On the shape of human red blood cells interacting with flat artificial surfaces--the 'glass effect'.

The morphology of the human red blood cell (RBC) contained between two flat artificial surfaces has been investigated. Shape transformation from the discocytic into various crenated (echinocytic) states was not caused solely by glass ('glass effect'). Various organic polymers, and mica, were effective, provided the distance (0.1 mm) between the two surfaces was carefully controlled. The discocytic state could only be preserved using moderately hydrophobic glass, extensive dimethylsilylation induced stomatocytes. With washed blood samples crenation occurred in a potassium chloride medium and in the presence of EDTA. Temperature-dependent transformation in the shape of human erythrocytes occurred between two glass surfaces 0.1 mm apart, e.g., in a hemacytometer. With cells in blood diluted directly 200-times with isotonic saline crenation appeared at 32-36 degrees C. A sphero-echinocytic state prevailed at 34 degrees C and outside the temperature range of 32-36 degrees C the RBCs retained the shape of a biconcave disk. Cells responding to the 'glass effect' even at temperatures below the transition region did not respond further at elevated temperatures. The 'glass effect' was found to be dependent on the RBC concentration (hematocrit). Raising this concentration reversibly decreased the degree of crenation. The amount of endogenous albumin present was estimated to be insufficient to cover the exposed glass surfaces with a protein monolayer. With washed cells over a wide concentration range, approximately the same total amount of albumin (serum) had to be present to avoid crenation, as long as observation was performed at a fixed low cell concentration. The effect of albumin was not abolished by gamma-globulin or anti-human albumin IgG. The discocyte-stabilizing influence of albumin is discussed.     

Crenation and cupping of the red cell: A new theoretical approach. Part I. Crenation

Crenation can be thought of as a surface instability caused by intrinsic precurvature of the membrane. Mathematical modeling, on the presupposition that the red blood cell is a thin shell consisting of a connected (coupled) bilayer having uniformly distributed elastic properties shows that crenation can be initiated by negative precurvature, that is, intrinsic curvature having its concavity directed towards the outside of the cell. This is contrary to the currently accepted view which attributes the effect to positive precurvature of an unconnected bilayer. Crenation and the biconcave shape can coexist in the red cell. This suggests that the bilayer must be connected even when the cell is crenated because the biconcave shape could not otherwise be maintained. The progressive development of crenation to more advanced stages, such as the echinocyte type III and the spheroechinocyte can be accounted for if the outer layer of the membrane is stressed beyond the range where strain is proportional to stress. This is consistent with the extremely small radius of curvature at the tips of the crenations.Certain small variations in the uncrenated biconcave shape of the red cell can be interpreted mathematically as due either to negative intrinsic curvature or to shear resistance. Since, however, a small amount of negative precurvature has been shown to be capable of inducing crenation, it is unlikely to be the cause of the variations in the biconcave shape. These must therefore be due to shear resistance.In the light of this new approach, membrane molecular models based on the assumption that crenation is due to positive precurvature need reconsideration.     

Effects of monovalent cations on red cell shape and size

Human erythrocytes were incubated in isotonic solutions of different monovalent cations. The apparent size of the red cells measured on scanning electron microscopic pictures decreases in the order Li+ greater than Na+ = K+ greater than Rb+. These differences in size are abolished after pretreatment with trypsin, which removes a large part of the charges associated with membrane glycoproteins. Shape alterations are also observed. Normal biconcave shapes are visible after Na+ or K+ incubation, whereas Li+ leads to flabby, flattened cells with a certain tendency to crenation, and Rb+ causes more pronounced biconcavity with a certain tendency to cupping. The overall effects of pretreatment with trypsin are similar to those of Li+. Our results provide evidence that the electrostatic repulsion of glycoproteins and other charged membrane components may play an essential role in maintaining red cell shape.     

Effects of membrane reference state on shape memory of a red blood cell

By using a three-dimensional continuum model, we simulate the shape memory of a red blood cell after the remove of external forces. The purpose of this study is to illustrate the effect of membrane reference state on cell behavior during the recovery process. The reference state of an elastic element is the geometry with zero stress. Since the cell membrane is composed of cytoskeleton and lipid bilayer, both the reference states of cytoskeleton (RSC) and lipid bilayer (RSL) are considered. Results show that a non-spherical RSC can result in shape memory. The energy barrier due to non-spherical RSC is determined by the ratio of the equator length to the meridian length of the RSC. Thus different RSCs can have similar energy barrier and leading to identical recovery response. A series of simulations of more intermediate RSCs show that the recovery time scale is inversely proportional to the energy barrier. Comparing to spherical RSL, a spheroid RSL contributes to the energy barrier and recovery time. Furthermore, we observe a folding recovery due to the biconcave RSL which is different from the tank treading recovery. These results may motivate novel numerical and experimental studies to determine the exact RSC and RSL.     

