Advanced consumable-free morphological analysis of intact red blood cells by a compact scanning flow cytometer.

Whereas modern automated blood cell analyzers measure the volume of individual red blood cells (RBCs), leading to four RBC indices (mean corpuscular volume, MCV; mean corpuscular hemoglobin, MCH; mean corpuscular hemoglobin concentration, MCHC; red cell distribution width, and RDW), the RBC shape has not been assessed by clinical screening tools. We applied the scanning flow cytometer (SFC) for complete characterization of intact RBC morphology in terms of diameter, maximal and minimal thicknesses, volume, surface area, sphericity index, spontaneous curvature, hemoglobin concentration, and content. The above-mentioned individual RBC characteristics were measured without fluorescent markers and other chemicals by a SFC equipped only with 660 nm laser for RBC illumination and single detector for measurement of angle-resolved light scattering. The distributions over all RBC characteristics were constructed and processed statistically to form the novel 31 RBC indices for 22 donor samples. Our results confirm the possibility of precise, label-free, enhanced morphological analysis of individual intact RBCs with compact single-detector flow cytometer. Detailed characterization of RBCs with high statistics and precision can be used to increase the value of screening examinations and to reveal pathologies accompanied by abnormality of RBC shape. © 2017 International Society for Advancement of Cytometry.

Fluorescence-free flow cytometry for measurement of shape index distribution of resting, partially activated, and fully activated platelets.

Whereas commercially available hematological analyzers measure volume of individual platelets, angle-resolved light-scattering provides unique ability to additionally measure their shape index. We utilized the scanning flow cytometer to measure light-scattering profiles (LSPs) of individual platelets taken from 16 healthy donors and the solution of the inverse light-scattering problem to retrieve the volume and shape index of each platelet. In normal conditions, the platelet shape index distribution (PSID) demonstrates three peaks, which relate to resting, partially activated, and fully activated platelets. We developed an algorithm, based on fitting PSID by a sum of three peak functions, to determine the percentage, mean platelet shape index, and distribution width of each platelet fraction. In total, this method gives eight additional parameters of platelet morphology and function to be used in clinical hematological analysis. We also stimulated the platelets with adenosine diphosphate (ADP) and measured the dependence of equilibrium PSID, including the total percentage of activated platelets, on ADP concentration. © 2016 International Society for Advancement of Cytometry.

Dynamic quantification of antigen molecules with flow cytometry.

Traditional methods for estimating the number of expressed molecules, based on the detection of target antigens bound with fluorescently labeled antibodies, assume that the antigen-antibody reaction reaches equilibrium. A calibration procedure is used to convert the intensity of the fluorescence signal to the number of target molecules. Along with the different limitations of every calibration system, this substantially limits the applicability of the traditional approaches especially in the case of low affinity antibodies. We address this problem here with studies in which we demonstrate a new approach to the antigen molecule quantification problem. Instead of using a static calibration system, we analyzed mean fluorescence values over time by flow cytometry during antibody-antigen binding. Experimental data obtained with an LSRII cytometer were fitted by a diffusion-reaction mathematical model using the Levenberg-Marquardt nonlinear least squares curve-fitting algorithm in order to obtain the number of target antigen molecules per cell. Results were compared with the Quanti-BRITE calibration system. We conclude that, instead of using experiment-specific calibration, the value of the binding rate constant for each particular antibody-antigen reaction can be used to quantify antigen molecules with flow cytometry. The radius of CD8 antibody molecule binding site was found, that allows recalculating the binding rate constant for other conditions (different sizes of reagent molecules, fluorescent label, medium viscosity and temperature). This approach is independent of specially prepared calibration beads, antibody reagents and the specific dye and can be applied to both low and high affinity antibodies, under both saturating and non-saturating binding conditions. The method was demonstrated on a human blood sample dataset investigating CD8α antigen on T cells in stable binding conditions.

Dynamic quantification of antigen molecules with flow cytometry.

