Hemolysis Pathways during Storage of Erythrocytes and Inter-Donor Variability in Erythrocyte Morphology.

BACKGROUND: Red blood cells (RBCs) stored for transfusions can lyse over the course of the storage period. The lysis is traditionally assumed to occur via the formation of spiculated echinocyte forms, so that cells that appear smoother are assumed to have better storage quality. We investigate this hypothesis by comparing the morphological distribution to the hemolysis for samples from different donors. METHODS: Red cell concentrates were obtained from a regional blood bank quality control laboratory. Out of 636 units processed by the laboratory, we obtained 26 high hemolysis units and 24 low hemolysis units for assessment of RBC morphology. The association between the morphology and the hemolysis was tested with the Wilcoxon-Mann-Whitney U test. RESULTS: Samples with high stomatocyte counts (p = 0.0012) were associated with increased hemolysis, implying that cells can lyse via the formation of stomatocytes. CONCLUSION: RBCs can lyse without significant echinocyte formation. Lower degrees of spiculation are not a good indicator of low hemolysis when RBCs from different donors are compared.

Nanoscale-Specific Reaction in a Precursor Film: Mixing Sodium Carbonate, Calcium Chloride, and an Organic Thiol to Produce Crystals of Calcium sulfate.

The ultrathin precursor film surrounding droplets of liquid on a solid surface is used here as a confined reaction medium in order to drive a reaction that would not occur in bulk fluid. Sodium carbonate and calcium chloride mixed together in the presence of the organic thiol dithiothreitol (DTT) produced crystals of gypsum, or calcium sulfate, instead of the otherwise expected calcium carbonate. The possible sources of sulfate in the system are contaminants in the DTT or the oxidation product of the DTT sulfhydryl. The amount of gypsum produced implies that contaminants do not account for the total sulfate present in the system, suggesting that the DTT could be oxidized. The reaction quotient may be skewed in favor of this unexpected reaction by a combination of efficient removal of sulfate by precipitation and the concentration of DTT at the leading edge of the precursor film through the coffee-ring effect during a brief drying step.

Lipid ratios as a marker for red blood cell storage quality and as a possible explanation for donor gender differences in storage quality.

BACKGROUND AND OBJECTIVES: Red blood cells that are stored for transfusions as red cell concentrates (RCCs) undergo changes during the storage period, culminating in the lysis of the cells. The goal of this work is to find markers that are linked to high haemolysis, in order to explain the inter-donor variability that is known to occur in storage quality, and also the known differences between RCCs from male and female donors. MATERIALS AND METHODS: The relative amounts of lipids at the end of the storage period were compared for one group of low haemolysis samples (24 units, all ≤0·15% haemolysis), and one group of high haemolysis samples (26 units, all ≥0·5% haemolysis). Representative lipids were analysed from different lipid classes, including cholesterol, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and ceramide. Whole membrane preparations were analysed with one mass spectrometry technique, and lipid extracts were analysed with a second mass spectrometry technique. RESULTS: The ratio of palmitoyl-oleoyl phosphatidylcholine (POPC) to sphingomyelin was different for the high and low haemolysis groups (P = 0·0001) and for the RCCs from male and female donors (P = 0·0009). The ratio of cholesterol to phospholipids showed only minimal links to haemolysis. Higher relative amounts of sphingomyelin were associated with lower haemolysis, and higher relative amounts of ceramides were associated with increased haemolysis. CONCLUSION: The level of sphingomyelinase activity and the resulting ratio of sphingomyelin to POPC is proposed as a possible marker for RCC storage quality.

The Blood Bag Plasticizer Di-2-Ethylhexylphthalate Causes Red Blood Cells to Form Stomatocytes, Possibly by Inducing Lipid Flip-Flop.

BACKGROUND: During storage of red blood cell (RBC) concentrates, the plasticizer di-2-ethylhexylphthalate (DEHP) that keeps the blood bags soft leaches out and can be taken up by the RBCs. DEHP is known to be beneficial for the RBC storage quality, but the molecular mechanisms of the action are unknown. METHODS: Aqueous suspensions of DEHP were added to RBCs in buffer. The morphological effects were observed on RBCs from 5 donors. Flow cytometry with annexin A5 binding was used to measure the exposed phosphatidylserine. RESULTS: DEHP induced the formation of stomatocytes at concentrations as low as ng/ml, provided that the cell suspension was also sufficiently dilute. Some spherocytes, which were susceptible to lysis, were also formed; after lysis, RBC ghosts were seen to continue the transition to the cup-shaped stomatocyte form. Incubation with DEHP increased the exposed phosphatidylserine, an effect that was also observed in the presence of vanadate, which inhibits the ATP-dependent translocases that maintain the membrane's lipid asymmetry. CONCLUSIONS: DEHP can have an active effect on RBC shape, instead of just preventing the storage-related shape changes. The effect appears to be mediated by increased flip-flop of lipids between the leaflets of the RBC membrane.