Role of the bilayer in the shape of the isolated erythrocyte membrane

Summary The determinants of cell shape were explored in a study of the crenation (spiculation) of the isolated erythrocyte membrane. Standard ghosts prepared in 5mm NaP_i (pH 8) were plump, dimpled disks even when prepared from echinocytic (spiculated) red cells. These ghosts became crenated in the presence of isotonic saline, millimolar levels of divalent cations, 1mm 2,4-dinitrophenol or 0.1mm lysolecithin. Crenation was suppressed in ghosts generated under conditions of minimal osmotic stress, in ghosts from red cells partially depleted of cholesterol, and, paradoxically, in ghosts from red cells crenated by lysolecithin. The susceptibility of ghosts to crenation was lost with time; this process was potentiated by elevated temperature, low ionic strength, and traces of detergents or chlorpromazine.In that ghost shape was influenced by a variety of amphipaths, our results favor the premise that the bilayer and not the subjacent protein reticulum drives ghost crenation. The data also suggest that vigorous osmotic hemolysis induces a redistribution of lipids between the two leaflets of the bilayer which affects membrane contour through a bilayer couple mechanism. Subsequent relaxation of that metastable distribution could account for the observed loss of crenatability.     

Bending Resistance and Chemically Induced Moments in Membrane Bilayers

Pure bending of a membrane bilayer is developed including different properties for each membrane half. Both connected and unconnected bilayer surfaces are treated. The bilayer bending resistance is the resultant of parallel surface compression “resistances.” The neutral surface is a function of the upper and lower surface compressibility moduli and does not necessarily coincide with the mid-surface. Alterations in the interfacial chemical free energy density (surface tension) on either face can create induced bending moments and produce curvature; even small changes can have a pronounced curvature effect. Chemically induced moments are considered as a possible mechanism for crenation of red blood cells.     

Interaction of added amphiphilic lipids with the membrane of intact human erythrocytes to induce change in the cell shape

Addition of an appropriate amount of amphiphilic lipid, such as fatty acid, lysophospholipid and medium-chain phospholipid, into a suspension of human erythrocytes (pH 7.4) at 37 degree C resulted in their incorporation into the membrane and induction of a cell shape change of crenation (echinocyte-spherocyte) type without causing hemolysis. The extent of the shape change was dependent on the amount of the lipid incorporated and the crenation disappeared on removing the incorporated molecules from the membrane. The crenation induced by acidic lipids was further altered drastically by resuspending the treated cells in media of pH 6, 7, and 8, whereas that induced by choline-phospholipid or -lysophospholipid was not so pH-dependent. Based on these results, the mechanism of this shape change is discussed.     

Effects of monovalent cations on red cell shape and size

Human erythrocytes were incubated in isotonic solutions of different monovalent cations. The apparent size of the red cells measured on scanning electron microscopic pictures decreases in the order Li⁺>Na⁺=K⁺>Rb⁺. These differences in size are abolished after pretreatment with trypsin, which removes a large part of the charges associated with membrane glycoproteins. Shape alterations are also observed. Normal biconcave shapes are visible after Na⁺ or K⁺ incubation, whereas Li⁺ leads to flabby, flattened cells with a certain tendency to crenation, and Rb⁺ causes more pronounced biconcavity with a certain tendency to cupping. The overall effects of pretreatment with trypsin are similar to those of Li⁺. Our results provide evidence that the electrostatic repulsion of glycoproteins and other charged membrane components may play an essential role in maintaining red cell shape.     

The RBC morphological dependence of the RBC disaggregability

The aim of this study was to determine the red blood cell (RBC) disaggregability dependence upon the RBC shape. The study concentrated on stored blood during bank storage and on suspensions of artificially induced echinocytes. Measurements was performed in autologous plasma of hematocrit 0.45 and at constant plasmatic content. Rheological studies using stationary viscometry, nonstationary viscometry and rheoscopy were made in order to assess different stages of the disaggregability process. Whatever the method of measurement used, the morphological interpretation of the results reveal that beyond 75% of echinocytes within the sample, the disaggregation process is altered. The shear stresses required to dissociate the echinocyte aggregates are significantly higher than those required to disaggregate normal RBC rouleaux.     

