Impact of Different Clinical Perfusates During Normothermic Ex Situ Liver Perfusion on Pig Liver Transplant Outcomes in a DCD Model

Abstract

Background: Human albumin/dextran (HA-D), bovine-gelatin (BG), and packed red blood cells plus plasma have been used in European and North-American clinical trials of normothermic ex situ liver perfusion (NEsLP). We compared the effects of these perfusates in a porcine model during NEsLP and after transplantation.

Methods: Porcine livers were retrieved 30 minutes after circulatory death. After 5 hours of NEsLP, grafts were transplanted. Three groups (n = 6) were assessed (HA-D vs BG vs whole blood [WB]). One group of static cold storage (SCS) was evaluated for comparison with the perfusion groups. Hemodynamic variables, liver and endothelial injury, and function were assessed during NEsLP and posttransplantation.

Results: Hepatic artery flow was higher since the beginning of NEsLP in the HA-D group (HA-D, 238 ± 90 mL/min vs BG, 97 ± 33 mL/min vs WB, 148 ± 49 mL/min; P = 0.01). Hyaluronic acid was lower in the HA-D at the end of perfusion (HA-D, 16.28 ± 7.59 ng/μL vs BG, 76.05 ± 15.30 ng/μL vs WB, 114 ± 46 ng/μL; P < 0.001). After transplant, aspartate aminotransferase was decreased in the HA-D group when compared with the rest of the groups (HA-D, 444 ± 226 IU/L vs BG, 1033 ± 694 IU/L vs WB, 616 ± 444 IU/L vs SCS, 2235 ± 1878 IU/L). At 5 hours after transplant, lactate was lower in the HA-D group (HA-D, 3.88 ± 1.49 mmol/L vs BG, 7.79 ± 2.68 mmol/L vs WB, 8.16 ± 3.86 mmol/L vs SCS, 9.06 ± 3.54 mmol/L; P = 0.04). International Normalized Ratio was improved in HA-D group compared to the rest of the groups (HA-D, 1.23 ± 0.30 vs BG, 1.63 ± 0.20 vs WB, 1.50 ± 0.31 vs SCS, 1.97 ± 1.55; P = 0.03) after transplantation. In contrast, BG displayed lower aspartate aminotransferase levels during NEsLP (HA-D, 183 ± 53 IU/L vs BG, 142 ± 52 IU/L vs WB, 285 ± 74 IU/L; P = 0.01) and less cleaved-caspase-3 staining (HA-D, 2.05 ± 0.73% vs BG, 0.95 ± 1.14% vs WB, 1.74 ± 0.54% vs SCS, 7.95 ± 2.38%) compared with the other groups. On the other hand, the bile from the WB showed higher pH (HA-D, 7.54 ± 0.11 vs BG, 7.34 ± 0.37 vs WB, 7.59 ± 0.18) and lower glucose levels (HA-D, 0.38 ± 0.75 mmol/L vs BG, 1.42 ± 1.75 mmol/L vs WB, 0 ± 0 mmol/L) by the end of perfusion.

Conclusions: Overall HA-D displayed more physiologic conditions during NEsLP that were reflected in less graft injury and improved liver function and survival after transplantation. Optimization of the perfusates based on the beneficial effects found with these different solutions would potentially improve further the outcomes through the use of NEsLP in marginal grafts.

FIGURE 1

Hemodynamic parameters during perfusion. HA flows were higher during the entire perfusion with significant difference found at the start of perfusion in the Steen vs Gelofusine vs WB group (238 ± 90 mL/min vs 97 ± 33 mL/min vs 148 ± 49 mL/min; P = 0.01) (A). Intraarterial resistance significantly increased at the beginning of the perfusion in the Gelofusine group compared with the rest of the groups (Steen, 0.23 ± 0.05 mm Hg·mL⁻¹·min⁻¹ vs Gelofusine, 0.52 ± 0.18 mm Hg·mL⁻¹·min⁻¹ vs WB, 0.32 ± 0.10 mm Hg·mL⁻¹·min⁻¹; P = 0.01) (B). PV flow was at physiological levels in the 3 groups since the start of the NEsLP (C). Results are expressed as mean ± SD. Six experiments were performed per group (n = 6). One-way ANOVA was used for analysis (A, B, C). ANOVA, analysis of variance; HA, human albumin; NEsLP, normothermic ex situ liver perfusion; PV, portal vein; SD, standard deviation; WB, whole blood.

