Simulation of ultrasound backscattering by red cell aggregates: effect of shear rate and anisotropy

Tissue characterization using ultrasound (US) scattering allows extraction of relevant cellular biophysical information noninvasively. Characterization of the level of red blood cell (RBC) aggregation is one of the proposed application. In the current paper, it is hypothesized that the microstructure of the RBCs is a main determinant of the US backscattered power. A simulation model was developed to study the effect of various RBC configurations on the backscattered power. It is an iterative dynamical model that considers the effect of the adhesive and repulsive forces between RBCs, and the effect of the flow. The method is shown to be efficient to model polydispersity in size, shape, and orientation of the aggregates due to the flow, and to relate these variations to the US backscattering properties. Three levels of aggregability at shear rates varying between 0.05 and 10 s(-1) were modeled at 40% hematocrit. The simulated backscattered power increased with a decrease in the shear rate or an increase in the RBC aggregability. Angular dependence of the backscattered power was observed. It is the first attempt to model the US power backscattered by RBC aggregates polydisperse in size and shape due to the shearing of the flow.     

Evaluation of the energy of red blood cell agglutination by monoclonal antibodies

The knowledge of the energy involved in cell-cell interactions has significant implications in biological and medical sciences. Red blood cells (RBC) become mutually adhered when specific (agglutinins) or nonspecific macromolecules bind adjacent cells in an irreversible or reversible form. Flow chamber technique with digital image processing was successfully applied to determine the partial separation, by shear stress, of two RBC agglutinated face to face (doublet) by specific monoclonal antibodies (MAb) and also to evaluate the shear stress required to attain this separation. The work done in separating adhered cells is then easily calculated and serves to evaluate the antibody affinity. In this work, this technique was applied to evaluate the affinity of a monoclonal antibody specific to the blood group A antigen. The specific disaggregation energy gamma (i.e., the work done by the shear stress) required to disrupt a unit of adhered membrane areas between agglutinated cells was calculated. On play back of the image analysis, measurements were taken to determine the force applied to the doublet and the relative separation between both RBC. Values of gamma(d) (from 2.70 to 4.61 x 10(-9) N/cm) were found to be proportional to the density (D = 27 to 55 molecules/microm(2)) of MAb molecules bound on the RBC membrane.     

A simple method for quantifying high density antigens in erythrocyte membrane by flow cytometry

RBC flow cytometric analysis is usually used to quantify antigen content. Calibration systems enable antigen content determination by relating mean fluorescence intensity with the number of bound antibody molecules (equivalent to the number of antigen molecules). For that reason, antibodies must be used at saturating concentration, which may lead to agglutination when working with high density antigens. Then, forward scattering, side scattering and fluorescence will be increased, thus obtaining wrong results. In this work, the simple Langmuir adhesion model was applied. Flow cytometry was used to quantify GPA, a transmembrane protein present at high density on RBC. The fluorescence intensity of samples at different anti-GPA sub-saturating concentrations was measured. Sometimes, agglutinates were present and two peaks of fluorescence were observed, the principal one corresponding to isolated cells and the secondary one corresponding to agglutinated cells. In those cases, the principal peak was taken into account for the analysis. The GPA antigen content obtained for nine analyzed samples ranged from 3 to 13 x 10(5) sites per cell, which is similar to those values found in literature. Therefore, the Langmuir adsorption model enables us to determine the antigen content for the anti-GPA/GPA system on RBC membrane. This model could be used to quantify high density antigens in RBC and in other cells.     

Aggregation of human red blood cells after moderate heat treatment

The aggregation behaviour of normal and heat treated (48.4 degrees C, 48.8 degrees C, 49.5 degrees C) red blood cells (RBCs) suspended in dextran-saline solutions (Dx 70, Dx 173) was investigated by a laser light reflectometric method over a wide range of bridging energies. The characteristic times of rouleau formation were found to be increased after RBC heat treatment. The disaggregation shear stress is not significantly different between normal RBCs and heat treated RBCs. The loss of cell deformability is nevertheless shown to improve slightly the dissociation efficiency of the flowing liquid in a shear flow resulting in a small reduction of the disaggregation shear rate after heat treatment. Heat treatment is also shown to alter the structure of RBC network at equilibrium. These results indicate that heat induced alterations of erythrocytes only affects the mechanical properties of the cell membrane without significant changes in the macromolecular bridging energy.     