Influence of storage and buffer composition on the mechanical behavior of flowing red blood cells

On-chip study of blood flow has emerged as a powerful tool to assess the contribution of each component of blood to its overall function. Blood has indeed many functions, from gas and nutrient transport to immune response and thermal regulation. Red blood cells play a central role therein, in particular through their specific mechanical properties, which directly influence pressure regulation, oxygen perfusion, or platelet and white cell segregation toward endothelial walls. As the bloom of in-vitro studies has led to the apparition of various storage and sample preparation protocols, we address the question of the robustness of the results involving cell mechanical behavior against this diversity. The effects of three conservation media (EDTA, citrate, and glucose-albumin-sodium-phosphate) and storage time on the red blood cell mechanical behavior are assessed under different flow conditions: cell deformability by ektacytometry, shape recovery of cells flowing out of a microfluidic constriction, and cell-flipping dynamics under shear flow. The impact of buffer solutions (phosphate-buffered saline and density-matched suspension using iodixanol/Optiprep) are also studied by investigating individual cell-flipping dynamics, relative viscosity of cell suspensions, and cell structuration under Poiseuille flow. Our results reveal that storing blood samples up to 7 days after withdrawal and suspending them in adequate density-matched buffer solutions has, in most experiments, a moderate effect on the overall mechanical response, with a possible rapid evolution in the first 3 days after sample collection.     

Effect of dehydration on the viscoelastic behavior of red cells.

Dehydration of red cells alters their rheological behavior and may contribute to the pathology of disorders such as sickle cell disease. We have measured the viscoelastic properties of individual human HbAA red cells after graded dehydration induced by incubation with valinomycin at different external K levels. With dehydration, the cells underwent progressive reduction in their rate of extensional shape recovery (i.e., after elongation by micropipette manipulation). Their rigidity remained unaffected until the mean cell hemoglobin concentration (MCHC) rose above 50 g/dl, but then increased about 100% as judged from the response of membrane tongues drawn into micropipettes. There was also a marked reduction in the shape recovery rate at this level of dehydration, and the cells no longer behaved elastically but rather showed unrecoverable residual deformation. Additionally, the cytoplasm took on solid-like properties. Changes in cell rigidity and shape recovery rate have been previously demonstrated for dense sickle cells; our results indicate that normal red cells can be induced to behave similarly, but that a greater degree of dehydration is required.     

Fatty acid composition of erythrocyte and vesicle total lipids during storage of human erythrocytes in protein-free media and in citrate-phosphate-dextrose

We have studied the fatty acid composition of total lipids of erythrocytes and vesicles either during storage of human erythrocytes in their own plasma under blood bank conditions or during incubation at 37 degrees C in protein-free media. Vesicles appear as a heterogeneous population with a diameter of about 130 nm and a varying content of hemoglobin. The fatty acid pattern of vesicle total lipids changes in respect to control erythrocytes, while in erythrocytes it remains fairly constant during the total ageing period. Our results indicate a marked rearrangement of the membrane components during blood storage.     

Erythrocyte concentrates, low in buffy coat: production, preservation and evaluation of its quality

Buffy coat-poor packed red cells were prepared from fresh ACD-, ACD-AG- and EDTA-blood, than resuspended with a preservation solution, containing glucose, adenine, guanosine, sucrose, citric acid and sodium citrate and stored at 4 degrees C for 6 weeks. The survival rate of resuspended red cells from ACD-AG-blood amounted to 77% after 6 weeks of storage. The ATP content of resuspended red cells was approximately 25% lower than in ACD-AG whole blood during storage caused probably by increased ATP consuming reactions at the red cell membrane. The P2G-content of resuspended red cells from ACD- and ACD-AG-blood decreased above 50% of the normal level during the first week, as fast as in ACD- and ACD-AG whole blood. The P2G-breakdown in red cells from EDTA-blood was delayed for a week due to the higher pH as in CPD blood. Additions of xylitol, inorganic phosphate, and bicarbonate in 6, 5 and 20 mM final concentrations in the red cell suspensions and an increased pH at the same time delayed the breakdown of ATP and P2G. Packed red cells can be administered fast enough at hematocrits to 0.60 that will be achieved by adding 50 to 100 ml preservation solution. Leukocytes and thrombocytes were reduceds to 70 to 80%. With increasing rate of reduction a higher loss of red cells occured. Buffy coat-poor red cell concentrate contains only few microaggregates. It diminishes the risc of febrile transfusion reactions and delays the appearance of alloimmunisation. The circulatory overload of patients is less frequent than after transfusions of red cell resuspensions containing a large resuspension volume.     

