## 1 Introduction  
Red blood cell (RBC) spontaneous aggregation and forced disaggregation processes strongly influence the microcirculation of blood and impact human health in general [1, 2]. The understanding of RBC aggregation mechanisms is still not clear and contradictory at some points [2]. Since the middle of last century, two main hypotheses of RBC aggregation mechanism were coexisting: the \"depletion\" theory and the \"bridging\" model [2, 3, 4]. In the \"depletion\" theory, the interaction between RBCs is described by osmotic forces that arise in the solution of macromolecules (e.g., proteins or synthetic macromolecules) surrounding the cells. In the \"bridging\" model, the interaction is described by the forces that arise due to the adsorption of macromolecules at the surface of RBC membrane and producing the bridges of these macromolecules between RBC membranes. To this day, there are strong arguments in favor of both models and there are some assumptions that both mechanisms influence the RBC aggregation [5].  
Aggregation properties of blood can change due to many factors: alterations of blood plasma composition, changes in blood temperature, pathological cell properties, RBC age, and many others [2; 6; 7; 8]. To date, the influences of these factors on RBC aggregation are not fully understood. Here, we focused on the investigation of RBC in vivo age.  
In a healthy individual, after being released from the bone marrow, RBCs remain in the circulation for approximately 115 days [9; 10] and their degradation is best described by a Weibull distribution [11]. Among individuals, the mean lifespan of 115 days varies by approximately \(\pm 15\%\)[9]. Old RBCs are removed from the blood circulatory system by the reticuloendothelial system. During aging, RBCs undergo intrinsic changes, such as the loss of membrane surface and increase in density, impaired deformation of membrane and other changes [12; 13; 14]. There are some in vitro separation techniques based on these changes. To fraction young and old RBCs from blood for in vitro studies, the separation of RBCs by cell density is usually performed using, e.g., high-speed centrifugation [15] or making use of density differences (self-forming gradient or layering) in Percoll (solution of colloidal silica particles of 15-30 nm diameter which have been coated with polyvinylpyrrolidone) in combination with centrifugation [16]. In addition, fractioning on the basis of differences in RBC volume using counterflow centrifugation or the combination of counterflow centrifugation and Percoll is used in many studies [17]. The use of Percoll has proven to be an efficient separation technique [13; 17; 18], however, it must be mentioned that there is no strict correlation between the RBC density and age [19]. In most studies, RBC separation can be described only in terms of older and younger cells without determining their exact age.  
The aim of this work was to investigate the in vitro differences in the RBC aggregation properties of RBCs of different ages at the cellular level using laser tweezers (LTs). LTs are a powerful laser technique allowing to trap living cells to manipulate them without mechanical contact [7; 20]. Recently, they proved to be a very useful tool for studying the characteristics of RBC interaction [21; 22], to quantify aggregation forces [23] as well as for the complementation of other methods [24; 25]. In this work, LTs were used to study the interaction of pair-aggregating RBCs in autologous plasma and in the dextran 70 kDa (50 mg/mL) solution.