Aggregation of red blood cells studied by ultrasound backscattering

The erythrocyte aggregation phenomenon is an important factor in capillary circulation. This phenomenon can be evaluated by a number of methods (microscopic observations, viscometry, light measurements) which cannot be applied simply to in vivo measurements. In contrast, ultrasound which propagates through soft tissues allows measurement of the mechanical properties of red blood cell (RBC) suspensions which depend on the aggregation phenomenon. We devised an apparatus in order to measure in vitro the ultrasonic backscattering intensity of RBC suspensions. First, with latex particles of different sizes, the ultrasonic backscattering coefficient has been measured in order to evaluate the apparatus response. Then, the ultrasonic backscattering coefficient of different aggregated erythrocyte suspensions has been measured and correlated with the erythrocyte sedimentation rate. Finally, the size of RBC aggregates of different suspensions has been evaluated.     

Non-chemotactic translocation of phagocytic cells mediated by a fibronectin-related human lymphokine

A fibronectin (FN)-related human lymphokine, macrophage agglutination factor (MAggF), agglutinates monocytes at femtomolar concentrations. Similar concentrations of MAggF translocate monocytes and neutrophils through artificial extracellular matrices by a non-chemotactic adhesive process not dependent on intracellular metabolism (matrix-driven translocation). As is the case with matrix-driven translocation mediated by other FN, MAggF-mediated translocation depends on interaction of the lymphokine amino-terminal heparin-binding domain with cell surface heparin-like molecules. In contrast, lymphokine-mediated agglutination involves interactions between the MAggF cell-binding domain and integrin FN receptors recognizing the Arg-Gly-Asp sequence. MAggF-mediated translocation and agglutination are also dependent on the lymphokine gelatin-binding domain. The extremely high activity of MAggF in translocating and agglutinating monocytes may result from cooperative interactions between multiple lymphokine domains and multiple classes of cell surface receptor molecules. We suggest that MAggF-mediated matrix-driven translocation could act independently of or in addition to chemotaxis in recruiting monocytes and neutrophils to a tissue site of T cell-mediated inflammation. Subsequent interaction of MAggF and monocyte FN receptor could then detain monocytes there.     

Size determination of red blood cell aggregates induced by dextran using ultrasound backscattering phenomenon.

Different methods are commonly used to study the red blood cell aggregation phenomenon. The major interest of the ultrasonic method presently discussed is to assess the mean size of red blood cell (RBC) aggregates by measuring ultrasonic intensity backscattered by blood. Applying Rayleigh theory of sound to blood medium, one can show that the scattered ultrasonic intensity is proportional to the 6th power of the size of the RBC aggregates. The ultrasonic method is used to evaluate the mean size of RBC aggregates induced by dextrans. RBCs are suspended at various hematocrits H, in solution of dextrans of different molecular weights M and at different weight concentrations Cw. Results are presented by using the ultrasonic backscattering coefficient chi which is a relevant quantity in a scattering experiment. For suspensions of RBCs aggregated with dextran of molecular weight 70,000 dalton (dextran 70) at concentration Cw = 40 g/l, variations of chi as a function of H are similar to those obtained for normal blood. At a fixed hematocrit, variation of chi versus Cw for dextran 70 exhibits a maximum at 40 g/l. In the case of RBCs suspended at hematocrit 20% and aggregated with dextrans of molecular weight M, 70,000 less than or equal to M less than or equal to 2,000,000, the variations of chi versus molar concentration Cm are similar to those of the microscopic aggregation index defined by Chien (1). Finally, a statistical model of the blood structure previously described (2) is applied to evaluate the mean size of the aggregates. According to this model, the mean size of aggregates is independent of hematocrit for H less than or equal to 40% and independent of the molecular weight of dextran for M greater than or equal to 150,000 dalton.     