Measurement of adenosine triphosphate and 2,3-diphosphoglycerate in stored blood with 31P nuclear magnetic resonance spectroscopy

Conditions for blood storage are chosen to assure adequate levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG). Because of the invasive nature of the techniques, biochemical assays are not routinely used to measure levels of these compounds in stored blood. However, 31P NMR spectroscopy measures phosphorylated intermediates in intact cells and could be used without disruption of the storage pack. We compared levels of ATP and 2,3-DPG measured by 31P spectroscopy and standard enzyme-linked biochemical assays in whole blood (WB) and packed red blood cells (PRBCs) at weekly intervals during a 35-day storage period. NMR demonstrated a marked decrease in 2,3-DPG and an increase in inorganic phosphate after the first week of storage. No significant differences in ATP concentrations were seen in WB during the storage period, but a significant decrease in ATP in PRBCs was documented. There was good agreement in levels of ATP and 2,3-DPG measured by NMR and biochemical techniques. 31P NMR spectroscopy is a noninvasive technique for measuring ATP and 2,3-DPG which has a potential use in quality assurance of stored blood.     

Blood banking-induced senescent modifications on red blood cells.

In the past few years significant progress has been made in the extension of storage time for red blood cells (RBCs). Albeit this, membrane still undergoes damage during conservation under blood bank conditions, reducing red cell viability following transfusion. Consequently in this study, we evaluated the presence of senescence indices (appearing on RBCs during in vivo aging) on blood bank stored RBCs, more precisely the 4.1a/4.1b ratio and the binding of autologous immunoglobulins. Although not significant, a slight increase (0.995 +/- 0.070 to 1.008 +/- 0.058) was observed in the 4.1a/4.1b ratio during the first three weeks of storage. A rapid and significant (p less than 0.001) increase in the number of cell-bound IgGs (91 +/- 22 to 913 +/- 92), following incubation with purified autologous IgGs, was observed in the first days of storage. Thus, following transfusion, binding of autologous IgGs to stored RBCs could influence post-transfusion viability.     

Changes in transferrin during the red cell replacement in amphibia

Transferrin, a plasma glycoprotein, carries iron from storage sites to immature erythroid cells for hemoglobin synthesis. The replacement of larval red cells by adult red cells, which occurs during metamorphosis in bullfrogs, requires extensive formation of hemoglobin and new red cells. Large changes in red cell iron storage also occur during the red cell replacement. Both the concentration and the level of iron saturation of plasma transferrin were measured during metamorphosis to determine if there were changes in plasma transferrin which coincided with the changes in red cell iron storage and ferritin content. Plasma transferrin concentrations increased from 0.96 to 2.6 mg/ml during the period when red cell storage iron and ferritin decreased. Plasma iron concentrations also increased when the transferrin concentration increased, suggesting that the additional transferrin may be involved in moving iron from the larval red cell stores. At the end of metamorphosis, the plasma iron concentration decreased to premetamorphic levels but the transferrin concentration remained high, resulting in a decrease in saturation to 18% compared to 45% in the larvae. In addition to differences in iron saturation, adult transferrin had different electrophoretic properties from larval transferrin. The results support the hypotheses that during early ontogeny plasma transferrin and red cell iron storage are coordinated to provide iron for the formation of the first generation of adult red cells and that transferrin may participate in the control of red cell ferritin synthesis.     

A new method to study shape recovery of red blood cells using multiple optical trapping.

In this new method for studying the shape recovery of deformed red blood cells, three optical traps ("optical tweezers") induce a parachute-shaped red cell deformation, which is comparable to the deformation in small capillaries. The shape recovery is recorded, and a relaxation time is obtained for each individual red blood cell. The sensitivity of this technique for the detection of differences in relaxation times is demonstrated on subpopulations of density-separated red blood cells: "young" cells have shorter (162 ms) and "old" cells have longer (353 ms) relaxation times compared with the total population (271 ms). The relaxation time is remarkably shorter (114 ms) when the plasma surrounding the cells is replaced by a phosphate-buffered saline solution. The main advantages of this technique are the relatively short measuring and preparation time and the physiological type of deformation and shape recovery in which all relevant cell properties play a role. Therefore, especially when automated further, the technique may be a powerful tool for the study of (sub)populations of pathological red blood cells.     

