Detailed results of the blood compatibility tests for the six catheter materials and the control run in the Chandler loop model are demonstrated in Table 2a and 2b.
Blood cell system
Erythrocytes
Hemolysis index
According to the international standard ASTM F756-17, a hemolytic index of > 5% is classified as “hemolytic.” All catheters except PU-2+CHSS showed “non-hemolytic” properties, with median hemolytic indexes ranging from 0.6% to 0.9%. Blood exposed to the PU-2+CHSS material had a median hemolytic index of 29%, which is significantly higher than the rates noted for the other five materials and the control loop (P = 0.005 for all comparisons). To determine whether the hemolysis was due to complement activation, we exposed the PU-2+CHSS material to the C5 inhibitor eculizumab (50ug/mL), which prevents formation of the membrane attack complex and complement-mediated lysis. However, the hemolytic index was still 27% to 36%, indicating that hemolysis was not due to complement activation but rather to a toxic effect of the PU-2+CHSS coating.
Leukocytes
Remaining leukocytes
Membrane disruption of leukocytes leads to a decrease in the count of such cells in a blood analysis. The leukocytes remaining after circulating in the Chandler loop system ranged from 93% to 100% in plasma exposed to the six catheter materials and the control loop. Only blood exposed to PU-2+CHSS showed a significantly lower level of remaining leukocytes compared to the control loop (P = 0.050). Also, PU-2+CHSS exposure led to a lower rate of remaining leukocytes compared to all the other materials except Si-1 (PU-2+CHSS vs. Si-1, P = 0.499; PU-2+CHSS vs. PU-1, P = 0.021; PU2+CHSS vs. PU-2, P = 0.012; PU2+CHSS vs. PU-3+BZC, P = 0.008; PU2+CHSS vs. PU-4+NbMC, P = 0.013).
L-selectin MPs
Activation of leukocyte membranes causes a release of the cell adhesion protein L-selectin MPs (CD62L) from the surface of the platelets. The median number of L-selectin MPs ranged from 17/mL to 282/mL in plasma exposed to the six catheter materials and 37/mL in the control loop. The release of MPs was significantly higher (282/mL) in plasma exposed to the PU-3+BZC material than in the control loop (P = 0.043). When comparing the different materials, PU-3+BZC induced significantly higher L-selectin levels than PU-2 (P =0.043) and PU-4+NbMC (P = 0.043).
Platelets and coagulation
Remaining platelets
Both the disruption of platelet membranes and the aggregation of platelets causes a decrease in platelet counts after circulation in the Chandler system. The median number remaining platelets ranged from 72% to 94% for all materials except PU2+CHSS; blood exposed to this catheter showed falsely higher platelets counts of 110% due to the presence of fragments of disrupted erythrocytes and leukocytes interfering with the platelet measurements. The uncoated catheters (Si-1, PU-1, and PU-2) all showed significantly lower levels of remaining platelets than the control loop (P = 0.005 for all comparisons). When comparing the tested materials, Si-1 resulted in significantly lower levels of remaining platelets than all the other materials except PU-2 (Si-1 vs. PU-1, P = 0.047; Si-1 vs. PU-2, P = 0.074; Si-1 vs. PU-2+CHSS, P = 0.005; Si-1 vs. PU-3+BZC, P = 0.005; Si-1 vs. PU4+NbMC, P = 0.005).
P-selectin MPs
Activation of platelet membranes causes release of the cell adhesion glycoprotein, P-selectin MPs (CD62P) from the surface of the platelets. The median number of P-selectin MPs ranged from 75/mL to 337/mL in plasma exposed to the six catheter materials and 31/mL in the control loop. The P-selectin levels were significantly higher in plasma exposed to all the materials except PU3+BZC in comparison with the control loop (P = 0.208 for PU3+BZC vs. the control loop but P = 0.012 for all other materials vs. the control). When comparing the materials, PU-2 + CHSS and Si-1 showed significantly higher levels of P-selectin MPs than all the other materials except PU-2 (PU-2+CHSS vs. PU-1, P = 0.036; PU-2+CHSS vs. PU-2, P =0.575; PU-2+CHSS vs. PU-3+BZC, P = 0.012; PU-2 + CHSS vs. PU-4+NbMC, P = 0.012; Si-1 vs. PU-1, P = 0.017; Si-1 vs. PU-2, P = 0.161; Si-1 vs. PU-3+BZC, P = 0.012; and Si-1 vs. PU-4+NbMC, P =0.012).
FXIIa activity
Factor XII (FXII) is the first of the plasma proteins to be activated by contact with a foreign material present in the blood, resulting in formation of FXIIa. The median activity of FXIIa (absorbance at 405 nm) ranged from 0.04 to 0.25 on the surface of the six catheter materials tested in our study. Measurement of FXIIa was not applicable in the control loop, because FXII is activated by the surface of a catheter. Upon exposure to plasma, FXIIa showed significantly higher activity on the surfaces of Si-1 (0.24) and PU-1 (0.25) compared to all other materials (P = 0.005 for all comparisons).