The effect of pyruvate on antibody interaction with group-specific erythrocyte antigens

Using the AB0 antibody-antigen model the influence of natural metabolite pyruvate on the antibody interaction with of erythrocyte antigens, defining their group specificity has been investigated. Before agglutination reaction erythrocytes of A (II)–AB (IV) blood groups, monoclonal anti-A and anti-B antibodies were incubated with sodium pyruvate. Visualization of agglutinates was performed by means of flow cytometry and laser scanning confocal microscopy. Computer-aided prediction of the spectrum of biological activity of pyruvate by a PASS program proposed major regulatory pathways, in which pyruvate may be involved. It has been demonstrated that pyruvate can regulate the intensity of antigen-antibody interaction. These results suggest the possibility of using small molecules, for example pyruvate, as molecular probes and prospects of the use of erythrocytes with antigenic determinants of the ABO system expressed on their membranes for studies of protein-protein interactions due to convenient visualization and possibility of quantitative evaluation of this process.     

Automatic blood group determination in the ABO- and Rh-system using conductometry]

A method was developed and tested on the basis of conductometry for determining blood group-specific agglutinates in the AB0 and Rh-system (D). By comparing the measured particle numbers before and after administration of specific antisera or test erythrocytes and objective evaluation can be made whether agglutination has occurred or not. The calculated degrees of agglutination were compared depending on the method, sex, and age of patients. The method can be automated.     

Dimeric ristocetin flocculates proteins, binds to platelets, and mediates von Willebrand factor-dependent agglutination of platelets

Ristocetin in aqueous solution dimerizes with an equilibrium dissociation constant of 5.0 x 10(-4) M, i.e. approximately 1.1 mg ml-1 (Waltho, J.P., and Williams, D. H. (1989) J. Am. Chem. Soc. 111, 2475-2480). At concentrations of about 1.0 mg ml-1 ristocetin flocculates many proteins, lyses platelets and, in the presence of von Willebrand factor, agglutinates both fresh and formalin-fixed platelets. Because ristocetin exists as both monomeric and dimeric species, we sought to determine which of these forms flocculates proteins and agglutinates platelets. We found that: 1) the initial rate of flocculation of certain proteins, 2) the initial rate of agglutination of formalin-fixed platelets, and 3) the binding of ristocetin to formalin-fixed platelets are higher order solely with respect to the concentration of ristocetin dimers. As to the operative mechanism, it appears that bifunctional dimers cross-link proteins that possess multiple copies of a common recognition site. Preliminary evidence indicates that a recognition site is a beta-turn of the form X-P-G-X'.     

Comparative evaluation of whole blood D-dimer test to plasma D-dimer test for diagnosis of disseminated intravascular coagulation

Three rapid D-dimer test methods were compared for the diagnosis of acute disseminated intravascular coagulation (DIC). These were (a) SimpliRED, an autologous red cell agglutination assay. (b) DIMERTEST latex agglutination assay, containing monoclonal antibody DD-3B6/22(6), and (c) D-DI latex agglutination assay containing mouse anti-human D-dimer monoclonal antibodies. The D-DI latex method having higher sensitivity (100%) and specificity (81%) in clinically acute DIC was postulated as the gold standard and compared with the other two methods. The results suggest that D-DI latex agglutination assay containing mouse anti-human D-Dimer monoclonal antibodies are the better assay methods amongst all the three kits analyzed. It is advisable to look for the nature of the antibody used to coat the latex particles in plasma based kits. In emergency setting RBC kits may be of some use as rapid diagnosis is advantageous.     

