RESUMEN
The analysis of individual particles with complex morphologies from light scattering is crucial in disperse systems studies, such as blood cells. Characterization, which assumes determining particle characteristics, has a higher likelihood of succeeding in solving the inverse light-scattering problem if an instrument provides enough light-scattering data. In this study, we demonstrate how we extend the operating angular interval for the 4π Scanning Flow Cytometer (4πSFC), which measures angle-resolved light-scattering profiles (LSPs) of individual particles. The angular interval is extended by additionally measuring light scattering for the backward hemisphere. Currently, the 4πSFC setup uses three lasers, a single optical cell, and three photomultipliers. It enables the measurement of the LSP of individual particles within the angular interval of 10 to 170° for polar angles with integration over azimuth angles, which covers the spatial angle of 98.5% of the 4π angle. We demonstrate the 4πSFC's performance in measuring LSPs from the analysis of polymer beads, mature and spherized erythrocytes, and platelets. The 4πSFC has the potential to be very useful in identifying platelet dimers and granulocytes without labels, characterizing lymphocytes, monocytes, and abnormal erythrocytes.
Asunto(s)
Plaquetas , Luz , Citometría de Flujo , Dispersión de Radiación , GranulocitosRESUMEN
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.
Asunto(s)
Plaquetas , Activación Plaquetaria , Simulación por Computador , Citometría de Flujo , Humanos , Reproducibilidad de los ResultadosRESUMEN
The well-known platelet shape change is the universal hallmark of activation. This review uncovers the biophysics underlying this rapid and dramatic transformation. We aim to give a broad vision of the interplay between different cytoskeletal subsystems, which is based on physical considerations and recent advances in mathematics and computational biology. These novel findings lead to the understanding that the ring of microtubules counterbalances cortical tension in the resting platelet, making it a "mechanically charged" system. Platelet activation breaks the balance via several mechanisms, triggering rapid ring buckling and cell rounding. Based on the review of known data concerning the relations between platelet shape and function, we hypothesize that disk-to-sphere transformation facilitates platelet adhesion under flow. Conclusions of the paper may be useful for the development of novel, cytoskeletal-based strategies of antiplatelet therapy.
Asunto(s)
Plaquetas/citología , Plaquetas/fisiología , Animales , Fenómenos Biofísicos , Forma de la Célula , Citoesqueleto/metabolismo , Humanos , Microtúbulos/metabolismo , Activación Plaquetaria , Adhesividad PlaquetariaRESUMEN
We present a simple physically based quantitative model of blood platelet shape and its evolution during agonist-induced activation. The model is based on the consideration of two major cytoskeletal elements: the marginal band of microtubules and the submembrane cortex. Mathematically, we consider the problem of minimization of surface area constrained to confine the marginal band and a certain cellular volume. For resting platelets, the marginal band appears as a peripheral ring, allowing for the analytical solution of the minimization problem. Upon activation, the marginal band coils out of plane and forms 3D convoluted structure. We show that its shape is well approximated by an overcurved circle, a mathematical concept of closed curve with constant excessive curvature. Possible mechanisms leading to such marginal band coiling are discussed, resulting in simple parametric expression for the marginal band shape during platelet activation. The excessive curvature of marginal band is a convenient state variable which tracks the progress of activation. The cell surface is determined using numerical optimization. The shapes are strictly mathematically defined by only three parameters and show good agreement with literature data. They can be utilized in simulation of platelets interaction with different physical fields, e.g. for the description of hydrodynamic and mechanical properties of platelets, leading to better understanding of platelets margination and adhesion and thrombus formation in blood flow. It would also facilitate precise characterization of platelets in clinical diagnosis, where a novel optical model is needed for the correct solution of inverse light-scattering problem.