Perfusion metrics
Resistance, lactate, oxygen consumption, weight change, ATP, and EC were monitored throughout the 6 hour perfusion to assess liver performance. RT LR flush livers with no SCS (group 1) had lowest mean intrahepatic resistance on NMP at 1 hour (0.18 ± 0.064 mmHg/(gr⋅ml/min)), 3 hours (0.15 ± 0.019 mmHg/(gr⋅ml/min)), and 6 hours (0.15 ± 0.033 mmHg/(gr⋅ml/min)) while no flush livers with SCS (group 4) had the highest mean intrahepatic resistance on NMP at 1 hour (0.47 ± 0.12 mmHg/(gr⋅ml/min)), 3 hours (0.34 ± 0.096 mmHg/(gr⋅ml/min)), and 6 hours (0.39 ± 0.042 mmHg/(gr⋅ml/min)). Compared to the clinical standard of cold UW flush livers with SCS (group 2), RT LR flush livers with SCS (group 3) had lower mean intrahepatic resistance at 3 hours (0.33 ± 0.027 vs 0.17 ± 0.017 mmHg/(gr⋅ml/min), p = 0.0007) and 6 hours (0.34 ± 0.042 vs 0.18 ± 0.043 mmHg/(gr⋅ml/min), p = 0.0075) (Fig. 2A).
RT LR flush livers with no SCS (group 1) had the lowest mean lactate levels on NMP at 1 hour (1.01 ± 0.69 mmol/L) and 6 hours (3.6 ± 0.48 mmol/L) while no flush livers SCS (group 4) had the highest mean lactate levels at 1 hour (2.9 ± 0.91 mmol/L), 3 hours (6.0 ± 0.89 mmol/L), and 6 hours (7.6 ± 0.31 mmol/L). Compared to the clinical standard of cold UW flush livers with SCS (group 2), RT LR flush livers with SCS (group 3) had significantly lower mean lactate levels at 3 hours (4.2 ± 0.82 vs 2.2 ± 0.3 mmol/L, p = 0.04) and 6 hours (7.6 ± 0.89 vs 3.9 ± 0.69 mmol/L, p = 0.003) (Fig. 2B).
RT LR flush livers with no SCS (group 1) had the highest mean level of oxygen consumption on NMP at 1 hour (278 ± 48 mmHg), 3 hours (297 ± 72 mmHg), and 6 hours (417 ± 28 mmHg). By 6 hours of NMP, RT LR flush livers with no SCS had a higher mean level of oxygen consumption (417 ± 28 mmHg) compared to group 2 livers (137 ± 78 mmHg, p = 0.01), group 3 livers (127 ± 68 mmHg, p = 0.005), and group 4 livers (185 ± 66, p = 0.01). There were no statistically significant differences in mean oxygen consumption between groups 2, 3, and 4 (Fig. 2C).
RT LR flush livers with no SCS (group 1) had the lowest mean percent weight increase (0.40 ± 0.58 %) while RT LR flush livers with SCS (group 3) had the highest (6.4 ± 5.4 %). There were no statistically significant differences in mean percent weight change before and after NMP between the 4 groups (Fig. 2D).
ATP and EC were calculated at T = 0 (before perfusion) and T = 6 hours (after perfusion). RT LR flush livers with no SCS (group 1) had the highest mean ATP levels at T = 0 (2.8 ± 2.17 ug/ml) while no flush livers with SCS (group 4) had the lowest mean ATP levels at T = 0 (0.26 ± 0.22 ug/ml). There were no statistically significant differences between the 4 groups before perfusion. At T = 6 hours, group 1 livers continued to have the highest mean ATP levels (4.9 ± 2.4 ug/ml) while group 4 livers continued to have the lowest levels (0.15 ± 0.16 ug/ml). There were no statistically significant differences between group 2 and 3 livers (Fig. 2E).