Signaling mechanisms in red blood cells: A view through the protein phosphorylation and deformability

Intracellular signaling mechanisms in red blood cells (RBCs) involve various protein kinases and phosphatases and enable rapid adaptive responses to hypoxia, metabolic requirements, oxidative stress, or shear stress by regulating the physiological properties of the cell. Protein phosphorylation is a ubiquitous mechanism for intracellular signal transduction, volume regulation, and cytoskeletal organization in RBCs. Spectrin-based cytoskeleton connects integral membrane proteins, band 3 and glycophorin C to junctional proteins, ankyrin and Protein 4.1. Phosphorylation leads to a conformational change in the protein structure, weakening the interactions between proteins in the cytoskeletal network that confers a more flexible nature for the RBC membrane. The structural organization of the membrane and the cytoskeleton determines RBC deformability that allows cells to change their ability to deform under shear stress to pass through narrow capillaries. The shear stress sensing mechanisms and oxygenation-deoxygenation transitions regulate cell volume and mechanical properties of the membrane through the activation of ion transporters and specific phosphorylation events mediated by signal transduction. In this review, we summarize the roles of Protein kinase C, cAMP-Protein kinase A, cGMP-nitric oxide, RhoGTPase, and MAP/ERK pathways in the modulation of RBC deformability in both healthy and disease states. We emphasize that targeting signaling elements may be a therapeutic strategy for the treatment of hemoglobinopathies or channelopathies. We expect the present review will provide additional insights into RBC responses to shear stress and hypoxia via signaling mechanisms and shed light on the current and novel treatment options for pathophysiological conditions.     

Localization and characterization of tissue changes by laser backscattering imaging and Monte Carlo simulation

Laser backscattering from biological tissues depends on their composition and blood flow. The onset of the tissue abnormalities is associated with the change in composition at a specific location which may affect laser backscattering. The objective of the present work is to detect the compositional changes in tissue-equivalent phantom of fat, prepared by mixing paraffin wax with wax colors, and to characterize these in terms of their optical parameters. For this purpose these phantoms are scanned by a multi-probe non-contact automatic laser scanning system and images of the normalized backscattered intensity (NBI) are constructed. By scanning the background subtracted image of the phantom the location of the abnormality and its size from the full width at half maximum (FWHM) are determined. The data obtained by ultrasonic technique for localization of the abnormalities are in agreement with that as obtained by the present method. The optical parameters of the abnormality are obtained by matching the measured surface profiles of the abnormality with that of the profile obtained by Monte Carlo simulation. This analysis shows the possibility of detection of changes at the onset stage in tissues as required for planning of the photodynamic therapy.     

Desmoglein 2 Is Less Important than Desmoglein 3 for Keratinocyte Cohesion

Desmosomes provide intercellular adhesive strength required for integrity of epithelial and some non-epithelial tissues. Within the epidermis, the cadherin-type adhesion molecules desmoglein (Dsg) 1–4 and desmocollin (Dsc) 1–3 build the adhesive core of desmosomes. In keratinocytes, several isoforms of these proteins are co-expressed. However, the contribution of specific isoforms to overall cell cohesion is unclear. Therefore, in this study we investigated the roles of Dsg2 and Dsg3, the latter of which is known to be essential for keratinocyte adhesion based on its autoantibody-induced loss of function in the autoimmune blistering skin disease pemphigus vulgaris (PV). The pathogenic PV antibody AK23, targeting the Dsg3 adhesive domain, led to profound loss of cell cohesion in human keratinocytes as revealed by the dispase-based dissociation assays. In contrast, an antibody against Dsg2 had no effect on cell cohesion although the Dsg2 antibody was demonstrated to interfere with Dsg2 transinteraction by single molecule atomic force microscopy and was effective to reduce cell cohesion in intestinal epithelial Caco-2 cells which express Dsg2 as the only Dsg isoform. To substantiate these findings, siRNA-mediated silencing of Dsg2 or Dsg3 was performed in keratinocytes. In contrast to Dsg3-depleted cells, Dsg2 knockdown reduced cell cohesion only under conditions of increased shear. These experiments indicate that specific desmosomal cadherins contribute differently to keratinocyte cohesion and that Dsg2 compared to Dsg3 is less important in this context.     

Spreading of wheat germ agglutinin-induced erythrocyte contact by formation of spatially discrete contacts.