The effect of stabilizer composition on the thrombocytes and thrombocyte function in stored whole blood. II. Results of the thrombocyte spreading test]

The behaviour of thrombocyte spreading in ACD, AcD-A and AcD-AG stabilized blood was examined in 18 apparently healthy test persons for a storage period of 9 days. Due to an improved energy metabolism of thrombocytes the addition of adenin or guanosin respectively in ACD stabilized blood will cause the thrombocyte spreading to be widely preserved during storage.     

Preservation of red blood cells with purines and nucleosides. II. Uptake and utilization of purines and nucleosides by stored red blood cells

The uptake of adenine, guanine, guanosine and inosine by stored red cells was investigated in whole blood and red cell resuspensions at initial concentrations of 0.25, 0.5 and 0.75 mM for adenine and 0.5 mM for the other additives using a rapid ion-exchange chromatographic microanalysis of purines and nucleosides in plasma and whole blood. Increasing adenine concentrations from 0.25 to 0.75 mM in blood elevated the adenine uptake from 0.3 up to 0.8 mmol/l red cells during 2 hours after collecting blood. The intra-/extracellular distribution ratio changed from 1 : 1.3 to 1: 1.7. Some 2 hours after withdrawing blood into CPD--solution with purines and nucleosides the uptake of adenine and guanine resulted in 40 per cent and 70 per cent respectively and of guanosine and inosine in 80 and 90 per cent respectively. The replacement of plasma by a resuspending solution gave the same uptake rates for purines and nucleosides. The nucleosides were rapidly split to purines and R-1-P and disappeared from blood during one week. Adenine and guanine were utilized to 80 to 90 per cent only after 3 weeks. During the same period the utilization of guanine was smaller by 40 per cent than that of adenine due to the different activity of the purine nucleoside phosphorylase for these substrates. The plasma of all analyzed blood samples contained hypoxanthine and inosine, but guanine and guanosine were detected only in those samples to which one of them was added. After 3 weeks of storage the highest concentration of hypoxanthine was found in CPD-AI blood with 600 microM in plasma and the highest concentration of synthesized inosine in CPD-AG blood with a concentration of 100 microM in plasma. Three ways of utilization of purines by stored red cells were discussed : the synthesis of nucleotide monophosphates, the formation of nucleosides, and the deamination. The portions of these ways change during storage. The most effective concentrations of adenine and guanosine in stored blood seems to be 0.25 and 0.5 mM respectively. The full utilization of the nucleoside requires the addition of inorganic phosphate.     

The miRNA Profile of Platelets Stored in a Blood Bank and Its Relation to Cellular Damage from Storage

Millions of blood products are transfused each year, and many lives are directly affected by transfusion. Platelet concentrate (PC) is one of the main products derived from blood. Even under good storage conditions, PC is likely to suffer cell damage. The shape of platelets changes after 5 to 7 days of storage at 22°C. Taking into consideration that some platelet proteins undergo changes in their shape and functionality during PC storage. Sixteen PC bags were collected and each PC bag tube was cut into six equal pieces to perform experiments with platelets from six different days of storage. Thus, on the first day of storage, 1/6 of the tube was used for miRNA extraction, and the remaining 5/6 was stored under the same conditions until extraction of miRNAs on each the following five days. Samples were sequenced on an Illumina Platform to demonstrate the most highly expressed miRNAs. Three miRNAs, mir127, mir191 and mir320a were validated by real-time quantitative PCR (RQ-PCR) in 100 PC bags tubes. Our method suggests, the use of the miRNAs mir127 and mir320a as biomarkers to assess the "validity period" of PC bags stored in blood banks for long periods. Thus, bags can be tested on the 5th day of storage for the relative expression levels of mir127 and mir320a. Thus, we highlight candidate miRNAs as biomarkers of storage damage that can be used as tools to evaluate the quality of stored PC. The use of miRNAs as biomarkers of damage is unprecedented and will contribute to improved quality of blood products for transfusions.