*3.1. Variation of PFH Concentration with Gradient Change of Shear Stress and Exposure Time*

PFH is a classic label which is first applied to evaluate the red blood cell damage caused by VADs. The world-recognized CF-VADs testing process (American Society of Testing Materials Standard Practices F1830, ASTM F1841) and evaluation index, including normalized index of hemolysis (NIH) and modified index of hemolysis (MIH), were established on the basis of PFH concentration measurement. According to the curve of PFH concentration variation, shown in Figure 2a, when the blood was exposed in a mechanical shear stress environment, within 5 min, the concentrations of PFH release under 25 to 100 Pa rose synchronously without notable differences. When the exposure time increased up to 10 min, the concentration of PFH rose rapidly in the group with 75 and 100 Pa shear stress. In particular, extremely high PFH releases were detected in the 75 and 100 Pa groups when the exposure time reached 15 min. Meanwhile, as shown in Figure 2b, in a low shear stress environment (25 Pa and 50 Pa), there were no significant changes of PFH concentration with the increasing of exposure time. However, in the high shear stress condition, especially under 100 Pa, PFH release rose extremely high with the extension of exposure time. Therefore, long exposure time under high shear stress environment would induce more damage to erythrocytes.

**Figure 2.** *Cont*.

**Figure 2.** The changes of plasma plasma free hemoglobin (PFH) concentration under different shear stress and exposure times. (**a**) With the increase of shear stress, the concentration of PFH released into plasma showed a slow rising trend within 5 min and 10 min, but with the extension of exposure time up to 15 min, the concentration of PFH showed a rapid increase trend, especially in the shear stress of 100 Pa. (**b**) With the increase of exposure time, the concentration of PFH released into plasma showed a slow rise trend at 25 and 50 Pa, but the shear stress up to 75 and 100 Pa, the concentration of PFH showed a rapid increase trend, especially in the exposure time 15 min.

## *3.2. LDH Release with Gradient Change of Shear Stress and Exposure Time*

Compared to PFH, LDH is a novel and more sensitive protein label to assess the trauma of the erythrocyte membrane. Differently to PFH release based on erythrocyte rupture, injuries to the red blood cell membrane will induce an increase of LDH concentration in plasma. As the results show in Figure 3a, there was no significant difference in LDH release between the three groups with the exposure times of 5, 10, and 15 min in a low shear stress environment within 50 Pa. The increase of LDH concentration was smooth and maintained a low level. However, when blood was exposed to a high shear stress condition (especially at 100 Pa), the concentration of LDH rose rapidly with the increase of exposure time. Meanwhile, as the curve shows in Figure 3b, LDH release was insensitive to the prolonging of exposure time at low shear stress conditions in 25 Pa and 50 Pa. However, the concentration of plasma LDH increased dramatically in a high shear stress environment, and the longer the exposure was, the greater the influence became. Therefore, high shear stress force was the main factor causing erythrocyte membrane injury, and the long exposure time could aggravate this trauma.