Traditional methods for estimating the number of expressed molecules, based on the detection of target antigens bound with fluorescently labeled antibodies, assume that the antigen-antibody reaction reaches equilibrium. A calibration procedure is used to convert the intensity of the fluorescence signal to the number of target molecules. Along with the different limitations of every calibration system, this substantially limits the applicability of the traditional approaches especially in the case of low affinity antibodies. We address this problem here with studies in which we demonstrate a new approach to the antigen molecule quantification problem. Instead of using a static calibration system, we analyzed mean fluorescence values over time by flow cytometry during antibody-antigen binding. Experimental data obtained with an LSRII cytometer were fitted by a diffusion-reaction mathematical model using the Levenberg-Marquardt nonlinear least squares curve-fitting algorithm in order to obtain the number of target antigen molecules per cell. Results were compared with the Quanti-BRITE calibration system. We conclude that, instead of using experiment-specific calibration, the value of the binding rate constant for each particular antibody-antigen reaction can be used to quantify antigen molecules with flow cytometry. The radius of CD8 antibody molecule binding site was found, that allows recalculating the binding rate constant for other conditions (different sizes of reagent molecules, fluorescent label, medium viscosity and temperature). This approach is independent of specially prepared calibration beads, antibody reagents and the specific dye and can be applied to both low and high affinity antibodies, under both saturating and non-saturating binding conditions. The method was demonstrated on a human blood sample dataset investigating CD8α antigen on T cells in stable binding conditions.

Mathematical modeling the kinetics of cell distribution in the process of ligand-receptor binding.

A statistical approach is presented to model the kinetics of cell distribution in the process of ligand-receptor binding on cell surfaces. The approach takes into account the variation of the amount of receptors on cells assuming the homogeneity of monovalent binding sites and ligand molecules. The analytical expressions for the kinetics of cell distribution have been derived in the reaction-limited approximation. In order to demonstrate the applicability of the mathematical model, the kinetics of binding the rabbit, anti-mouse IgG with Ig-receptors of the murine hybridoma cells has been measured. Anti-mouse IgG was labeled with fluorescein isothiocyanate (FITC). The kinetics of cell distribution on ligand-receptor complexes was observed during the reaction process by real-time measuring of the fluorescence and light-scattering traces of individual cells with the scanning flow cytometer. The experimental data were fitted by the mathematical model in order to obtain the binding rate constant and the initial cell distribution on the amount of receptors.

Individual Escherichia coli cells studied from light scattering with the scanning flow cytometer.

BACKGROUND: Flow cytometry is a powerful tool for the analysis of individual particles in a flow. Differential light scattering (an indicatrix) was used for many years to obtain morphologic information about microorganisms. The indicatrices play the same role for individual particle recognition as a spectrum for substance characterization. We combined two techniques to analyze the indicatrix of the cells for the purpose of developing a database of light-scattering functions of cells. METHODS: The scanning flow cytometer (SFC) allows the measurement of the entire indicatrix of individual particles at polar angles ranging from 5 degrees to 100 degrees. In this work, light-scattering properties of Escherichia coli have been studied both experimentally and theoretically with the SFC and the T-matrix method, respectively. The T-matrix method was used because of the nonspherical shape of E. coli cells, which were modeled by a prolate spheroid. RESULTS: The indicatrices of E. coli cells were stimulated with T-matrix method at polar angles ranging from 10 degrees to 60 degrees. The absolute cross-section of light scattering of E. coli has been determined comparing the cross section of polystyrene particles modeled by a homogeneous sphere. The E. coli indicatrices were compared for logarithmic and stationary phases of cell growth. CONCLUSIONS: The indicatrices of E. coli were reproducible and could be used for identification of these cells in biologic suspensions. The angular location of the indicatrix minimum can be used in separation of cells in logarithmic and stationary phases. To use effectively the indicatrices for that purpose, the light-scattering properties of other microorganisms have to be studied.

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.

Particle classification from light scattering with the scanning flow cytometer.

BACKGROUND: The differential light-scattering pattern, an indicatrix, provides the most complete characterization of the optical properties of a particle. Particle classification can be performed on the basis of particle parameters retrieved from the indicatrices. This classification extends the ability of flow cytometry in particle recognition. METHODS: The scanning flow cytometer (SFC) permits an acquisition of traces of light scattering signals, i.e., native SFC traces, from single particles. The acquired native SFC traces are transformed into indicatrices. The performance of the SFC in measurements of indicatrices has been demonstrated for the following particles: lymphocytes, erythrocytes, polystyrene particles, and milk-fat particles. RESULTS: The structure and profile of the indicatrix for each particle type have been found to be unique. Classification of polystyrene particles has been performed on the basis of the map formed by particle refractive index and size. The polystyrene particles were classified using this map into different size categories ranging from 1.4-7 microm, with a size deviation of 0.07 microm. CONCLUSIONS: The method based on analysis of native SFC traces shows better performance in particle classification than the method based on the particle refractive index and size map. The classification performance of the SFC will be useful, for example, for particle sorting and particle identification, and with additional fluorescent measurements may have applications in multiparameter particle-based immunoassay.

