Calibration-free method to determine the size and hemoglobin concentration of individual red blood cells from light scattering.

At present, hemoglobin concentration and the volume of an erythrocyte can be determined from the intensities of light scattered by an individual cell at fixed angular intervals. This method is used in modern hemoglobin analyzers, but it requires calibration of optical and electronic units by certified particles of known size and refractive index. We describe a method that is based on the parametric solution of an inverse light-scattering problem and does not require a calibration procedure. The method is based on the use of parameters of the entire angular light-scattering pattern, called an indicatrix here. These parameters do not depend on the absolute intensity of light scattering. The indicatrix parameters form approximating equations that relate these parameters to the size and the phase-shift parameters of the particle. The applicability of the method is demonstrated by measurement of the indicatrices of individual sphered erythrocytes. The indicatrices of the individual erythrocytes were measured with a scanning flow cytometer at an angular range of from 15 to 55 deg. The volume and the hemoglobin concentration have been calculated by use of the developed method and by fitting of the experimental indicatrices to the indicatrices calculated from the Mie theory.