FIGURE 2

Hepatocytes and ECs injury and function during NEsLP. AST showed significantly lower levels in the Gelofusine group compared to the rest of the groups (Steen, 183 ± 53 vs Gelofusine, 142 ± 52 vs WB, 285 ± 74; P = 0.01) (A). Lactate clearance was similar for the 3 groups at all timepoints (B). Hyaluronic acid was significantly lower in Steen vs Gelofusine vs WB group at 1 hour (3.09 ± 1.48 ng/μL vs 32.63 ± 3.55 ng/μL vs 18.89 ± 13.48 ng/μL; P < 0.001) and 5 hours (16.28 ± 7.59 ng/μL vs 76.05 ± 15.30 ng/μL vs 113.62 ± 46.43 ng/μL; P < 0.001) of perfusion. Results are expressed as mean ± SD. Six experiments were performed per group (n = 6). One-way ANOVA (A, B) and Student t test (C) were used for analysis.ANOVA, analysis of variance; AST, aspartate aminotransferase; EC, endothelial cell; NEsLP, normothermic ex situ liver perfusion; SD, standard deviation; WB, whole blood.

FIGURE 3

OC and bile production during NEsLP. OC was stable since the 1 hour of perfusion without differences between the groups (A). Bile production was significantly higher in the WB group since 2 hours of NEsLP (B). Significantly higher bile pH was found in the WB at 1 hour of perfusion compared to the rest of the groups (Steen, 7.37 ± 0.13 vs Gelofusine, 7.16 ± 0.12 vs WB, 7.49 ± 0.05; P < 0.01) (C). Results are expressed as mean ± SD. Six experiments were performed per group (n = 6). One-way ANOVA was used for all the analyses (A, B, C). ANOVA, analysis of variance; NEsLP, normothermic ex situ liver perfusion; OC, oxygen consumption; SD, standard deviation; WB, whole blood.

FIGURE 4

Bile bicarbonate concentration and bile/serum ratio. Bile HCO3- concentration showed significantly higher values in the WB group for the majority of the timepoints (A). WB group showed also higher bile/perfusate HCO3- ratio when compared to Steen and Gelofusine groups (B). Results are expressed as mean ± SD. Six experiments were performed per group (n = 6). Student t test was used for analysis (A, B). Note: BL bile and serum samples represent samples taken directly from the bile duct and central line of donor pigs before any intervention, respectively.SD, standard deviation; WB, whole blood.

FIGURE 5

Bile electrolytes. Bile analysis showed higher Na⁺ concentration in Steen group since the 2 hours of perfusion compared with Gelofusine and WB group (A). The bile/perfusate Na+, Ca++ and K+ ratios also showed higher values in the Steen group compared with Gelofusine and WB groups at the majority of the timepoints (B, C, D). Results are expressed as mean ± SD. Six experiments were performed per group (n = 6). Student t test was used to perform all the analyses. Note: BL bile and serum samples represent samples taken directly from the bile duct and central line of donor pigs before any intervention, respectively; SD, standard deviation; WB, whole blood.

FIGURE 6

Bile markers of cellular injury. Analysis of LDH and gamma-glutamyl transferase showed a decrease in the levels of these markers by the end of the perfusion in all the groups. Steen vs Gelofusine vs WB group showed a trend to significantly lower levels of LDH (562 ± 490 IU/L vs 905 ± 450 IU/L vs 694 ± 435 IU/L; P = 0.30) and gamma-glutamyl transferase (292 ± 111 IU/L vs 576 ± 294 IU/L vs 934 ± 552 IU/L; P = 0.07) at 1 hour after the liver was placed on pump (A, B). The bile glucose levels were lower in the WB group compared to Steen and Gelofusine groups at all timepoints without reaching significant difference (C). Results are expressed as mean ± SD. Six experiments were performed per group (n = 6). Student t test was used for analysis. Note: BL bile samples represent pig serum samples taken directly from the bile duct of donor pigs before any intervention; SD, standard deviation; WB, whole blood.