Light-scattering properties of individual erythrocytes.

We have studied the light-scattering properties of human erythrocytes both experimentally and theoretically. In the experimental study measurements were performed with a Scanning Flow Cytometer (SFC). The SFC can measure the light-scattering pattern (indicatrix) of an individual particle over an angular range of 10-60 degrees. We have observed polymorphism in the measured set of indicatrices. To understand the reason for this polymorphism, we have made a theoretical study of the scattering properties of erythrocytes. The Wentzel-Kramer-Brillouin approximation has been employed to calculate indicatrices of individual erythrocytes in different orientations relative to the incident light beam. The indicatrices were calculated over an angular range of 15-35 degrees. A comparison of the experimentally measured and theoretically calculated indicatrices shows that the polymorphism is due mainly to the different orientation of the erythrocytes in the flow. The effect caused by the Poiseuille profile of the flow on an individual erythrocyte within the SFC cuvette capillary was studied theoretically by use of the Stokes approximation. Rotation of an erythrocyte was predicted by this theoretical analysis, and this prediction was further verified by comparison with experimental results.

A new design of the flow cuvette and optical set-up for the scanning flow cytometer.

We introduce a new design for the optical cuvette and a new optical lay-out for the Scanning Flow Cytometer (SFC) that permits measurement of the angular dependency of the scattered light from individual moving particles. The improved optical scheme of the SFC allows measurement of the angular scattering pattern of individual particles at polar angles from 10 degrees to 120 degrees with integration at azimuthal angles from 0 degrees to 360 degrees and with angular resolution of better than 0.5 degrees. The performance of the SFC is demonstrated using certified polystyrene particles as reference material The aim of this work is to develop a flow cytometer, which, by recording the entire light scattering pattern of individual biological particles, would provide more information about the particle structure than the ordinary wide angle, forward and side scattering concepts.

Parametric solution of the inverse light-scattering problem for individual spherical particles.

We analyze the relation between spherical particle parameters (size parameter ? and relative refractive index m ) and light-scattering indicatrix parameters (fringe pitch and visibility) for ?and m ranging from 7 to 88 and from 1.025 to 1.2, respectively. We consider formation of the indicatrix structure under variation of size parameter ? and phase-shift parameter ? =2 ?(m - 1). We show that indicatrix visibility depends basically on the phase-shift parameter whereas fringe pitch depends on the size parameter. An analysis of these dependencies allows us to determine the region in the ? x ? map where particle parameters can be derived unambiguously from indicatrix parameters. We clarify errors in determination of particle characteristics on the basis of the approximating equations that relate particle size and phase shift to indicatrix parameters.

Absolute real-time measurement of particle size distribution with the flying light-scattering indicatrix method.

The flying light-scattering indicatrix (FLSI, angular dependency of the intensity of light scattered by a moving individual particle) method, based on a scanning flow cytometer (SFC) that permits measurement of individual particle characteristics from light-scattering data, has been used for the determination of size distribution of the following particles: polystyrene latex, milk fat, and spores (Penicillium levitum, Aspergillus pseudoglaucus). The optical system of the SPC and empirical equations provided absolute sizing at the rate of 50 particles/s. Size distributions obtained with the FLSI method and a best-fit procedure using Mie scattering theory have been compared.

Measurement of scattering properties of individual particles with a scanning flow cytometer.

A hydrofocusing head with an optical cuvette has been developed for the flow cytometer to generate complete scatter patterns of single particles at scattering angles ranging from 10° to 120°. The scatter signal has been measured as a function of the angle (a flying indicatrix) by the use of particle motion within a scanning system of the flow cytometer by the use of a single photomultiplier. Scattering data measured with the flow cytometer have been compared with those calculated from Mie theory for latex particles. A calculation algorithm has been used to estimate the size and the refractive index of spherical particles from the scattering data measured.