## 4 Discussion  
The results (Figure 4a,b) show an enhancement of AF in the process of RBC doublet formation with the RBC age both in autologous plasma and in the dextran 70 kDa solution at the concentration 50 mg/mL. For RBCs in autologous plasma, the youngest cells exhibit AF = \(2.4\pm 0.2\) pN (M \(\pm\) SE), while the oldest ones--AF = \(3.8\pm 0.1\) pN (M \(\pm\) SE); for RBCs in the dextran 70 kDa solution, the youngest cells exhibited AF = \(3.0\pm 0.2\) pN (M \(\pm\) SE), while the oldest ones--AF = \(4.4\pm 0.1\) pN (M \(\pm\) SE). AF monotonously increases with the density fraction for RBCs in autologous plasma, while for RBCs in dextran solution, the AF differs only between the first and all other fractions. Figure 4: The dependence of the AF on the cells’ age (the higher the layer number the older the cells) for (**a**) RBCs in autologous plasma and (**b**) RBCs in the dextran 70 kDa solution at the concentration 50 mg/mL. Data from 5 healthy volunteers are presented. Bars here represent standard deviations. (I) \(p=0.003\), (II) \(p=0.002\), (III) \(p=0.004\), (IV) \(p=2\times 10^{-5}\). Previously, using a high-speed centrifugation technique and a Myrenne aggregometer, an increase in RBC aggregation (increase in aggregation indexes) with cell age was shown both in autologous plasma and dextran solution [33]. Other studies also confirmed the higher aggregation with cell age [2]. When older RBCs have diminished deformability [13; 34], it may contribute to changes of RBC aggregation properties. Decrease in aggregation is expected with the decrease in RBC deformability. However, the inverse pattern was observed. It seems that other important cellular factors [35; 36; 37], changing throughout the life span of RBC, are relevant to the higher aggregation of older cells. Previously, the higher aggregation of old RBCs was related to decreased levels of membrane sialic acid [38], which is the main factor contributing to the RBC surface charge. As far as the surface charge is one the main factors of RBC aggregation in the \"depletion\" theory [2; 4], the change of membrane sialic acid with RBC aging is one the key factors responsible for the aggregation differences of RBCs of different ages. Certainly, there are other age-related cellular factors that influence the aggregation, since the difference between younger and older cells still exists after removing sialic acid from the RBC surface [39]. Furthermore, it was shown that the old RBCs have a larger depletion layer compared to the young ones [40]. It could be explained either by a decrease in the RBC membrane glycocalyx thickness or a similar decrease in the polymer penetration into their glycocalyx considering the \"depletion\" theory [2; 4]. A larger depletion layer leads to the enhancement of old RBC aggregation. The \"bridging\" model does not satisfy the obtained results, to the extent that the older the cells, the less receptors on RBC membrane [13] and the less \"bridges\" can be produced. As for the \"bridging\" model, receptor(s) responsible for the bridging is (are) not identified. It is known that the older the cells, the less functional receptors are on a RBC membrane [13]. However, \"bridging\" receptors could be lipids or non-functional receptors. For example, the putative increase in phosphatidylserine in the outer membrane leaflet with increasing RBC age could be an appealing concept in favor of the \"bridging\" model [36]. Based on the results, the ratio of RBCs of different ages can influence the rheology of blood, as far as the RBC aggregation highly influences the blood flow rate of capillaries and veins [2]. In numerous diseases, such as diabetes mellitus and many others, the lifespan of RBCs is decreased, and the ratio between RBCs of different ages varies compared to the healthy donors [41]. This means that senescent RBCs in case of pathology may not achieve maximum AF. Additionally, it means that the ratio of senescent and young RBCs could be one of the factors that determine the rheology of blood.