The correlation of human erythrocyte shape with dark-field light scattering intensity]

This study was concerned with the quantitative evaluation of dark field light scattering by sedimented erythrocytes of banked human blood samples. Due to considerable variability of both appearance and amount of scattered light the discocyte group had to be subdivided into discocyte I and discocyte II. The mean intensity of scattered light increased about three fold from discocyte II to echinocytes I, II, III, sphaeroechinocyte, and sphaerocyte. On the other hand the average light scattering intensity of discocytes I exceeded that of discocytes II about 2.5 times, with individual data varying over a wide range. There was a rapid disappearing of discocytes I correlated with time of storage. Therefore it is concluded that discocytes I represent the initial stage of erythrocytes transforming under banking conditions.     

The effect of phorbol esters on human erythrocyte morphological discocyte-echinocyte transitions

12-O-Tetradecanoylphorbol-13-acetate (TPA) (100 nM) when incubated with human erythrocytes under conditions of ATP depletion, delayed the onset of the morphological transition from discocytes to echinocytes so that at 2 h, when control incubations were estimated to contain 65% echinocytes, those treated with TPA contained 23% echinocytes. TPA did not alter the subsequent rate of the transition which was complete by 3 h in control cells and 5 h in TPA-treated cells. Addition of 100 nM TPA to ATP-depleted erythrocytes at 2.5 h (greater than 80% echinocytes) for 0.5 h at 37 degrees C resulted in 17% reversal to a discocyte morphology, but as the time of incubation under conditions of ATP depletion was extended, the level of the reversal fell. TPA had no significant effect on the fall in ATP concentrations over the time course of the experiments (5 h). Preincubation of discocytes with TPA for 10 min also prevented, by approx. 50%, the echinocytosis induced by the calcium (0.2 mM) loading of discocytes using 5 microM A23187. TPA was unable to reverse the echinocyte morphology of calcium-loaded cells back to discocytes. The less potent tumour promotor 4-phorbol-12,13-didecanoate had no effect on this discocyte-echinocyte transition. Incubation of discocytes with the diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) (1-10 microM) had complex effects on morphology, and the ATP-induced morphological transition, ranging from stomatocyte formation to echinocyte formation, depending upon the concentration of the agent and the time of incubation.     

Nano- and microscale mechanical properties of erythrocytes in hereditary spherocytosis

Hereditary spherocytosis (HS), an erythrocyte membranopathy, is a heterogeneous disease, even at the level of the erythrocyte population. The paper aims at studying the mechanical properties (the Young’s modulus, median and RMS roughness of friction force maps; fractal dimension, lacunarity and spatial distribution parameters of lateral force maps) of the cell surface layer of the erythrocytes of two different morphologies (discocytes and spherocytes) in HS using atomic force microscopy. The results of spatial-spectral and fractal analysis showed that the mechanical property maps of the HS spherocyte surface were more structurally homogeneous compared to the maps of HS discocytes. HS spherocytes also had a reduced RMS roughness and lacunarity of the mechanical property maps. The Young’s modulus and averaged friction forces over the microscale HS spherocyte surface regions were approximately 20% higher than that of HS discocytes. The revealed significant difference at the nano- and microscales in the structural and mechanical properties of main (discoidal and spheroidal) morphological types of HS erythrocytes can potentially cause blood flow disturbance in the vascular system in HS.     

Factors of the shape change of human erythrocytes induced with lidocaine

We studied the molecular mechanism of the shape change of erythrocytes with a local anesthetic, lidocaine. The shape of human erythrocytes changed from discocytes to stomatocytes in the presence of lidocaine when ATP was present. But, the shape of resealed cells which were prepared with 10 mM Tris-HCl buffer (pH 7.4) containing 2 mM ATP-MgCl2 and various substances was not changed from discocytes to stomatocytes with lidocaine. When intact cells and resealed cells which were prepared with various concentrations of Tris-HCl buffer (pH 7.4) were incubated with various concentrations of lidocaine and their membrane proteins were analyzed by SDS-PAGE, the densities of bands 62K, 28K and 22K depended on lidocaine concentration: in particular, that of band 28K changed remarkably. These membranous 62K-, 28K- and 22K-proteins agreed with cytoplasmic 62K-, 28K- and 22K-proteins in molecular weight. We propose that not only ATP but also the 62K-, 28K- and 22K-proteins in the cytoplasm are concerned with the shape change of human erythrocytes induced with lidocaine.     

