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23OPTICAL TECHNIQUES FOR ASSESSING BLOOD MICRORHEOLOGY: RED BLOOD CELLS DEFORMABILITY, AGGREGATION AND THEIR INTERRELATION
ANDREI LUGOVTSOV12, ANASTASIA MASLYANITSINA1, PETR ERMOLINSKIY1 AND ALEXANDER
PRIEZZHEV1'2
1Physics Department, Lomonosov Moscow State University, Russia 2International Laser Center, Lomonosov Moscow State University, Russia
Abstract
The state of human organism largely depends on blood microcirculation that, in turn, depends on the microrheologic properties of red blood cells (RBCs), in particular, the RBC intrinsic properties of deformability and aggregation that are supposed to be interdependent [1]. The RBCs have the ability to reversibly deform in the blood flow. Usually they elongate in the direction of the flow but, also, they can change their shape dramatically in the vessels that are smaller than the size of the RBCs, for example, capillaries with diameters from 3 to 5 ^m [2]. A considerable contribution to the deformability comes from the elasticity of the cell membrane, as well as from the viscosity of hemoglobin solution inside the cell [3]. RBC deformability plays a significant role in the blood circulation. In particular, RBC filtering in narrow circulatory pathways in the human spleen is based on their impaired deformability. Another important process that influences the blood flow is the aggregation of RBCs [4]. It is a reversible process of formation of linear and more complex structures of RBCs. The aggregation happens predominantly inside large vessels. However, the aggregates can become quite large, and if not their ability to disaggregate to single cells due to shear stress, the blood flow would be impaired.
Socially significant diseases such as arterial hypertension, diabetes mellitus and others are associated with serious changes in the RBC deformability [4-6]. At the same time a significant change in the aggregation parameters may happen [4]. For example, RBC aggregates in the blood of patients suffering from arterial hypertension are stronger and form faster than in the blood of healthy people [6]. Moreover, these pathologies are accompanied by an alteration in the number of RBC involved in the process of spontaneous aggregation. This can be caused by many reasons: a change in the protein composition of plasma, cell membrane changes, different rigidity and age of the cells, as well as the average patient age and their medication, etc. [4].
The aim of this work is to identify the relationship between the deformability of RBCs and their aggregation properties, both of which are the key factors for the blood flow. Laser diffractometry, diffuse light scattering and laser tweezers were implemented for in vitro measurements.
Laser diffractometry performed with the RheoScan diffractometer (RheoMeditech, Korea) was used to obtain the shear induced deformation parameters of RBCs by processing the light intensity distribution in the diffraction pattern [7]. This pattern is based on diffraction of a laser beam on a highly diluted RBC suspension in a flow channel in vitro. The dimensions of the channel are 0.2 mm high x 4.0 mm wide x 40 mm long. We measured the RBC deformability index (DI) that describes the average elongation of the cells by shear stress. The elongation of the cells corresponds to the elongation of the diffraction pattern. Different shear stresses from 20 Pa to 0.5 Pa were applied to the RBC suspension in order to change the shear stress and, consequently, the elongation of the cells.
Laser aggregometry was performed using the RheoScan aggregometer (RheoMeditech, Korea) [8]. It is based on diffuse light scattering and is applied to whole blood samples in order to retrieve a number of the RBC aggregation properties. By analyzing the scattered light intensity as a function of time during the process of RBC spontaneous aggregation we can evaluate the aggregation index (AI), which characterizes the relative number of aggregated cells during the first 10 sec of the aggregation process [14]. Besides that, the critical shear stress (CSS) that characterizes the balance of aggregation and disaggregation processes was measured. In order to do it, the blood flow conditions were created in vitro with varying shear stress, and the light scattered backwards was analyzed.
Different osmolalities of plasma (150-500 mOsm/l) and concentrations of glutaraldehyde (GA) (up to 0.004%) were used to change the deformability of the RBCs in vitro. The measurements were performed at 37 °C. The study was conducted on the blood of 2 healthy donors. The values of AI, DI and the forces of RBC interaction were measured 5 times for the same sample. The results were then averaged and the standard deviations from the mean values were calculated.
The RBC deformability changes are presented in the Fig. 1. The results in the Fig. 1a show a decrease in the DI with the increase of GA concentration. Secondly, in a separate experiment, the osmolality of the suspension was also shown to influence RBC deformability as seen in Fig. 1b.
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Figure 1: The deformability index as a function of the shear stress (a) at different glutaraldehyde concentrations
and (b) at different osmolarities.
The results obtained by laser aggregometry for the samples treated with GA are shown in Fig. 2a. The AI significantly drops for samples with low DI, which corresponds to high GA concentration. Namely, the control measurement (0% GA) yields DI equal to 0.522 ± 0.006 and AI equal to 39 ± 4%. For 0.004% GA concentration DI decreases to 0.426 ± 0.006 and AI decreases to 9 ± 3%. This means that at high GA concentrations the process of spontaneous aggregation almost stops. For the samples suspended in plasma at different osmolarities the laser aggregometry method shows similar results (Fig. 2b). Lower deformability corresponds to smaller AI.
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Deformability Index at 20 Pa Deformability Index at 20 Pa
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Figure 2: The aggregation index as functions of the deformability index for different glutaraldehyde concentrations (a)
and osmolarities (b).
The method of laser diffractometry confirmed that with the addition of glutaraldehyde and with a large change in the osmolality of the solution, RBCs become more rigid. Secondly, the methods of laser aggregometry and laser tweezers gave consistent results: with the decreased ability of RBCs to deform the formation of aggregates becomes impaired. However, the critical shear stress and the disaggregation force measured with laser tweezers remain mostly unchanged. This means that the RBC aggregate formation is dependent on the deformability of the membrane, while the connection to disaggregation is less pronounced and more complicated in nature.
Acknowledgement: This work was supported by the Russian Foundation for Basic Research grant #19-5251015.
References
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[6] P. Ermolinskiy, A. Lugovtsov, A. Maslyanitsina, A. Semenov, L. Dyachuk, and A. Priezzhev, Interaction of erythrocytes in the process of pair aggregation in blood samples from patients with arterial hypertension and healthy donors: measurements with laser tweezers, Journal of Biomedical Photonics & Engineering 4(3), 030303, 2018.
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[8] S. Shin, Y. Yang, and J. Suh, Measurement of erythrocyte aggregation in a microchip stirring system by light transmission, Clinical Hemorheology and Microcirculation 41(3), 197-207, 2009.