Flow morphometry to assess the red blood cell storage lesion.

We present a novel automated system for morphology analysis of red blood cells (RBC) under flow. RBC concentrates collected by blood banks for transfusions are stored for periods of up to several weeks, during which time a number of changes occur, collectively termed the storage lesion. Typically the extent of hemolysis is the defining criterion to determine the acceptability of the RBCs for transfusions. Morphological changes are related with biochemical alteration during the storage of RBCs. The typical blood smear procedure for determining such changes is a labor-intensive and potentially biased manual process. The advantage of the flow morphometry system presented here is that it provides fully automated morphological classification of RBCs with large sample numbers in a short time. Our system uses a commercially available flow cell and flow conditions that prevent adhesion of RBCs, thus eliminating the need for blocking agents such as albumin that affect the distribution of cell shapes. Our morphometry results are validated by comparison with standard biochemical assays (hemolysis, ATP) for blood from 17 donors stored under blood bank conditions for 13 weeks. We show that the percentage of spherocytes present can be used to estimate the status of RBC concentrates. © 2017 International Society for Advancement of Cytometry.

Chain Length and Grafting Density Dependent Enhancement in the Hydrolysis of Ester-Linked Polymer Brushes.

Poly(N,N-dimethylacrylamide) (PDMA) brushes with different grafting density and chain length were grown from an ester group-containing initiator using surface-initiated polymerization. Hydrolysis of the PDMA chains from the surface was monitored by measuring thickness of the polymer layer by ellipsometry and extension length by atomic force microscopy. It was found that the initial rate of cleavage of one end-tethered PDMA chains was dependent on the grafting density and chain length; the hydrolysis rate was faster for high grafting density brushes and brushes with higher molecular weights. Additionally, the rate of cleavage of polymer chains during a given experiment changed by up to 1 order of magnitude as the reaction progressed, with a distinct transition to a lower rate as the grafting density decreased. Also, polymer chains undergo selective cleavage, with longer chains in a polydisperse brush being preferentially cleaved at one stage of the hydrolysis reaction. We suggest that the enhanced initial hydrolysis rates seen at high grafting densities and high chain lengths are due to mechanical activation of the ester bond connecting the polymer chains to the surface in association with high lateral pressure within the brush. These results have implications for the preparation of polymers brushes, their stability under harsh conditions, and the analysis of polymer brushes from partial hydrolysates.

Simultaneous measurement of mechanical and surface properties in thermoresponsive, anchored hydrogel films.

Hydrogel films have been used extensively in the preparation of biosensors and biomedical devices. The characteristics of the aqueous interface of the polymer layer are significant for the biosensor or device function; likewise, the changing mechanical properties of thermoresponsive polymers are an important feature that affects the polymer behavior. Atomic force microscopy was used here to characterize both the surface and the mechanical properties of polymeric hydrogel films prepared from a thermoresponsive terpolymer of N-isopropylacrylamide and acrylic acid with benzophenonemethacrylate as a photoreactive cross-linker comonomer. The force-distance curves thus obtained were analyzed to assess both the surface forces and the mechanical response that were associated with the hydrogel. These properties were investigated as a function of temperature, in water and in Tris buffer, for different degrees of polymer cross-linking. For samples in water, the distance over which the surface forces were effective was found to remain constant as the temperature was increased from 26 to 42 °C, even though the mechanical response indicated that the samples had been heated past the lower critical solution temperature, or LCST. The bulk of the polymer becomes less soluble above the LCST, although this does not seem to affect the surface properties. This may be due to the segregation of the acrylic acid-rich polymer segments near the gel surface, which is in agreement with reports for related systems.

Relative activity of cholesterol in OPPC/cholesterol/sphingomyelin mixtures measured with an acoustic sensor.

Acoustic devices are sensitive to the mole fraction of cholesterol present in liposomes adsorbed to the device surface as a result of the different mechanical properties of the liposomes. This fact was exploited to develop an acoustic assay to determine the relative affinity of cholesterol for different lipid mixtures. In the assay described here, the initial rate of beta-cyclodextrin-induced removal of cholesterol was measured for liposomes having a range of compositions. The initial rate of cholesterol removal was found to be directly proportional to the concentration of beta-cyclodextrin (betaCD) present over the range of 0-7.5 mg/ml (0-6.6 mM), consistent with other assays measuring the betaCD-accelerated transfer of cholesterol between liposomes. The affinity of cholesterol for 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) liposomes with a sphingomyelin mole fraction, chi(SPM), of 0.2 was found to be 1.4x higher than that for pure OPPC liposomes. For liposomes composed only of OPPC and cholesterol in varying ratios, the initial rate of cholesterol removal was determined as a function of cholesterol mole fraction (chi(C)). The initial rate of removal showed an increase at chi(C) = 0.13, consistent with phase diagrams showing the start of liquid ordered domain formation, but no such increase at chi(C) = 0.25, in contrast to the predictions of the umbrella model for OPPC/cholesterol interactions.