Similar to ATP levels, at T = 0 group 1 livers had the highest mean EC (0.15 ± 0.062) while group 4 livers had the lowest (0.68 ± 0.065). There were no statistically significant differences between the 4 groups before perfusion. At T = 6 hours, group 1 livers continued to have the highest mean EC (0.20 ± 0.058) while group 4 livers continued to have the lowest (0.042 ± 0.021). There were no statistically significant differences between group 2 and 3 livers (Fig. 2F).
Perfusion injury markers
AST, ALT, and perfusate potassium levels were recorded to assess the degree of hepatic injury during perfusion. RT LR flush livers with no SCS (group 1) had the lowest mean AST levels on NMP at 1 hour (16 ± 5.0 units/L), 3 hours (25 ± 8.1 units/L), and 6 hours (48 ± 5.7 units/L) while cold UW flush livers with SCS (group 2) actually had the highest mean AST levels at 1 hour (106 ± 47 units/L), 3 hours (279 ± 65 units/L), and 6 hours (629 ± 121 units/L). Both group 1 and group 3 livers had mean AST levels within transplantable criteria during 6 hours of NMP while group 2 and group 4 livers did not meet AST transplantable criteria by 6 hours.[25, 31] Compared to cold UW flush livers with SCS (group 2), RT LR flush livers with SCS (group 3) had lower mean AST levels at 6 hours of NMP (629 ± 121 vs. 351 ± 97 units/L, p = 0.046) (Fig. 3A).
RT LR flush livers with no SCS (group 1) had the lowest mean ALT levels on NMP at 1 hour (9.7 ± 4.8 units/L), 3 hours (14 ± 7.0 units/L), and 6 hours (25 ± 13 units/L) while cold UW flush livers with SCS (group 2) had the highest mean ALT levels at 1 hour (69 ± 37 units/L) and 3 hours (189 ± 63 units/L). At 6 hours of NMP, group 4 livers had the highest mean ALT levels (586 ± 135 units/L). Both group 1 and group 3 livers had mean ALT levels within transplantable criteria during 6 hours of NMP while group 2 and group 4 livers did not meet ALT transplantable criteria by 6 hours.[25, 31] The differences in mean ALT levels between group 2 and 3 livers did not reach statistical significance by 6 hours (492 ± 98 vs. 278 ± 91 units/L, p = 0.069) (Fig. 3B).
RT LR flush livers with no SCS (group 1) had the lowest mean perfusate potassium levels on NMP at 1 hour (5.8 ± 0.082 mmol/L) and 6 hours (5.9 ± 0.096 mmol/L) while no flush livers with SCS (group 4) had the highest mean potassium levels at 3 hours (7.9 ± 1.3 mmol/L) and 6 hours (8.4 ± 1.0 mmol/L). There were no statistically significant differences in mean perfusate potassium levels between group 2 and 3 livers (Fig. 3C).
Finally, on gross appearance after 6 hours of NMP, RT LR flush livers with no SCS (Fig. 3D) and RT LR flush livers with SCS (Fig. 3F) had similar viable appearance devoid of infarcted lobes or cellular washout. Cold UW flush livers experienced significant cellular washout, giving them a pale appearance (Fig. 3E), and no flush livers had large patchy areas of infarction and necrosis (Fig. 3G).
Histology
Liver parenchyma histology with H&E, Reticulin, TUNEL, and PAS-diastase were done to assess cellular artichecture, quantify the number of retained peripheral cells, and assess for DNA damage after 6 hours of NMP. Group 1 livers which underwent a RT LR flush and had no SCS had preserved cellular architecture (Fig. 4A). Periportal hepatocytes however did show persistent edema in addition to marked sinusoidal dilation that is expected due to the constant, passive perfusion pressure (Fig. 4B, D). This resulted in some hepatocyte drop-out due to the widening of the subendothelial spaces. Finally, there was minimal TUNEL staining compared to the other 3 flush groups (Fig. 4C) showing focal hepatocyte apoptotic changes.