Membrane bilayer balance and erythrocyte shape: a quantitative assessment

When human erythrocytes are incubated with certain phospholipids, the cells become spiculate echinocytes, resembling red cells subjected to metabolic starvation or Ca2+ loading. The present study examines (1) the mode of binding of saturated phosphatidylcholines and egg lysophosphatidylcholine to erythrocytes and (2) the quantitative relationship between phospholipid incorporation and red cell shape. We find that the phospholipids studied become intercalated into erythrocyte membranes, not simply adsorbed to the cell surface. Spin-labeling and radiolabeling data show that the incorporation of (4 +/- 1) X 10(6) molecules of exogenous phosphatidylcholine per cell converts discocytes to stage 3 echinocytes with about 35 conical spicules. This amount of lipid incorporation is estimated to expand the red cell membrane outer monolayer by 1.7% +/- 0.6%. Calculations of the inner and outer monolayer surface areas of model discocytes and stage 3 echinocytes yield an estimated difference of 0.7% +/- 0.2%.     

Inverse pH-dependent shape changes of erythrocytes in the presence of albumin

A comparative study on the shape of human erythrocytes suspended in 7 different media showed, contrary to the well-known albumin-free case, an enhancement of the number of discocytes and stomatocytes for pH rising in all HSA containing media applied. At the same time, the transmembrane potential as determined by extra- and intracellular pH was lowered in all of 6 media tested. Consequently, there is no simple relationship between the pH-dependent behaviour of cell shape and corresponding changes of transmembrane potential.     

Na+/H+ exchange is increased in sickle cell anemia and young normal red cells

Summary Red cell volume regulation is important in sickle cell anemia because the rate and extent of HbS polymerization are strongly dependent on initial hemoglobin concentration. We have demonstrated that volume-sensitive K:Cl cotransport is highly active in SS whole blood and is capable of increasing MCHC. We now report that Na⁺/H⁺ exchange (Na/H EXC), which is capable of decreasing the MCHC of erythrocytes with pH_i<7.2, is also very active in the blood of patients homozygous for HbS. The activity of Na/H EXC (maximum rate) was determined by measuring net Na⁺ influx (mmol/liter cell·hr=FU) driven by an outward H⁺ gradient in oxygenated, acidloaded (pH_i 6.0), DIDS-treated SS cells. The Na/H EXC activity was 33±3 FU (mean±se) (n=19) in AA whites, 37±8 FU (n=8) in AA blacks, and 85±15 FU (n=14) in SS patients (P<0.005). Separation of SS cells into four density-defined fractions by density gradient revealed mean values of Na/H EXC four to five times higher in reticulocytes (SS1), discocytes (SS2) and dense discocytes (SS3), than in the fraction containing irreversibly sickled cells and dense discocytes (SS4). In contrast to K:Cl cotransport, which dramatically decreases after reticulocyte maturation, Na/H EXC persists well after reticulocyte maturation. In density-defined, normal AA red cells, Na/H EXC decreased monotonically as cell density increased. In SS and AA red cells, the magnitude of stimulation of Na/H EXC by cell shrinkage varied from individual to individual. We conclude that Na/H EXC is highly expressed in SS and AA young red cells and decays slowly after reticulocyte maturation.     

