Erythrocyte lysis in isotonic solution of ammonium chloride: theoretical modeling and experimental verification.

A mathematical model of erythrocyte lysis in isotonic solution of ammonium chloride is presented in frames of a statistical approach. The model is used to evaluate several parameters of mature erythrocytes (volume, surface area, hemoglobin concentration, number of anionic exchangers on membrane, elasticity and critical tension of membrane) through their sphering and lysis measured by a scanning flow cytometer (SFC). SFC allows measuring the light-scattering pattern (indicatrix) of an individual cell over the angular range from 10 degrees to 60 degrees . Comparison of the experimentally measured and theoretically calculated light scattering patterns allows discrimination of spherical from non-spherical erythrocytes and evaluation of volume and hemoglobin concentration for individual spherical cells. Three different processes were applied for erythrocytes sphering: (1) colloid osmotic lysis in isotonic solution of ammonium chloride, (2) isovolumetric sphering in the presence of sodium dodecyl sulphate and albumin in neutrally buffered isotonic saline, and (3) osmotic fragility test in hypotonic media. For the hemolysis in ammonium chloride, the evolution of distributions of sphered erythrocytes on volume and hemoglobin content was monitored in real-time experiments. The analysis of experimental data was performed in the context of a statistical approach, taking into account that parameters of erythrocytes vary from cell to cell.

Experimental and theoretical study of light scattering by individual mature red blood cells by use of scanning flow cytometry and a discrete dipole approximation.

Elastic light scattering by mature red blood cells (RBCs) was theoretically and experimentally analyzed by use of the discrete dipole approximation (DDA) and scanning flow cytometry (SFC), respectively. SFC permits measurement of the angular dependence of the light-scattering intensity (indicatrix) of single particles. A mature RBC is modeled as a biconcave disk in DDA simulations of light scattering. We have studied the effect of RBC orientation related to the direction of the light incident upon the indicatrix. Numerical calculations of indicatrices for several axis ratios and volumes of RBC have been carried out. Comparison of the simulated indicatrices and indicatrices measured by SFC showed good agreement, validating the biconcave disk model for a mature RBC. We simulated the light-scattering output signals from the SFC with the DDA for RBCs modeled as a disk-sphere and as an oblate spheroid. The biconcave disk, the disk-sphere, and the oblate spheroid models have been compared for two orientations, i.e., face-on and rim-on incidence, relative to the direction of the incident beam. Only the oblate spheroid model for rim-on incidence gives results similar to those of the rigorous biconcave disk model.

Single-particle sizing from light scattering by spectral decomposition.

A Fourier transform was applied to size an individual spherical particle from an angular light-scattering pattern. The position of the peak in the amplitude spectrum has a strong correlation with the particle size. A linear equation retrieved from regression analysis of theoretically simulated patterns provides a relation between the particle size and the location of the amplitude spectrum's peak. The equation can be successfully applied to characterize particles of size parameters that range from 8 to 180 (corresponding to particle sizes that range from 1.2 to 27.2 microm at a wavelength of 0.633 microm). The precision of particle sizing depends on the refractive index and reaches a value of 60 nm within refractive-index region from 1.35 to 1.70. We have analyzed four samples of polystyrene microspheres with mean diameters of 1.9, 2.6, 3.0, and 4.2 microm and a sample of isovolumetrically sphered erythrocytes with a scanning flow cytometer to compare the accuracy of our new method with that of others.