measuring cell biophysical properties which are important for clinical and research purposes. This is the first study to analyze RBCs membrane charges during storage. In addition, we herein also measured the elasticity of RBCs also collected in CPD-SAGM. In conclusion, the zeta potential decreased 42% and cells were 134% less deformable at the end of storage. The zeta potential from leukodepleted units had a similar profile when compared to units stored without leukoreduction, indicating that leukocyte lyses were not responsible for the zeta potential decay. Flow cytometry measurements of reactive oxygen species suggested that this decay is due to membrane oxidative damages. These results show that measurements of zeta potentials provide new insights about RBCs storage lesion for transfusion purposes.