Prothrombin fragment F1+2
Prothrombin fragment F1+2 is a peptide released from prothrombin during its activation. The median generation of F1+2 ranged from 514 to 1,078 pmol/mL in plasma exposed to the six catheter materials and was 300 pmol/mL in the control loop. The Si-1, PU-2, and PU4+NbMC materials all resulted in significantly higher levels of F1+2 than noted in the control loop. We found no significant difference in generation of F1+2 between the six materials (P = 0.071).
TAT
TAT is an indicator of activation of the coagulation cascade and is generated when thrombin neutralizes anti-thrombin. The median generation of TAT in the blood in contact with the six catheter materials ranged from 131 to 370 µg/L and was significantly higher than the low level of 48 µg/L observed in the control loop (P = 0.005 for all comparisons). The PU-1 catheter led to the highest TAT level of 370 µg/L, which was significantly higher than compared to all the other materials (P = 0.005 for all comparisons) except Si-1 (P = 0.285). PU-2+CHSS resulted in a TAT level of 251 µg/L, which was significantly higher than the levels noted for PU-2 (P = 0.005), PU-3+BZC (P = 0.005), and PU-4+NbMC (P = 0.037).
Molecular markers of the innate immune system
Activation of the complement system
Activation of the complement system via the cellular system (leukocytes and platelets) or via protein absorption on the material surface was assessed by analyzing the increase in inflammatory anaphylatoxin C3a and soluble terminal complement complex sC5b-9, a marker of cell membrane lysis, after exposure to the six catheter materials and the control loop.
C3a anaphylatoxin
The median generation of C3a ranged from 834 to 1528 ng/mL in plasma exposed to the six catheter materials and was 854 ng/mL in the control loop. The PU-2+CHSS and Si-1 materials generated significantly higher C3a compared to the control loop (P = 0.007 and P = 0.047, respectively). The median value for Si-1 was low (873 ng/mL) but varied markedly (range 665–1,926 ng/mL), resulting in statistical significance in paired comparisons. The PU-2+CHSS catheter generated the highest C3a levels (1,528 ng/mL), which were significantly higher than compared to the control loop and all the materials except Si-1 (PU-2+CHSS vs. loop, P = 0.007; PU-2+CHSS vs. PU-1, P = 0.005; PU-2+CHSS vs. PU-2, P = 0.007; PU-2+CHSS vs. PU-3+BZC, P = 0.005; PU-2+CHSS vs. PU-4+NbMC, P = 0.005; and PU-2+CHSS vs. Si-1, P = 0.13).
sC5b-9 marker
The results of the analysis of sC5b-9 showed a trend similar to that observed in the C3a analysis, which was expected. As for C3a, the PU-2+CHSS catheter generated the highest median level of sC5b-9 of 1218 ng/mL, which was significantly higher than noted for all the other materials (P = 0.005 for all comparisons) except Si-1 (P = 0.114). In contrast to C3a, all catheters generated significantly higher levels of sC5b-9 than the control loop (P = 0.005 for all comparisons).
Acute inflammatory reaction: IL-8, TNF-α, and VEGF
To assess the acute inflammatory reaction caused by leukocyte and thrombocyte activation or membrane disruption, we analyzed IL-8, TNF-α, and VEGF released from leukocytes into plasma upon exposure to the six different catheter materials and the control loop.
Interleukin 8
The median generation of IL-8 was 2.09 pg/mL in the control loop and ranged from 2.41 to 12.5 pg/mL after exposure to the six catheter materials. Compared to the control loop, the Si-1, PU-2, PU2+CHSS, and PU4+NbMC materials generated significantly higher IL-8 levels (loop vs. Si-1, P = 0.037; loop vs. PU-2, P = 0.047; loop vs. PU2+CHSS, P = 0.005; loop vs. PU4+NbMC, P = 0.005). Also, PU-2+CHSS generated significantly higher IL-8 than all the other materials except Si-1 (PU-2+CHSS vs. PU-1, P = 0.005; PU-2+CHSS vs. PU-2, P = 0.009; PU-2+CHSS vs. PU-3+BZC, P = 0.005; PU-2+CHSS vs. PU-4+NbMC, P = 0.005; PU-2+CHSS vs Si-1, P = 0.114).
TNF-α
The median level of TNF-a was 1.54 pg/mL in the control loop and ranged from 1.25 to 6.64 pg/mL after exposure to the six catheter materials. The PU-2+CHSS catheter generated significantly higher TNF-a than noted for the control loop and all the other materials (P = 0.007 for PU-2+CHSS vs. PU-3+BZC, but P = 0.005 for all other comparisons).
VEGF
Median generation of VEGF was 36 pg/mL in the control loop and ranged from 33 to 71 pg/mL after exposure to the six catheter materials. Compared to the control loop, the Si-1 material, PU-2, and PU-2+CHSS catheters generated significantly higher VEGF levels (loop vs. Si-1, P = 0.018; loop vs. PU-2, P = 0.018; loop vs. PU-2+CHSS, P = 0.043). The Si-1 generated the highest VEGF levels, which were significantly higher than the levels induced by PU-1, PU-2, PU-3+BZC, and PU-4+NbMC but not PU-2+CHSS (Si-1 vs. PU-1, P = 0.012; Si-1 vs. PU-2, P = 0.018; Si-1 vs. PU-3+BZC, P = 0.012; Si-1 vs. PU-4+NbMC, P = 0.012; Si-1 vs. PU-2+CHSS, P = 0.401).