The time dependence of agglutination and cell-cell contact spreading in human erythrocytes exposed to wheat germ agglutinin (WGA) was characterized by light and electron microscopy. Cells (3 x 10(7)/mL) had a threshold lectin concentration in the range of 0.6-2.0 micrograms/mL for initial cell contact. Spreading was essentially completed within 60 and 2 min in undisturbed and gently agitated suspensions, respectively. The cells in large WGA agglutinates retained features of their initial disk form in contrast to the convex outlines of polycation or polyethylene glycol-induced agglutinates. Spreading of contact area was accompanied by development of a pattern of discrete contact regions separated by a distance of the order of 1 micron. Freeze fracture electron microscopy and studies with ferritin-labeled WGA showed no significant aggregation of intramembrane particles or specific lectin receptors under conditions when contact spreading occurred. It is argued that flow stress effects on cells in suspended agglutinates give rise to a situation where opposite membranes, at the leading edge of cell contact, are separated by a thin aqueous layer. When this intercellular water layer exceeds a critical length, it becomes unstable. The layer breaks up by surface wave development to form an array of intracellular water spaces. Formation of the aqueous spaces causes opposite membrane regions to move synchronously toward each other. Lectin molecules crosslink the wave crests to give spatially periodic contact points.     

Protection of erythrocytes from sub-hemolytic mechanical damage by nitric oxide mediated inhibition of potassium leakage

The effects of mechanical stress on red blood cell (RBC) deformability were evaluated by subjecting cells to a uniform fluid shear stress of 120 Pa for 15-120 seconds at 37 degrees C. This level of stress induced significant impairment of RBC deformability as assessed by ektacytometry, with the degree of impairment independent of extracellular calcium concentration. Inhibition of RBC nitric oxide (NO) synthesis by a competitive inhibitor of NO synthases (N-omega-nitro-L-arginine methyl ester, L-NAME) had no effect on deformability after exposure to mechanical stress. The NO donor sodium nitroprusside (SNP) prevented the deterioration of RBC deformability in a dose-dependent manner with 10(-4) M being the most effective concentration. A similar protective effect by the non-selective potassium channel blocker, tetraethylammonium chloride (TEA) suggests that the effect of NO might be mediated by the inhibition of potassium leakage from RBC. These results suggest that NO may prevent mechanical deterioration of RBC exposed to high shear stresses. While RBC are not exposed to such high levels of shear stresses for prolonged periods under normal circulatory conditions, comparable levels of mechanical stress can be encountered under certain situations (i.e., artificial organs, extracorporeal circulation) and may result in subhemolytic damage and hemorheological alterations.     

Blood group B-specific lectin of Plecoglossus altivelis (Ayu fish) eggs

A lectin that agglutinates human blood group B erythrocytes but not blood group A and O erythrocytes was isolated from eggs of Ayu sweet fish (Plecoglossus altivelis). The lectin also agglutinates Ehrlich ascites carcinoma cells but not rat ascites hepatoma AH109 or rat sarcoma 150 cells tested. The lectin agglutination was most effectively inhibited by monosaccharides with the first type of configuration, i.e., L-rhamnose, L-mannose and L-lyxose at a concentration of 0.03 mM. The lectin agglutination was moderately inhibited by monosaccharides with the second type of configuration, i.e., D-galactose, D-fucose and D-galacturonic acid at a concentration of 0.4 mM. However, the agglutination was not inhibited by various other monosaccharides and oligosaccharides that have other types of configuration. The basis for an apparent B-specific hemagglutination may be due to the steric similarity of the C2 and C4 of the galactosyl series, the B-specific determinant, and the L-rhamnosyl-Sepharose column and was characterized as a homogeneous low molecular weight protein (Mr 14000) with an abundance of hydrophobic amino acids and dicarboxylic amino acid.     

