Effect of optical clearing agent glycerol on RBC aggregation and deformation: in vitro study by optical techniques

Abstract

Red blood cells (RBCs) aggregation is a process of spontaneous reversible formation of two-dimensional linear and complex three-dimensional RBC structures in the blood flow and in stasis conditions. The ability of RBCs for reversible deformation is characterized by another intrinsic property of RBC, their deformability. Both these processes play a crucial role in rheology of blood because of their strong impact on blood viscosity. Optical clearing agents (OCAs) are special substances that are widely used in vivo and in vitro in different biomedical applications for the enhancement of imaging tissues and organs [1]. The mechanism of the OCA action is based on their diffuse penetration into the tissues resulting in the reduction of the amount of multiple light scattering by equalizing the relative refractive index of the light scattering nonhomogeneities inside the investigated tissue. This makes the tissue optically more homogeneous as a result increasing the depth and resolution of imaging [2]. In a wide number of methods, e.g., in vivo laser speckle imaging of blood flow in cerebral capillaries or digital capillaroscopy of the nail bed capillaries, or OCT, the applied OCAs penetrate up to the blood vessels. Most of the OCAs are osmotically active and can interact with RBCs by changing their deformability and aggregation properties. This can lead to alteration in the scattering signal from the tissue containing the blood and affect the results of a research. Despite this fact, the effect of OCAs on RBCs aggregation and deformation has not been studied yet. The main goal of this work was to investigate the effect of glycerol as an OCA on RBC aggregation and deformability properties by optical techniques in vitro.
In this work, such RBCs aggregation parameters as the RBC aggregation rate and the hydrodynamic strength of RBC aggregates were measured using the diffuse light scattering technique [3]. This method is based on the registration of time dependences of the intensity of laser light (633 nm) scattered forward or backward by the blood samples. With this method we can measure the critical shear stress (CSS) that characterizes the hydrodynamic strength of aggregates and the aggregation index (AI) that shows the per cent number of aggregated RBCs during the first 10 seconds of spontaneous aggregation process in whole blood samples. [3] The deformability index, which characterizes the ability of RBC to change their shape in the blood flow was measured by laser diffractometry technique. It is based on processing the light intensity distributions in the diffraction patterns at different shear induced stresses in the flow. These patterns are produced due to the diffraction of a laser beam on a highly diluted suspension of RBC in a flow vessel in vitro. Visual control of the RBC morphology after their incubation with glycerol or after adding similar aliquots of distilled water was performed by optical microscopy of the samples using Zeiss AxioVert 200M microscope with 64x magnification.
All experiments were carried out with the samples of whole blood that were freshly drawn from healthy volunteers and incubated with the OCA glycerol at the concentrations of 10% and 20%. The control samples were prepared by mixing the blood with the aliquots of distilled water.
The obtained results show that there are no statistically significant differences in CSS parameter at all glycerol concentrations, while AI is reduced with an increase in glycerol concentration. For example, AI of the sample with 20% of glycerol is reduced by 66 ± 11%, in comparison with that of the whole blood sample, while in the control sample with distilled water AI drops only by 35 ± 12%. Also, the RBC deformability index becomes significantly lower (by 29 ± 13%) in the samples with glycerol, which we cannot observe in the control samples.
Basing on the results of our experiments, we can conclude that with an increase in the glycerol concentration the reduction of RBC aggregation is pronounced stronger than in the blood samples diluted with distilled water. This means that there is a marked effect of glycerol on RBC aggregation. A negative effect of glycerol on deformability takes place as well. At the same time the morphology of RBC changes neither with the addition of glycerol at the studied concentrations, nor with the addition of distilled water. No lysis of RBC was noticed in the latter case. We assume that our results confirm the statements from the work [4] that glycerol penetrate into the RBCs through the cells membrane. As the result, deformability decreases due to rise in the viscosity of intracellular contents. This leads to reduction of the deformability and as consequence entails the AI impairs.
This work was supported by the Russian Science Foundation (Grant No. 23-45-00027).
[1] E.A. Genina, A.N. Bashkatov, Yu.P. Sinichkin, I.Yu. Yanina, V.V. Tuchin. “Optical clearing of biological tissues: prospects of application in medical diagnostics and phototherapy”, Journal of Biomedical Photonics and Engineering 1(1):22-58 (2015).
[2] Luis M.C. Oliveira, V.V. Tuchin. “The Optical Clearing Method”, Springer Briefs in Physics (2019).
[3] A.E. Lugovtsov, Yu.I. Gurfinkel, P.B. Ermolinskiy, A.I. Maslyanitsina, L.I. Dyachuk, and A.V. Priezzhev, “Optical assessment of alterations of microrheologic and microcirculation parameters in cardiovascular diseases”, Biomedical Optics Express 10 (8):3974-3986 (2019).
[4] B H. Shaz, C D. Hillyer, J C. Zimring, T C. Abshire. «Frozen Blood Products». In: “Transfusion Medicine and Hemostasis: Clinical and Laboratory Aspects”, Elsevier, 2009, chapter 39.


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Moldon Pavel
Faculty of Physics, Lomonosov Moscow State University, Moscow, Russia
Russia

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