## 4 Discussion
[18] observed that the blood salvage reduced the RBC's deformability but did not affect the RBC aggregation. Thus, we can conclude that there is no unequivocal conclusion in the literature about the effect of mechanical stress on the properties of RBCs. The characteristics of the applied stress will most probably determine the result of mechanical action, but it remains unknown whether the cells' initial (pre-exposure) properties themselves are a factor influencing the observed effect. In a previous study, we tried to assess the effect of the processing of donated blood into a packed RBC unit (in which the RBCs are subjected to shear stress induced by centrifugation and filtration) on the RBC deformability [58]. We found that the change in the percentage of undeformable cells (%UDFC) was bidirectional, depending on its initial level. The percentage of UDFC increased when its initial level was low and decreased when it was high [58]. In the present study, we set out to determine whether that relationship is specific to the preparation of storage units or whether it can be extended to a broader range of situations in which cells are subjected to moderate mechanical stress. For that purpose, we have analyzed the distribution of RBC deformability following the application of moderate mechanical stress, either in vitro (by rolling RBCs with metal beads) or during blood salvage, focusing on the percentage of undeformable cells, as described in Methods. For both procedures, in all study groups, we observed, in accord with our previous findings, that the \(\Delta\)%UDFC was bidirectional, depending on its initial level (Figures 2 and 7); the %UDFC increased when its initial level was low and decreased when it was high. This phenomenon can be explained by the results presented by [PERSON] et al. [32] and [PERSON] et al. [77], who demonstrated that in the blood (from Holstein calves or pigs) sheared by rotary pumps, an increase in the MCV of erythrocytes and a decrease in MCHC were observed. The authors [77] speculated that the likely mechanism is that aged erythrocytes with smaller volumes and higher cytosol concentrations of hemoglobin were primarily destroyed, leaving younger cells with higher volumes and lower Hb concentrations. Thus, they assume that the selective destruction of the aged RBCs occurred by removing more fragile cells possessing a lower hemolytic threshold. Due to the aged RBCs' higher fragility [62], i.e., lower resistance to shear stress, their selective destruction leads to an elevation of average MCV and a reduction of MCHC following blood pumping [77; 32]. In light of the cited publications [77; 32], we can provide the following explanation for our results: Exposure of red cells to moderate mechanical stress initiates a deterioration in the deformability of each cell. However, due to the RBC population being a mixture of cells of different ages and damage level, their exposure to mechanical stress induces differential impairment in cell deformability and selective destruction of \"aged\"/defective cells. Thus, in our system, we simultaneously observe the results of two processes: the decrease in cell deformability and the \"destruction\" of non-deformable cells. Therefore, the overall effect depends on the proportion of undeformable cells in the RBC sample before treatment. With a significant amount of UDFC in the original sample, applying mechanical stress will result in partial hemolysis of these cells and, as a result, a decrease in their proportion in the RBC population. In an inverse situation, an increase in the fraction of undeformable cells is observed.