Dmitry I. Strokotov

Is there a difference between T- and B-lymphocyte morphology?

We characterize T- and B-lymphocytes from several donors, determining cell diameter, ratio of nucleus to cell diameter, and refractive index of the nucleus and cytoplasm for each individual cell. We measure light-scattering profiles with a scanning flow cytometer and invert the signals using a coated sphere as an optical model of the cell and by relying on a global optimization technique. The main difference in morphology of T- and B-lymphocytes is found to be the larger mean diameters of the latter. However, the difference is smaller than the natural biological variability of a single cell. We propose nuclear inhomogeneity as a possible reason for the deviation of measured light-scattering profiles from real lymphocytes from those obtained from the coated sphere model.

Accurate measurement of volume and shape of resting and activated blood platelets from light scattering

Abstract. We introduce a novel approach for determination of volume and shape of individual blood platelets modeled as an oblate spheroid from angle-resolved light scattering with flow-cytometric technique. The light-scattering profiles (LSPs) of individual platelets were measured with the scanning flow cytometer and the platelet characteristics were determined from the solution of the inverse light-scattering problem using the precomputed database of theoretical LSPs. We revealed a phenomenon of parameter compensation, which is partly explained in the framework of anomalous diffraction approximation. To overcome this problem, additional a priori information on the platelet refractive index was used. It allowed us to determine the size of each platelet with subdiffraction precision and independent of the particular value of the platelet aspect ratio. The shape (spheroidal aspect ratio) distributions of platelets showed substantial differences between native and activated by 10 μM adenosine diphosphate samples. We expect that the new approach may find use in hematological analyzers for accurate measurement of platelet volume distribution and for determination of the platelet activation efficiency.

Polarized light-scattering profile—advanced characterization of nonspherical particles with scanning flow cytometry

We instrumentally, theoretically, and experimentally demonstrate a new approach for characterization of nonspherical individual particles from light scattering. Unlike the original optical scheme of the scanning flow cytometer that measures an angle‐resolved scattering corresponding in general to S11 element of the light‐scattering matrix, the modernized instrument allows us to measure the polarized light‐scattering profile of individual particles simultaneously. Theoretically, the polarized profile is expressed by the combination of a few light‐scattering matrix elements. This approach supports us with additional independent data to characterize a particle with a complex shape and an internal structure. Applicability of the new method was demonstrated from analysis of polymer bispheres. The bisphere characteristics, sizes, and refractive indices of each sphere composing the bisphere were successfully retrieved from the solution of the inverse light‐scattering problem. The solution provides determination of the Eulerian angles, which describe the orientation of the bispheres relative to the direction of the incident laser beam and detecting polarizer of the optical system. Both the ordinary and polarized profiles show a perfect agreement with T‐matrix simulation resulting to 50‐nm precision for sizing of bispheres.

Super‐resolved calibration‐free flow cytometric characterization of platelets and cell‐derived microparticles in platelet‐rich plasma

Importance of microparticles (MPs), also regarded as extracellular vesicles, in many physiological processes and clinical conditions motivates one to use the most informative and precise methods for their characterization. Methods based on individual particle analysis provide statistically reliable distributions of MP population over characteristics. Although flow cytometry is one of the most powerful technologies of this type, the standard forward‐versus‐side‐scattering plots of MPs and platelets (PLTs) overlap considerably because of similarity of their morphological characteristics. Moreover, ordinary flow cytometry is not capable of measurement of size and refractive index (RI) of MPs. In this study, we 1) employed the potential of the scanning flow cytometer (SFC) for identification and characterization of MPs from light scattering; 2) suggested the reference method to characterize MP morphology (size and RI) with high precision; and 3) determined the lowest size of a MP that can be characterized from light scattering with the SFC. We equipped the SFC with 405 and 488 nm lasers to measure the light‐scattering profiles and side scattering from MPs, respectively. The developed two‐stage method allowed accurate separation of PLTs and MPs in platelet‐rich plasma. We used two optical models for MPs, a sphere and a bisphere, in the solution of the inverse light‐scattering problem. This solution provides unprecedented precision in determination of size and RI of individual spherical MPs—median uncertainties (standard deviations) were 6 nm and 0.003, respectively. The developed method provides instrument‐independent quantitative information on MPs, which can be used in studies of various factors affecting MP population.