Erythrocyte morphology reflects the transbilayer distribution of incorporated phospholipids

The transbilayer distribution of exogenous phospholipids incorporated into human erythrocytes is monitored through cell morphology changes and by the extraction of incorporated 14C-labeled lipids. Dilauroylphosphatidylserine (DLPS) and dilauroylphosphatidylcholine (DLPC) transfer spontaneously from sonicated unilamellar vesicles to erythrocytes, inducing a discocyte-to-echinocyte shape change within 5 min. DLPC-induced echinocytes revert slowly (t1/2 approximately 8 h) to discocytes, but DLPS-treated cells revert rapidly (10-20 min) to discocytes and then become invaginate stomatocytes. The second phase of the phosphatidylserine (PS)-induced shape change, conversion of echinocytes to stomatocytes, can be inhibited by blocking cell protein sulfhydryl groups or by depleting intracellular ATP or magnesium (Daleke, D. L., and W. H. Huestis. 1985. Biochemistry. 24:5406-5416). These cell shape changes are consistent with incorporation of phosphatidylcholine (PC) and PS into the membrane outer monolayer followed by selective and energy-dependent translocation of PS to the membrane inner monolayer. This hypothesis is explored by correlating cell shape with the fraction of the exogenous lipid accessible to extraction into phospholipid vesicles. Upon exposure to recipient vesicles, DLPC-induced echinocytes revert to discoid forms within 5 min, concomitant with the removal of most (88%) of the radiolabeled lipid. On further incubation, 97% of the foreign PC transfers to recipient vesicles. Treatment of DLPS-induced stomatocytes with acceptor vesicles extracts foreign PS only partially (22%) and does not affect cell shape significantly. Cell treated with inhibitors of aminophospholipid translocation (sulfhydryl blockers or intracellular magnesium depletion) and then incubated with either DLPS or DLPC become echinocytic and do not revert to discocytic or stomatocytic shape for many hours. On treatment with recipient vesicles, these echinocytes revert to discocytes in both cases, with concomitant extraction of 88-99% of radiolabeled PC and 86-97% of radiolabeled PS. The accessibility of exogenous lipids to extraction is uniformly consistent with the transbilayer lipid distribution inferred from cell shape changes, indicating that red cell morphology is an accurate and sensitive reporter of the transbilayer partitioning of incorporated exogenous phospholipids.     

The elliptocyte: a study of the relationship between cell shape and membrane structure using the camelid erythrocyte as a model

1. The elliptocytic shape of the camelid erythrocyte is very stable and has a high resistance to modification by drugs and treatment which alter the shape of the discocytic erythrocytes of scimitar-horned oryx and man. 2. Differences in the erythrocyte membrane proteins have been found which indicate that proteins play an important role in stabilisation of the camelid elliptocyte. 3. The organisation of the cytoskeletal network in camelid elliptocytes differs from that established for human discocytes.     

Morphofunctional and biochemical properties of erythrocytes in early postnatal ontogenesis in rats in norm and after prenatal stress

Morphofunctional and biochemical properties of erythrocyte membrane were investigated in early postnatal ontogenesis in rats in norm and after prenatal immobilization stress. The transient decrease of erythrocyte membranes stability was revealed in the control rats. The ability to erythrocyte transformation and the concentration of lipid peroxidation products are increased. It has been shown by an increase of percentage discocytes and lower lipid peroxidation level that the erythrocyte membrane of the rats after prenatal stress is more stable.     

Physical, chemical and functional changes following platelet activation in normal and "giant" platelets

