## 2 Materials and Methods
### Measurements of the Interaction Forces between Individual RBCs  
After placing the measuring chamber with RBCs over the objective, two single RBCs were trapped and brought together until they touched each other. The aggregation force (AF) as an aggregation parameter characterizing the spontaneous aggregation of two RBCs was measured. AF is the minimum force required to prevent the spontaneous aggregation of two interacting RBCs. The protocol of the AF measurements was described in detail in our previous papers [22,27,28]. The step-by-step protocol of the measurement of the AF is presented in Figure 3 and an example of the AF measurement is presented in Supplementary Video S1.  
Figure 2: Schematic layout of the holographic laser tweezers for the RBC interaction force measurements. LED—light-emitting diode; SLM—spatial light modulator.  
Two single RBCs were initially trapped using four optical traps (red crosses on Figure 3). After trapping, two RBCs were brought to overlap by moving the optical traps and the two middle traps are switched off. As far as the trapping force (F\({}_{\text{trap}}\)) exceeded the AF, trapping prevented the spontaneous aggregation process. Then, the laser beam power (i.e., F\({}_{\text{trap}}\)) started to decrease. The distance between the two peripheral optical traps (the cross-section of RBC overlapping) remained unchanged during the measurement whilst F\({}_{\text{trap}}\) exceeded the AF (F\({}_{\text{trap}}\)\(>\) AF). At some point in time, the F\({}_{\text{trap}}\) was not sufficient to prevent aggregation and the RBCs started to aggregate. The laser beam power and corresponding F\({}_{\text{trap}}\) was measured at this particular time-point and it equaled the AF as the minimum force required to prevent the spontaneous aggregation of two interacting RBCs.  
In order to compare the laser beam power and F\({}_{\text{trap}}\), the calibration procedure based on the comparison of the F\({}_{\text{trap}}\) with the viscous Stokes force was performed. The calibration procedure is fully presented in our previous paper [24].  
All experiments were performed within 6 h after blood withdrawal. During this time, the AF may undergo some changes, however, we performed the experiments (data not presented) on one healthy volunteer measuring the AF of fractionated RBCs in different orders of these fractions (e.g., 1-2-3-4, 4-3-2-1 etc.). No significant differences of the AF of the same fractions measured at different times were found.  
All experiments were performed at the controlled room temperature (22 \({}^{\circ}\)C). As it is known, RBC aggregation is a temperature-dependent process [6; 29]. The influence of temperature on RBC aggregation is related to the increase in plasma (or dextran) viscosity with decreased temperature and to the RBC membrane changes [30; 31]. These changes (the membrane shear modulus and membrane viscosity) mainly lead to the membrane rigidity alterations. It was found that below 25 \({}^{\circ}\)C, the RBC deformability decreased as the temperature decreased, whilst there was no decrement in RBC deformability between 25 and 37 \({}^{\circ}\)C [30]. In this work, the RBCs were studied under 22 \({}^{\circ}\)C, as far as the heating effect of optical trapping for RBCs was negligible [32]. Additionally, it was shown that the temperature-dependent change of the AF is nearly absent for the temperatures of 20 and 38 \({}^{\circ}\)C [28].