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

Abstract

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.

Mature red blood cells: from optical model to inverse light-scattering problem

“…In spite of statistically significant number of measured RBCs, it ignores shape and hemoglobin-concentration differences between measured cells introducing uncertain inaccuracy. A similar technique is the flow cytometry, based on measurement of two-angle light scattering by single cells [4] or angleresolved light scattering [5]. The main limitation of these methods is the need to chemically spherize RBCs prior to their characterization.…”

“…In this study we modernize the angle-resolved light-scattering approach based on the scanning flow cytometer (SFC) [5,9,10] for complete and rapid characterization of RBCs from light-scattering profiles (LSP). The new method does not require chemical spherization of RBCs and utilizes the solution of the inverse light-scattering (ILS) problem, using the optical model of a mature RBC.…”

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

“…The RBC volume is the most important hematological index measured with modern automatic hematology analyzers. 3 Flow cytometry 25 and Coulter's cell 3 are the instrumental solutions utilized for measurement of RBC volume distribution. The modified indicatrices of biconcave discs calculated for three volumes with a typical diameter of 6.75 µm are shown in Fig.…”

A mathematical erythrocyte model based on weak solutions of integral equations

“…This task is performed by flow cytometers, devices that measure the intensity of light scattered by a single cell to determine the cell parameters. So far, effective schemes of cytometers capable of measuring scattered radiation in the incident light propagation direction have been designed and implemented [1,2]. On the other hand, the interpretation of measurement results calls for rigorous mathematical models that can be used in analyzing the scattering characteristics of complex-shape particles in a wide parameter range.…”