Wall shear stress-based model for adhesive dynamics of red blood cells in malaria

Red blood cells (RBCs) infected by the Plasmodium falciparum (Pf-RBCs) parasite lose their membrane deformability and they also exhibit enhanced cytoadherence to vascular endothelium and other healthy and infected RBCs. The combined effect may lead to severe disruptions of normal blood circulation due to capillary occlusions. Here we extend the adhesion model to investigate the adhesive dynamics of Pf-RBCs as a function of wall shear stress (WSS) and other parameters using a three-dimensional, multiscale RBC model. Several types of adhesive behavior are identified, including firm adhesion, flipping dynamics, and slow slipping along the wall. In particular, the flipping dynamics of Pf-RBCs observed in experiments appears to be due to the increased stiffness of infected cells and the presence of the solid parasite inside the RBC, which may cause an irregular adhesion behavior. Specifically, a transition from crawling dynamics to flipping behavior occurs at a Young's modulus approximately three times larger than that of healthy RBCs. The simulated dynamics of Pf-RBCs is in excellent quantitative agreement with available microfluidic experiments if the force exerted on the receptors and ligands by an existing bond is modeled as a nonlinear function of WSS.     

Interaction energies in lectin-induced erythrocyte aggregation

Two N-acetylgalactosamine-reactive lectins, Helix pomatia (HPA) and Dolichos biflorus (DBA), were used to study the energies involved in cell-cell interactions through the specific binding of these lectins to their membrane receptors on genotype AO human erythrocytes (red blood cells) (RBCs). The energy required to dissociate a unit of aggregated membrane area (gamma d) of two RBCs bridged by lectin molecules was determined from the shear force needed to dissociate two-cell aggregates in a flow channel. When HPA were used as bridging molecules, gamma d (0.4 X 10(-4) to 3.8 X 10(-4) dyn/cm) was proportional to the density (D = 175 to 1,060 molecules/micron 2) of HPA molecules bound on the RBC membrane. A similar gamma d/D ratio was also obtained for DBA. These results indicate that the number of lectin molecules bound on the interface plays an important role in determining the energy required for cell-cell dissociation. The aggregation energy per unit membrane area (gamma a) in lectin-induced aggregates was calculated from the degree of encapsulation of a lectin-bound, heat-sphered human RBC by a normal discoid RBC. A minimum of approximately 1,800 HPA molecules/micron 2 on the spheres was required to form stable aggregates with the RBC. By using spheres having a surface HPA density of 1,830 to 2,540 molecules/micron 2, or 1.1-1.5 X 10(12) combining sites/cm2, the gamma a value for HPA-induced aggregation was found to be 2.2 X 10(-3) dyn/cm. This higher value of gamma a than gamma d has been explained on the basis of several differences in aggregation and disaggregation processes. The gamma a value for DBA-induced aggregation was not obtainable by the sphere encapsulation method because of the relative low D values. A comparison of the present results with the published value of the free energy change of 5 kcal/mol for the interactions of HPA and DBA with their ligands suggests that only a small fraction of the lectin molecules bound to RBC surface participate in the bridging of adjacent cells.     

A monoclonal antibody specific for the Thomsen-Friedenreich cryptic T antigen

A monoclonal antibody (49H.24) is described that was made after immunization of BALB/c mice with human neuraminidase-treated erythrocytes (NE-RBC). 49H.24 is an IgM and reacts with NE-RBC but not untreated, normal human RBC. As little as 100 pg of antibody protein produced detectable direct agglutination of, or binding to, NE-RBC. The fine specificity of 49H.24 was determined by using a series of synthetic sugar haptens as inhibitors of agglutination or binding of 49H.24 to NE-RBC. Only synthetic T hapten (beta Gal(1 leads to 3)alpha GalNAc) produced complete inhibition of agglutination or binding, and no inhibition was produced by several other closely related haptens. Synthetic T hapten-coated silica beads (Synsorb) were used to affinity purify 49H.24. Affinity-purified antibody was radiolabeled with 125I and used in a sensitive competition assay to detect natural T antigen associated with cell membranes or in soluble form. The potential use of this or similar monoclonal antibodies as probes for an important human tumor marker is discussed.