Erythrocyte lysis and angle-resolved light scattering measured by scanning flow cytometry result to 48 indices quantifying a gas exchange function of the human organism.

Molecular/cell level of gas exchange function assumes the accurate measurement of erythrocyte characteristics and rate constants concerning to molecules involved into the CO2 /O2 transport. Unfortunately, common hematology analyzers provide the measurement of eight indices of erythrocytes only and say little about erythrocyte morphology and nothing about rate constants of cellular function. The aim of this study is to demonstrate the ability of the Scanning Flow Cytometer (SFC) in the complete morphological analysis of mature erythrocytes and characterization of erythrocyte function via measurement of lysing kinetics. With this study we are introducing 48 erythrocyte indices. To provide the usability of application of the SFC in clinical diagnosis, we formed four categories of indices which are as follows: content/concentration (9 indices), morphology (26 indices), age (5 indices), and function (8 indices). The erythrocytes of 39 healthy volunteers were analyzed with the SFC to fix the first-ever reference intervals for the new indices introduced. The essential measurable reliability of the presented method is expressed in terms of errors of characteristics of single erythrocytes retrieved from the solution of the inverse light-scattering problem and errors of parameters retrieved from the fitting of the experimental kinetics by molecular-kinetics model of erythrocyte lysis.

Blood platelet quantification by light scattering: from morphology to activation.

Analysis of blood platelets encounters a number of different preanalytical issues, which greatly decrease the reliability and accuracy of routine clinical analysis. Modern hematology analyzers determine only four parameters relating to platelets. Platelet shape and dose-dependent activation parameters are outside the scope of commercial instruments. We used the original scanning flow cytometer for measurement of angle-resolved light scattering and the discrete dipole approximation for simulation of light scattering from a platelet optical model, as an oblate spheroid, and global optimization with two algorithms: the DATABASE algorithm to retrieve platelet characteristics from light scattering and the DIRECT algorithm to retrieve dose-dependent activation parameters. We developed the original sampling protocol to decrease spontaneous platelet activation. The new protocol allows us to keep most of the platelets in resting and partially activated states before analysis. The analysis delivers 13 content and morphological parameters of the platelets. To analyze platelet shape change during ADP activation we developed a phenomenological model. This model was applied to the analysis of ADP activation of platelets to give 8 dose-dependent activation parameters. To demonstrate the applicability of the developed protocol and analytical method, we analyzed platelets from five donors. This novel approach to the analysis of platelets allows the determination of 21 parameters relating to their content, morphology and dose-dependent activation.

High-precision characterization of individual E. coli cell morphology by scanning flow cytometry.

We demonstrate a flow-cytometric method to measure length and diameter of single Escherichia coli cells with sub-diffraction precision. The method is based on the original scanning flow cytometer that measures angle-resolved light-scattering patterns (LSPs) of individual particles. We modeled the shape of E. coli cells as a cylinder capped with hemispheres of the same radius, and simulated light scattering by the models using the discrete dipole approximation. We computed a database of the LSPs of individual bacteria in a wide range of model parameters and used it to solve the inverse light-scattering problem by the nearest-neighbor interpolation. The solution allows us to determine length and diameter of each individual bacterium, including uncertainties of these estimates. The developed method was tested on two strains of E. coli. The resulting precision of bacteria length and diameter measurements varied from 50 nm to 250 nm and from 5 nm to 25 nm, respectively. The measured distributions of samples over length and diameter were in good agreement with measurements performed by optical microscopy and literature data. The described approach can be applied for rapid morphological characterization of any rod-shaped bacteria.

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.