Quantitative determination of size and shape of surface-bound DNA using an acoustic wave sensor.

DNA bending plays a significant role in many biological processes, such as gene regulation, DNA replication, and chromosomal packing. Understanding how such processes take place and how they can, in turn, be regulated by artificial agents for individual oriented therapies is of importance to both biology and medicine. In this work, we describe the application of an acoustic wave device for characterizing the conformation of DNA molecules tethered to the device surface via a biotin-neutravidin interaction. The acoustic energy dissipation per unit mass observed upon DNA binding is directly related to DNA intrinsic viscosity, providing quantitative information on the size and shape of the tethered molecules. The validity of the above approach was verified by showing that the predesigned geometries of model double-stranded and triple-helix DNA molecules could be quantitatively distinguished: the resolution of the acoustic measurements is sufficient to allow discrimination between same size DNA carrying a bent at different positions along the chain. Furthermore, the significance of this analysis to the study of biologically relevant systems is shown during the evaluation of DNA conformational change upon protein (histone) binding.

Atomic force microscopy and cells: Indentation profiles around the AFM tip, cell shape changes, and other examples of experimental factors affecting modeling.

We use atomic force microscopy in conjunction with a fluorescence microscope capable of optical sectioning to acquire images of white blood cells while force is applied with the AFM tip. The indentation profile within the cell is compared to the profile of the AFM tip: examples are shown for indentations at the center of the cell which are reasonable matches to the tip profile, and an additional example is shown for an indentation that is on the tilted side of a highly rounded cell and that differs from the tip shape. We also demonstrate that the AFM tip can interact with internal cell structures, we show that the contact area between the cell and the substrate can increase under applied pressure, that the main body of the cell can fuse with the extended lamellipodium, and that the cell can be displaced laterally by the AFM tip. The features illustrated here are relevant to the interpretation of indentation experiments that measure cell elasticity properties, as is discussed briefly.

Pulse mode shear horizontal-surface acoustic wave (SH-SAW) system for liquid based sensing applications.

In this work, we describe a novel pulse mode shear horizontal-surface acoustic wave (SH-SAW) polymer coated biosensor that monitors rapid changes in both amplitude and phase. The SH-SAW sensors were fabricated on 36 degrees rotated Y-cut X propagating lithium tantalate (36 YX.LT). The sensitivity of the device to both mass loading and visco-elastic effects may be increased by using a thin guiding layer of cross-linked polymer. Two acoustic modes are excited by the electrodes in this crystalline direction. Metallisation of the propagation path of the 36 YX.LT devices allows the two modes to be discriminated. Successive polymer coatings resulted in the observation of resonant conditions in both modes as the layer thickness was increased. Using the 36 YX.LT devices, we have investigated the application of a novel pulse mode system by sensing a sequence of deposition and removal of a biological layer consisting of vesicles of the phospholipid POPC. A continuous wave system was used to verify the accuracy of the pulse mode system by sensing a series of poly(ethylene glycol) (PEG) solutions. The data clearly demonstrates the ability of the 36 YX.LT pulse mode system to provide rapid measurements of both amplitude and phase for biosensing applications.

A silicate gel for promoting deposition of lipid bilayers.

A simple method for modifying a polymer surface to induce lipid bilayer formation by vesicle fusion is described. A silicate gel was prepared by condensation of tetraethyl orthosilicate (TEOS) in the presence of acid. When applied to a poly(methylmethacrylate) substrate, either a rough or a smooth layer could be produced, depending on the method used for the application. The smooth surface induced formation of a supported lipid bilayer by fusion of lipid vesicles; the rough silicate surface induced adsorption of a vesicle layer. A high-frequency acoustic waveguide device was used to follow the initial adsorption of vesicles, the transition from a vesicle layer to a bilayer, and the formation of a complete bilayer; the time required to form a bilayer was determined as a function of lipid concentration in suspension. The presence of a bilayer on the smooth silicate surface was confirmed by fluorescence recovery after photobleaching. An additional procedure is described to modify a gold surface to induce bilayer formation.

