Rodolfo J. Rasia

Shear deformation measurement of suspended particles: Application to erythrocytes

Laser diffraction produced by a single layer of erythrocytes in suspension is applied to assess the cell deformation in the erythrodeformeter. It is well known that erythrocytes become elliptically deformed when a fluid shear stress is applied to them. In the erythrodeformeter the red cell suspension fills the narrow gap between two parallel disks of glass, horizontally positioned, the lower of which rotates at a controlled speed while the upper is fixedly supported. A laser beam traversing the sample layer at r distance from the center of disks, displays a diffraction pattern ‘‘at infinity.’’ At no shear stress the cells have discoidal shapes and the diffraction pattern is circular. Shear‐induced elongation of cells leads to an elliptical diffraction pattern, its geometric characteristics being directly related to those of deformed cells. This deformation can be evaluated in terms of a deformation index calculated from the width and the length of the diffraction pattern ellipse. In the erythrodeformeter ...

Complex viscoelasticity of normal and lectin treated erythrocytes using laser diffractometry

A new method to find directly complex viscoelastic parameters (CVP) of red blood cells (RBC) is presented in this paper. Experimental determinations were carried out in an Erythrodeformeter (Rasia et al., 1986) operating in oscillating mode (0.5 to 3.5 Hz). The Erythrodeformeter performs direct determination of CVP of erythrocytes undergoing sinusoidal shear stresses by laser diffractometry. The measurements lead to the determination of mean values of the four components of erythrocyte complex viscoelasticity. The influence of the alterations induced on erythrocyte membrane by vegetable lactins (Ulex europaeus, wheat germ agglutinin and Enterolobium contorticilicum seeds) was analyzed to verify the sensitivity of this method. Differences observed between the CVP parameters of treated cells and the ones corresponding to control samples (non treated cells) are analyzed. Results obtained from cells treated with wheat germ agglutinin agree with observations published by Smith and Hochmuth (1982). Determinations of RBC complex viscoelasticity carried out by laser diffractometry could become an important tool to understand the influence of the factors associated with alterations of the rheologic properties of RBC membrane, which can affect the in vivo blood flow.

Quantification of glycophorin A and glycophorin B on normal human RBCs by flow cytometry

BACKGROUND: The quantification of antigens and proteins on RBCs has been achieved by different approaches. Flow cytometry allows the results of the earliest studies to be to reappraised because it offers the possibility of measuring the immunofluorescence intensity of single cells and integrating the individual data of a large number of cells within a very short time.

Viscoelasticity: Fractal parameters studied on mammalian erythrocytes under shear stress

In this work, experimental determinations are carried out using a home-made device called an erythrodeformeter, which has been developed and constructed for rheological measurements on red blood cells subjected to definite fluid shear stress. A numerical method formulated on the basis of the fractal approximation for ordinary and fractionary Brownian motion1 is proposed to evaluate the viscoelastic behavior of mammalian erythrocyte membranes. The diffraction pattern, which is circular when the mammalian erythrocyte membranes are at rest, becomes elliptical when the cells undergo shear stress. Photometric readings of light intensity variation along the major axis of the elliptical diffraction pattern are recorded during the creep and recovery process. These data series are used to calculate, fractal rheological parameters of self-affine Brownian motion on the erythrocytes, averaged over several millions of cells. Three different parameters over the time dependent process could be obtained, which are: correlation coefficient , correlation integral, andK2-entropy, and very different results were obtained.

Kinetic study of antibody adhesion on a silicon wafer by laser reflectometry

Abstract Antibody adhesion kinetic in real time has been studied by laser reflectometry technique. An ellipsometer is used to measure the light intensity reflected by a silicon wafer. Light intensity reflected by the wafer presents a minimum at the pseudo-Brewster angle. Then, the reflectance increases as the antibodies (monoclonal anti- AB ) adhere on interface. Mathematical analysis of reflectance curves versus time verifies that the antibody adhesion at the interface follows Langmuir kinetics (Prog. Biomed. Opt. Imaging 1(5) (2000) 19) for low antibody concentrations. Parameters obtained allow to carry out a detailed study of the antibody adsorption and the antigen–antibody interaction. This conduces to development of an optical immunosensor for detection and quantification of soluble antigens, and a novel method for commercial antiserum quality control. This technique does not require labeled antibodies, being also independent of cellular factors. Also, this technique is quicker and sensible than the conventional immunohematology methods.

Manual quantitative method for the study of red cell agglutination using light diffraction by suspended particles

Abstract. A new manual technique, called the diffractometric technique, for quantitation of hemagglutination is described in this paper. After the equilibrium has been attained in the reaction, the whole product is resuspended in a dense transparent medium in order to obtain a stable suspension. The relative optical extinction produced by this suspension depends on the number and sizes of the suspended particles and on the photodetector geometry. Hence the agglutination percentage of the reaction can be monitored by using a visible light transmittance photometer. The results of this technique compare very well with the ones obtained by applying two other methods, the Dybkjaer and the Ropars methods.

Determination of the complex viscoelastic parameters of human red blood cells by laser diffractometry

A new method to find directly complex viscoelastic parameters (CVP) of human red blood cells is presented in this paper. Experimental determinations were carried out in an Erythrodeformeter operating in oscillating mode (0.5 to 3.5 Hz). The Erythrodeformeter performs direct determination of CVP of erythrocytes undergoing sinusoidal shear stresses by laser diffractometry.

Evaluation of the influence of hematocrit on red blood cell deformability

The present preliminary communication is to show variation in the deformability of red blood cells of Wistar rats when changes were induced in their hematocrit

Determination of adhesive specific energy of erythrocyte agglutination by laser retrodiffusion

Abstract The quality of an antibody is related to its affinity expressed by the equilibrium constant. The biological activity of an antibody can be estimated by the strength of antigen–antibody bridges. An optical method is proposed to estimate the specific binding energy using the dissociation behavior of suspended RBC agglutinates in a shear flow and measuring laser backscattering. A constant increase of intensity was observed when the shear stress raises, pointing to a progressive dissociation of RBC agglutinates into smaller ones. The final break-up of two-cell rouleaux is produced at critical shear stress ( τ c ), which reflects the mechanical action required to dissociate the molecular bridges between cells. The critical shear stress permitted to define the specific surface adhesive energy ( Γ ) by using the Derjaguin relation. The determination of τ c provides a good way to assess the functional characterization of specific immunoglobulins, which could be very useful for antibody quality control.

Analysis of the 3D structure og agglutinated erythrocyte using CellScan and confocal microscopy: characterization by FLIM-FRET

We report the adhesion of human erythrocyte membranes mediated by monoclonal antibodies anti-glycophorin. The distribution of the linked antibodies on membrane was identified with selective fluorescence labels. To analyze the antibody distribution on interfacial region between two cells agglutinated and on its surface, three types of fluorescence marked strategy were evaluated. The 3D images were obtained in a CellScan and Confocal Laser Scanning Microscopy CLSM. We considered the FRET signal to characterize the agglutination of Red Blood Cells (RBC) by specific monoclonal antibodies (anti-glycophorin A or B). The fluorescence labeling demonstrated that distribution of antibody on erythrocyte membranes is not homogeneous. The fluorescence intensity on contact region in the agglutinated is bigger than the intensity on exterior surface. Tentatively, we interpreted these intensity differences in terms of the mobility of antibody linked to the glycocalix on cell surface. Such mobility has a large consequence in the morphology of cellular agglutinated.