FIGURE 7

Percentage of gain in liver weight at the end of perfusion. Analysis of liver weight before and after NEsLP demonstrated a significantly lower percentage of weight gain in the grafts perfused with Steen and WB groups compared with the Gelofusine group (Steen, 4.62 ± 5.75%, Gelofusine, 14.73 ± 5.95%, WB, 5.73 ± 7.58%; P = 0.01). Results are expressed as mean ± SD. Six experiments were performed per group (n = 6). Student t test was used for analysis.NEsLP, normothermic ex situ liver perfusion; SD, standard deviation; WB, whole blood.

FIGURE 8

Hepatic injury and apoptosis after transplantation. After transplantation Steen group showed lower AST levels compared with Gelofusine, WB, and SCS groups (day 1, 1250 ± 742 IU/L vs 1957 ± 1188 IU/L vs 1425 ± 9571 IU/L vs 2097 ± 1121 IU/L; P = 0.48), (day 2, 444 ± 226 IU/L vs 1033 ± 694 IU/L vs 616 ± 444 IU/L vs 2235 ± 1878 IU/L; P = 0.05) (day 3, 180 ± 89 IU/L vs 608 ± 554 IU/L vs 219 ± 145 IU/L vs 652 ± 549 IU/L; P = 0.15) (A). Lower levels of ALP and TBil were also found in Steen group compared with Gelofusine, WB, and SCS groups (B, C). Cleaved caspase-3 analysis showed significantly lower levels of positive staining in Gelofusine, Steen, and WB groups compared with SCS group (P < 0.001) (D). Results are expressed as mean ± SD. Six experiments were performed per group (n = 6). One way ANOVA (A, B, C) and Student t test were used for statistical analysis (D). ALP, alkaline phosphatase; ANOVA, analysis of variance; AST, aspartate aminotransferase; SCS, static cold storage; SD, standard deviation; TBil, total bilirubin; WB, whole blood.

FIGURE 9

Hepatic necrosis and EC integrity after transplantation. CD31 integrity score showed higher score in the Steen vs Gelofusine vs WB groups compared with SCS group (3.33 ± 0.51 vs 3.16 ± 0.75 vs 3.16 ± 1.16 vs 2 ± 0.63; P < 0.01) (A). Hyaluronic acid levels were significantly lower in the Steen, Gelofusine, and SCS groups when compared with the WB group (1257 ± 358 ng/μL vs 1501 ± 143 ng/μL vs 1280 ± 151 ng/μL vs 2925 ± 912 ng/μL; P = 0.01) (B). Of note, hematoxylin and eosin analysis demonstrated a significantly lower percentage of necrosis at POD3 in Steen group compared with Gelofusine (12.5 ± 6% vs 20 ± 4.5%; P = 0.03) and SCS group (12.5 ± 6% vs 25 ± 5%; P < 0.01) (C). All the animals in the perfusion groups showed preserved bile duct structure and epithelium (Steen vs Gelofusine vs WB). In contrast, 2 of 6 animals showed alternated areas of epithelium loss and distorted structure in the bile duct (D). Six experiments were performed per group (n = 6). Student t test was used for all the analyses. EC, endothelial cell; SCS, static cold storage; WB, whole blood.

FIGURE 10

Liver function and animal survival after transplantation. The INR values were lower in the Steen, Gelofusine, and SCS groups when compared with the WB group at 3 hours after PV reperfusion (Steen, 1.60 ± 0.26 vs Gelofusine, 1.65 ± 0.16 vs WB, 1.98 ± 0.47 vs SCS, 1.49 ± 0.26; P = 0.07) after transplant (A). Lactate clearance was significantly improved in the Steen group when compared to the Gelofusine, WB and SCS groups at 5 hours after transplantation (Steen, 3.88 ± 1.49 mmol/L vs Gelofusine, 7.79 ± 2.68 mmol/L vs WB, 8.16 ± 3.86 mmol/L vs SCS, 9.06 ± 3.54 mmol/L; P = 0.04) (B). All animals in the Steen group survived, whereas 1, 2, and 2 pigs from the Gelofusine, WB, and SCS groups, respectively were euthanized due to PNF (C). Results are expressed as mean ± SD. Six experiments were performed per group (n = 6). Student t test (A, B) and Kaplan Meier method with log-rank test (C) were used for analysis. INR, International Normalized Ratio; PNF, primary nonfunction; PV, portal vein; SCS, static cold storage; SD, standard deviation; WB, whole blood.