Rationalized approach to the determination of contact point in force-distance curves: application to polymer brushes in salt solutions and in water.

In this work, we present two methods to determine the contact point in force-distance curves obtained with the atomic force microscope. These procedures are compared with the typical determination of contact point by a visual assessment of the data. One method, based on the assumption that the sample shows linear elastic behavior, provides results similar to those obtained by a visual assessment of the data, and will be suitable for determining the contact point in cases where ionic repulsion is not significant. The second method is based on a series of measurements in which the sample deformation is measured at increasing values of applied load; the contact point is determined by extrapolation to zero load. Because this method is based on extrapolation of measurements made in the contact regime, it is not subject to long-range repulsion. It is thus suitable for the analyses of the contact point even in cases where ionic repulsions will affect the point at which individual force curves deviate from the baseline or zero-force regime. The methods described here are demonstrated with a glycopolymer brush compressed with a colloidal silica particle on the tip of the AFM cantilever.

Poly(dimethylsiloxane)-coated sensor devices for the formation of supported lipid bilayers and the subsequent study of membrane interactions.

The development of smooth hydrophilic surfaces that act as substrates for supported lipid bilayers (SLBs) is important for membrane studies in biology and biotechnology. In this article, it is shown that thin films of poly(dimethylsiloxane) (PDMS) formed on a sensor surface can be used as a substrate for the deposition of reproducible and homogeneous zwitterionic SLBs by the direct fusion of vesicles. Poly(dimethylsiloxane) solution (1% w/v) was spin coated on Love acoustic wave and surface plasmon resonance devices to form a thin PDMS layer. Acoustic, fluorescence, and contact angle measurements were used for the optimization of the PDMS film properties as a function of plasma etching time; parameters of interest involve the thickness and hydrophilicity of the film and the ability to induce the formation of homogeneous SLBs without adsorbed vesicles. The application of PDMS-coated sensor devices to the study membrane of interactions was demonstrated during the acoustic and fluorescence detection of the binding of melittin and defensin Crp4 peptides to model supported lipid bilayers.

Probing mechanical properties of liposomes using acoustic sensors.

Acoustic devices were employed to characterize variations in the mechanical properties (density and viscoelasticity) of liposomes composed of 1-oleoyl-2-palmitoyl- sn-glycero-3-phosphocholine (POPC) and cholesterol. Liposome properties were modified in three ways. In some experiments, the POPC/cholesterol ratio was varied prior to deposition on the device surface. Alternatively, the ratio was changed in situ via either insertion of cholesterol or removal of cholesterol with beta-cyclodextrin. This was done for liposomes adsorbed directly on the device surface and for liposomes attached via a biotin-terminated poly(ethylene glycol) linker. The acoustic measurements make use of two simultaneous time-resolved signals: one signal is related to the velocity of the acoustic wave, while the second is related to dissipation of acoustic energy. Together, they provide information not only about the mass (or density) of the probed medium but also about its viscoelastic properties. The cholesterol-induced increase in the surface density of the lipid bilayer was indeed observed in the acoustic data, but the resulting change in signal was larger than expected from the change in surface density. In addition, increasing the bilayer resistance to stretching was found to lead to a greater dissipation of the acoustic energy. The acoustic response is assessed in terms of the possible distortions of the liposomes and the known effects of cholesterol on the mechanical properties of the lipid bilayer that encloses the aqueous core of the liposome. To aid the interpretation of the acoustic response, it is discussed how the above changes in the lipid bilayer will affect the effective viscoelastic properties of the entire liposome/solvent film on the scale of the acoustic wavelength. It was found that the acoustic device is very sensitive to the mechanical properties of lipid vesicles; the response of the acoustic device is explained, and the basic underlying mechanisms of interaction are identified.

Interaction of cytolytic toxin CytB with a supported lipid bilayer: study using an acoustic wave device.

An acoustic technique was used to monitor the interaction of the pore-forming cytolytic toxin CytB with a positively charged supported lipid bilayer. The acoustic device, which is based on a waveguide geometry, is sensitive to changes in the mass of the supported bilayer. The specificity of the interaction, rate and extent of the association, reversibility and effect of previous depositions of toxin were investigated. The CytB was found to bind irreversibly to the lipids at all fractional coverages even when the protein-to-lipid ratio was high enough to imply that the protein was associating with the external surface of the bilayer. The CytB formed stable structures with the bilayer at high protein surface concentrations and did not appear to disrupt the bilayer in the manner of a detergent. The rate of association with the bilayer was found to be directly proportional to the solution concentration of CytB at higher concentrations but appeared to be low at a CytB solution concentration of 5 microg mL(-1), leading to relatively low amounts of CytB being associated with the bilayer.