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

We propose a method for characterization of mature red blood cells (RBCs) morphology, based on measurement of light-scattering patterns (LSPs) of individual RBCs with the scanning flow cytometer and on solution of the inverse light-scattering (ILS) problem for each LSP. We considered a RBC shape model, corresponding to the minimal bending energy of the membrane with isotropic elasticity, and constructed an analytical approximation, which allows rapid simulation of the shape, given the diameter and minimal and maximal thicknesses. The ILS problem was solved by the nearest-neighbor interpolation using a preliminary calculated database of 250,000 theoretical LSPs. For each RBC in blood sample we determined three abovementioned shape characteristics and refractive index, which also allows us to calculate volume, surface area, sphericity index, spontaneous curvature, hemoglobin concentration and content.

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.

Influence of magnesium sulfate on HCO3/Cl transmembrane exchange rate in human erythrocytes

Magnesium sulfate (MgSO4) is widely used in medicine but molecular mechanisms of its protection through influence on erythrocytes are not fully understood and are considerably controversial. Using scanning flow cytometry, in this work for the first time we observed experimentally (both in situ and in vitro) a significant increase of HCO3(-)/Cl(-) transmembrane exchange rate of human erythrocytes in the presence of MgSO4 in blood. For a quantitative analysis of the obtained experimental data, we introduced and verified a molecular kinetic model, which describes activation of major anion exchanger Band 3 (or AE1) by its complexation with free intracellular Mg(2+) (taking into account Mg(2+) membrane transport and intracellular buffering). Fitting the model to our in vitro experimental data, we observed a good correspondence between theoretical and experimental kinetic curves that allowed us to evaluate the model parameters and to estimate for the first time the association constant of Mg(2+) with Band 3 as KB~0.07mM, which is in agreement with known values of the apparent Mg(2+) dissociation constant (from 0.01 to 0.1mM) that reflects experiments on enrichment of Mg(2+) at the inner erythrocyte membrane (Gunther, 2007). Results of this work partly clarify the molecular mechanisms of MgSO4 action in human erythrocytes. The method developed allows one to estimate quantitatively a perspective of MgSO4 treatment for a patient. It should be particularly helpful in prenatal medicine for early detection of pathologies associated with the risk of fetal hypoxia.

Additivity of light-scattering patterns of aggregated biological particles

Abstract. The paper is focused on light scattering by aggregates of optically soft particles with a size larger than the wavelength, in particular, blood platelets. We conducted a systematic simulation of light scattering by dimers and larger aggregates of blood platelets, each modeled as oblate spheroids, using the discrete dipole approximation. Two-dimensional (2-D) light scattering patterns (LSPs) and internal fields showed that the multiple scattering between constituent particles can be neglected. Additionally, we derived conditions of the scattering angle and orientation of the dimer, under which the averaging of the 2-D LSPs over the azimuthal scattering angle washes out interference in the far field, resulting in averaged LSPs of the aggregate being equal to the sum of that for its constituents. We verified theoretical conclusions using the averaged LSPs of blood platelets measured with the scanning flow cytometer (SFC). Moreover, we obtained similar results for a model system of aggregates of polystyrene beads, studied both experimentally and theoretically. Finally, we discussed the potential of discriminating platelet aggregates from monomers using the SFC.

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.

Light-scattering gating and characterization of plasma microparticles

Abstract. Flow cytometry method (FCM) is widely used for analysis of cell-derived microparticles (MPs). Numerous efforts are currently aimed to standardize these measurements among different instruments. We push the FCM characterization of MPs to the limit based on rigorous simulation of measured signals. We measured forward- and side-scatter (FSC/SSC) signals and angle-resolved light-scattering profiles (LSPs) of polystyrene microspheres and MPs, including their aggregates, using a scanning flow cytometer (SFC). We used the Mie theory to (1) accurately evaluate instrument detection limits; (2) construct FSC/SSC gates for MPs in absolute scales of size and refractive index (RI); and (3) determine size and RI of individual spherical MPs. LSPs were used for advanced characterization, including differentiation of spherical and nonspherical particles. The proposed absolute FSC/SSC gating is naturally standardized for any FCM instrument, given the knowledge of its optical system and leads to instrument-independent analysis of MPs. The inverse Mie problem has a unique solution only for some regions of size and RI and uncertainties rapidly increase with decreasing size and RI. The developed methods are applicable to any flow cytometer, but are limited by assumption of particle sphericity. The latter can be relaxed only if additional signals, such as LSP, are measured.