Unactivated discocytes in healthy human donors have mean volumes of approximately 6.0 microns3 (range 3.8-7.5 microns3), while mean values for similarly-shaped discocytes obtained from donors with the hereditary "giant" platelet syndromes were either normal (one Bernard-Soulier syndrome (BSS) and all five members of a family with the Montreal platelet syndrome (MPS) or, on average, up to twice normal (range 6.4-13.8 microns3). This apparent heterogeneity is complicated by the much more consistent and significant observation that both BSS and MPS platelets undergo a defective hypervolumetric shape change following activation which is prolonged indefinitely, in contrast to a transient hypervolumetric change measureable in 1-5 s following ADP addition to normal platelets. It is suggested that the hypervolumetric shape change in both normal and "giant" platelets is accompanied by an increase in externalized plasma membrane surface area, with the most probable source being surface-connected canalicular system. Membrane glycoprotein I abnormalities were not detectable in platelets for 2/3 sibling MPS donors. The precise relation of these membrane changes to altered platelet functions is compared for normal and "giant" platelets, but largely remains to be experimentally determined. Early shape change appears tightly associated with early microscopically-measured aggregation (PA), with both PA and turbidimetrically-measured macroaggregation generally appearing normal to elevated for "giant" platelets.     

Atomic force microscopy study of red blood cell membrane nanostructure during oxidation‐reduction processes

The morphology and functional state of red blood cells (RBCs) mainly depends on the configuration of the spectrin network, which can be broken under the influence of intoxication because of oxidation processes in the cells. Measurement of these processes is a complex problem. The most suitable and prospective method that resolves this problem is atomic force microscopy (AFM). We used AFM to study the changes in the spectrin matrix and RBC morphology during oxidation processes caused by ultraviolet (UV) irradiation in RBC suspension. The number of discocytes decreased from 98% (in control) to 12%. We obtained AFM images of the spectrin matrix in RBC ghosts. Atomic force microscopy allows for the direct observation and quantitative measurement of the disturbances in the structure of the spectrin matrix during oxidation processes in RBCs. The typical section size of the spectrin network changed from approximately 80 to 200 nm (in control) to 600 nm and even to 1000 nm after UV irradiation. An AFM study showed that incubation of RBCs with Cytoflavin® after UV irradiation preserved the forms of RBCs almost at control levels; 89% of the cells remained as discocytes. To quantify the intensity of the oxidation‐reduction processes, the percentage of haemoglobin derivatives was measured. The content of methaemoglobin varied in the range of 1% to 70% during the experiments. These evidence‐based studies are important for the fundamental research of interactions during redox processes in RBCs at the molecular level.     

ATP-dependent transport of phosphatidylserine analogues in human erythrocytes

The plasma membrane of most cells contains a number of lipid transporters that catalyze the ATP-dependent movement of phospholipids across the membrane and assist in the maintenance of lipid asymmetry. The most well-characterized of these transporters is the erythrocyte aminophospholipid flippase, which selectively transports phosphatidylserine (PS) from the outer to the inner monolayer. Previous work has demonstrated that PS and to a lesser extent phosphatidylethanolamine (PE) are substrates for the flippase and that other phospholipids move across the membrane only by passive flip-flop. The present study re-evaluates these results. The incorporation and transbilayer movement of a number of short-chain (dilauroyl) phospholipid analogues in human erythrocytes was measured by observing lipid-induced changes in cell morphology, and the effect of an ATPase inhibitor (vanadate) and a sulfyhdryl reagent (N-ethylmaleimide) was determined. Incubation of cells with these lipids causes the rapid formation of echinocytes, because of the accumulation of the lipid in the outer monolayer. While dilauroylphosphatidylcholine-treated cells retained this shape, cells treated with sn-1,2-DLP-l-S, sn-1,2-DLP-d-S, or N-methyl-DLPS rapidly changed morphology to stomatocytes, which is consistent with the transport and accumulation of the lipid in the inner monolayer. A similar, although slower, stomatocytic shape change was induced by sn-2,3-DLP-l-S. Other lipids that were tested (dilauroylphosphatidylhydroxypropionate, dilauroylphosphatidylhomoserine, DLPS-methyl ester, or sn-2,3-DLP-d-S) reverted to discocytes only. In all cases, pretreatment with vanadate or N-ethylmaleimide inhibited the conversion of echinocytes to discocytes or stomatocytes. This is the first report of a protein- and energy-dependent pathway for the inwardly directed transbilayer movement of lipids other than PS and PE in the erythrocyte membrane and suggests that the flippase has broader specificity for substrates or that other lipid transporters are present.     

