A theoretical approach of the measurement of osmotic fragility of erythrocytes by optical transmission.

The osmotic fragility of the erythrocyte membrane to hypotonic solutions is investigated theoretically. The fragility curves exhibit a strong transmittance rise. This variation is assumed to result from changes in the scattering properties of erythrocytes under dialysis resulting from swelling and hemolysis. The refractive indices of erythrocytes are obtained through the Lorentz-Lorenz relation based on hemoglobin and water contents. The scattering cross sections (needed to calculate the collimated transmittance) and the forward scattered intensity (needed to calculate the incoherent transmittance) are expressed according to the simple algebraic relations of the anomalous diffraction approximation. It is shown that swelling (or shrinking) has no influence on the collimated transmittance. Hemolysis alone causes the abrupt sigmoidal increase of the collimated transmittance with time. The possible transmittance increase (decrease) observed during swelling (shrinking) is due to incoherent transmittance and depends on the detecting solid angle value of the experimental setup.

Validation of a test of the red cell membrane osmotic resistance.

The aim of the present work was to validate a new technique for the measurement of resistance of the red blood cell membrane using an automated apparatus called a Fragilimeter. Its principle lies in the measurement of the extinction of a laser beam projected through a red blood cell suspension subjected, by diffusion, to a variation of salinity from an isotonic equilibrium (154 mM NaCl) to, a hypotonic one, 25 mM NaCl. The variation of osmotic pressure induces on the cells a progressive lysis and a modification of the extinction of the transmitted light. The validation of the method was based on the comparison between results obtained with the Fragilimeter and those obtained using the reference DACIE technique. Analyses were based on blood samples from healthy donors. The determination of the initial, the 50% and the full haemolysis thresholds allowed observation of the fragility of the cell, through its membrane resistance. The physical phenomenon measured in these cells when subjected to various ionic strengths is discussed on the basis of observations realised by means of an optical microscope.

Osmotic fragility of the erythrocyte membrane: characterization by modeling of the transmittance curve as a function of the NaCl concentration.

The theoretical extinction of blood suspensions submitted to a slow dialysis is analyzed in terms of their NaCl concentration. The model involves two adjustable parameters, chi and K, related to swelling and hemolysis. During swelling, the erythrocyte volume is supposed to vary linearly with the saline concentration. During hemolysis, an exponential decay of the hemoglobin concentration in the erythrocyte is used. The theoretical transmittance curves are consistent with the measurements carried out at a wavelength of 0.808 microm on native and incubated blood samples. Chi and K are relevant parameters to characterize quantitatively the fragility of the erythrocyte membrane. The effect of a non ideal character of the hemoglobin solutions and of normal distributions of chi and K is also discussed.

Deformation of erythrocytes under shear: a small-angle light scattering study.

In this study, sharp small-angle light scattering (SALS) images of erythrocytes under increasing shear stresses in a Couette flow were obtained, and accurate measurements of the angular positions of the two first minima and maxima have been carried out. The deformed cells were assumed to be three-axis ellipsoids of constant volume for all shear stresses. Application of the Physical Optics Approximation (POA) then permitted the determination of the cell dimensions as a function of the applied shear stress. Our results agree with determinations obtained by other methods.

Light scattering by ellipsoids in a physical optics approximation.

A physical optics approximation based on Presnel's laws is developed to calculate the intensity of light scattered by a three-axis ellipsoid of any orientation and any refractive index. Some results concerning totally reflecting spheres and dielectric spheroids are presented. An approach suitable for large scatterers is particularly good for small scattering angles. The angular intensities, i(1) and i(2), are then plotted versus θ for large axially oriented ellipsoids of various thicknesses. Theoretical small-angle light-scattering patterns are also presented and discussed. The data from one of them correspond to red cells in a shear flow.

Small angle light scattering by large spheroids. Comparison with patterns from erythrocytes under shear.

The physical optics approximation has been used to compute small angle light scattering patterns from spheroids regarged as models for deformed red cells. According to their orientation, diagrams have been obtained whose fine structure can be very different from that resulting of the classical diffraction (existence of maxima and minima of intensity along the rings). Some experimental results for red cells suspensions under shear are shown which exhibit features very similar to the theoretical ones.

Deformation and orientation of red blood cells in a simple shear flow. Theoretical study and approach at small angle light scattering.

On the basis of a simple theoretical model representing the rheological behaviour of the erythrocyte membrane, the authors have proceeded with a theoretical and experimental investigation on the light-scattering diagrams at small angles of normal red blood cells subjected to Couette flow. The results show that the light-scattering diagrams take into account not only cell deformation, but also cell orientation. The prospects for applying this method to the analytical separation of cells with different deformabilities are considered.