ИСТИНА

Aggregation of red blood cells (RBCs) is an intrinsic property of blood, which has direct effect on blood viscosity and blood circulation throughout the body. However until now the aggregation mechanism is not fully clear. Measuring interaction forces between RBCs within a single aggregate and/or between two aggregates in the presence of various macromolecules in the solution that induce or inhibit aggregation with an optical trap allows for assessing the features of aggregation, and better understanding the mechanism of this important process [1]. The average forces required to separate two RBCs in an aggregate are strongly dependent on the environment and are in range from few pN to tens of pN. In this work, we used a double-channel optical trap to measure the interaction forces and kinetics between RBCs in single aggregates before and after their density separation. RBCs circulate in vivo during around 120 days and their aggregation properties change significantly during this period. The investigation of aggregation kinetics of density separated RBCs would allow more precise measurement of the aggregation parameters at single cell level. Density separation (age separation) of RBCs was performed using freshly drawn human blood samples at 50% hematocrit in autologous plasma using 1 hour-long centrifugation at 30,000g and 30 °C. After the centrifugation, the old and young cells were drawn from the 5% bottom and 5% top parts of the tube correspondingly. All experiments were performed in autologous plasma within 8 hours after drawing blood from a human individual. Age separated RBCs showed significantly different aggregation strength and aggregation speed. The same effect was found by other authors on large ensembles of cells using the light scattering aggregometry technique [2]. Aggregation strength of RBC doublets was measured as drag velocity required to separate two cells trapped in a single optical trap. The drag velocities of separation were 78.9 ± 6.1 µm/s for young cells (n = 9) and 110 ± 13 µm/s for old cells (n = 13). The aggregation time defined as the time required for two adjacent RBCs to spontaneously overlap starting from a small area of contact after being released from the optical trap. The measured values of the aggregation time were 7.6 ± 1.2 seconds for young cells (n=42) and 5.53 ± 0.7 (n = 22) for old cells showing significant differences between the fractions. These differences would raise significant deviations in single measurements on cells without age separation and where large statistics and sampling is difficult. The trapping force for age separated RBCs found to be different. Velocities required for RBCs to escape from optical trap were 74 ± 10 µm/sec for young cells and 84 ± 10 µm/sec for old cells at the same trapping power. (measurements were performed at 10 µm/sec steps). This should be accounted for in further RBC interaction force measurements with optical tweezers.