Ultrastructural observations on the transformations of osmotically stressed and electrically pulsed red cell carriers at stages during their formation

Although annealed red blood cell (RBC) carriers, when made by different methods, encapsulate similar quantities of methotrexate (MTX) molecules, the mechanisms by which the carrier cells are formed and by which molecules are taken up may be very different as evidenced by electron microscopy (EM) of the cells at various stages during their formation. Scanning electron microscopy (SEM) revealed that dialysis- and preswell-prepared carriers, both procedures which involve exposure to hypotonic buffer, initially transform from discocytes to echinocytes, but later exhibit morphologically heterogeneous cell types. In contrast, electroporated carriers, formed under isotonic conditions, uniformly show an initial discocyte-spheroechinocyte transformation. Transmission electron microscopy (TEM) of osmotically stressed RBC carriers demonstrates that uptake of molecules can be facilitated both by endocytosis and passive diffusion, whereas electrically pulsed carriers encapsulate material only by passive diffusion. Such experiments provide evidence that methods can be refined potentially for producing and isolating carriers that would have more predictable properties in vivo.     

Alkaline hemolysis fragility is dependent on cell shape: results from a morphology tracker.

BACKGROUND: The morphometric analysis of red blood cells (RBCs) is an important area of study and has been performed previously for fixed samples. We present a novel method for the analysis of morphologic changes of live erythrocytes as a function of time. We use this method to extract information on alkaline hemolysis fragility. Many other toxins lyse cells by membrane poration, which has been studied by averaging over cell populations. However, no quantitative data are available for changes in the morphology of individual cells during membrane poration-driven hemolysis or for the relation between cell shape and fragility. METHODS: Hydroxide, a porating agent, was generated in a microfluidic enclosure containing RBCs in suspension. Automatic cell recognition, tracking, and morphometric measurements were done by using a custom image analysis program. Cell area and circular shape factor (CSF) were measured over time for individual cells. Implementations were developed in MATLAB and on Kestrel, a parallel computer that affords higher speed that approaches real-time processing. RESULTS: The average CSF went through a first period of fast increase, corresponding to the conversion of discocytes to spherocytes under internal osmotic pressure, followed by another period of slow increase until the fast lysis event. For individual cells, the initial CSF was shown to be inversely correlated to cell lifetime (linear regression factor R=0.44), with discocytes surviving longer than spherocytes. The inflated cell surface area to volume ratio was also inversely correlated to lifetime (R=0.43) but not correlated to the CSF. Lifetime correlated best to the ratio of cell inflation volume (Vfinal-Vinitial) to surface area (R=0.65). CONCLUSIONS: RBCs inflate at a rate proportional to their surface area, in agreement with a constant flux model, and lyse after attaining a spherical morphology. Spherical RBCs display increased alkaline hemolysis fragility (shorter lifetimes), providing an explanation for the increased osmotic fragility of RBCs from patients who have spherocytosis.     

Investigation of erythrocyte shape, plasma membrane fluidity and conformation of haemoglobin haemoporphyrin under the influence of long-term space flight

The investigation of long-term space flight (SF) effect on the blood cells function is of great importance for modern space biology and medicine. We established that the number of discocytes decreased in the period of early rehabilitation after long-term SF. After SF plasma membrane fluidity and phospholipid content decreased and cholesterol content increased. After SF the amount of haemoglobin decreased and the parameters characterizing haemoglobin haemoporyphyrin (HH) conformation changed. We suppose that erythrocyte shape, membrane fluidity and HH conformation are among factors affecting oxygen transfer during and after space flight.     

The evaluation of the sizes of erythrocytes of the hibernating ground squirrel Citellus undulatus Pallas

A topographical image of individual erythrocytes of the ground squirrel Citellus undulatus Pallas, in unfixed unstained smears was first obtained by scanning probe microscopy for two states of the animal: hibernation and the active state. The scannig of single discocytes, i.e. erythrocytes having a typical discal form, was fulfilled. For the active male, the diameter of the discocyte was found to be approximately 6500 nm. For the hibernating female, the diameter is approximately 6000 nm. According to the data of light microscopy, the discocyte diameters are: 6610 +/- 100 nm for the active state of animal and 6430 +/- 160 nm for the hibernating state.     

Study of the relationship between shape and aggregation change in human erythrocytes

The relationship between shape and spontaneous and fibrinogen-induced aggregation change in human erythrocytes was studied. Spontaneous and fibrinogen-induced erythrocyte aggregation was investigated using a rheoscope designed according to the method of H. Schmid-Schonbein et al. (1973). The erythrocyte shape was studied by means of light microscopy. It was shown that plasma enriched with lysophosphatidic acid and ATP depletion of erythrocytes led to the change of erythrocyte shape: discocytes transformed into echinocytes. It was found that spontaneous aggregation of such cells was considerably decreased. Aggregation of erythrocytes, treated with lysophosphatidic acid, was diminished more markedly. Fibrinogen-induced aggregation of echinocytes, obtained after treatment with lysophosphatidic acid and produced by ATP depletion, was also greatly reduced.     

Theoretical model of thalassemic erythrocyte shape transformation

Our earlier model of reticulocyte shape transformation [Pawlowski, P.H., Burzynska, B., Zielenkiewicz, P., 2006. Theoretical model of reticulocyte to erythrocyte shape transformation. J. Theor. Biol. 243, 24-38] was applied to explain the morphological properties of thalassemic erythrocytes. Modification of the standard set of parameters of the model, describing minimal cell volume, membrane bending rigidity, and membrane tension, allowed for simulation of development of α- and β-thalassemic cells from splenectomized and nonsplenectomized individuals. This resulted in observation of thin rim discocytes, tailed erythrocytes and oval forms, as well as in differentiation of time of the cell shape metamorphosis. A comparative analysis of the susceptibility of thalassemic and normal erythrocytes to undergo deformation as well of their stability was performed.     

Morphology of glutaraldehyde-fixed preserved red blood cells and 24-hr post-transfusion survival

Liquid-stored red blood cells and washed, previously frozen red blood cells were studied to determine whether a correlation existed between morphology and post-transfusion survival. Red cell concentrates were stored at 4 °C in citrate-phosphate-dextrose (CPD) for 21 days or in CPD-adenine (CPDA-1, CPDA-2, or CPDA-3) for as long as 35 days as liquid-preserved red cells. Both nonrejuvenated and rejuvenated red blood cells were frozen with 40%$\text{w}\text{v}$ glycerol at ?80 °C and were washed prior to testing.Samples of fresh, liquid-stored, and washed, previously frozen red blood cells were fixed with a 2% veronal glutaraldehyde solution. Phase, light, and electron microscopy were used to measure the numbers of discocytes, discoechinocytes, echinocytes, echinospherocytes, and spherocytes in each sample. A morphology score was assigned, with 100 representing all discocytes and 500 all spherocytes. In all samples phase and light microscopy gave nearly identical scores (r = 0.94), and phase and electron microscopy gave highly similar scores (r = 0.83).The morphology score proved to be a good indicator of 24-hr post-transfusion survival in liquid-stored red blood cells but not in washed, previously frozen red blood cells. Red blood cells stored in the liquid state at 4 °C in CPD, CPDA-1, CPDA-2, or CPDA-3 showed a significant inverse correlation between morphology and 24-hr post-transfusion survival (r = ?0.611) and a significant correlation between red cell ATP and 24-hr post-transfusion survival (r = 0.742). We saw no significant correlation between morphology scores and 24-hr post-transfusion values or between ATP levels and post-transfusion survival values in nonrejuvenated or rejuvenated washed, previously frozen red blood cells.