Group 2 livers which underwent a cold UW flush followed by 24 hours of SCS had significant damage after perfusion which was appreciated on histology. Liver sinusoids were distended by heme pigments (due to fragmented RBCs) (Fig. 4E, F). There was exaggerated zonal differentiation with dilatation of pericentral sinusoids due to passive perfusion pressure. Periportal hepatocytes were preserved while those in pericentral area showed focal steatosis. TUNEL analysis of these livers showed that periportal liver sinusoidal endothelial cells (LSECs) had apoptotic changes with cytoplasmic staining in addition to hepatocytes with focal apoptotic changes (Fig. 4G). The sinusoids also showed remnant RBCs and lymphocytes with mostly preserved LSECs (Fig. 4H). Additional myeloperoxidase staining (Supplemental Fig. 1A) showed many fragmented granular cells with free granules in the liver sinusoids, contributing to sinusoidal distension.
Group 3 livers underwent a RT LR flush followed by 24 hours of SCS and had significantly better histology compared to group 2 livers with a cold UW flush. H&E showed physiologic architecture with preserved hepatocytes and mid-zone focal steatosis but no cellular dropouts (Fig. 4I). The sinusoids were mostly clear with preserved LSECs (Fig. 4J). TUNEL analysis showed a differential distribution as hepatocytes showed good viability but apoptotic bodies were seen within intra-sinusoidal dead granular leukocytes (Supplemental Fig. 1B), and extra-sinusoidal LSECs (Fig. 4K). There were a few leukocytes and lymphocytes noted inside the sinusoids and Kupffer cells near the portal areas (Fig. 4L).
Group 4 livers underwent no flush followed by 24 hours of SCS, and had the worst histology compared to the other 3 groups. Following 24 hours of SCS and before perfusion, H&E showed marked congestion of the liver sinusoids with stagnant RBCs, diffuse edema, and hepatocytes exhibited periportal hydropic changes indicating early cellular degeneration (Supplemental Fig. 1C). After 6 hours of NMP, H&E still showed diffuse edema and marked congestion of the liver sinusoids due to stagnant RBCs (Fig. 4M), indicating failure of NMP to remove these RPCs from the liver parenchyma. There were also focal hepatocyte emboli seen in the central veins (Supplemental Fig. 1D), indicative of hepatocyte detachment during perfusion. Reticulin stains revealed a focally disrupted reticular meshwork around the central veins (Fig. 4N). TUNEL analysis showed several intra-sinusoidal dead cells and apoptotic bodies. The overall TUNEL pattern was diffuse, patchy hepatocyte apoptotic staining (Fig. 4O). Monocytes and neutrophils were noted inside the sinusoids in the mid-zone and periportal area while lymphocytes appeared distributed within liver lobules. Kupffer cells were prominent in close proximity to portal areas (Fig. 4P).
Correlations between retained peripheral cells and perfusion parameters of liver function/injury
We hypothesized that the type of flush technique would influence the number of RPCs in a liver graft after perfusion and possibly affect liver function during NMP. Because the flush solutions were acellular, all RPCs seen on histology were retained from procurement and not deposited during perfusion. Our results found that LR-flush livers had the lowest number of RPCs compared to UW and no flush livers. Also, the duration of SCS (LR + 24 hour SCS vs. LR with no SCS) did not seem to affect the ability to flush out the retained peripheral cells during NMP (Supplemental Fig. 2).
Next, resistance, lactate, EC, and potassium were corelated with the presence of RPCs per 20 HPFs on histology after 6 hours of NMP using Pearson correlation coefficients (r) to measure the strength of association. Resistance (r = 0.69, p = 0.006), lactate (r = 0.73, p = 0.03), and potassium (r = 0.93, p = < 0.0001) all had significant positive correlations with the presence of RPCs in a liver allograft after 6 hours of NMP, indicating that more RPCs in a liver graft were associated with higher intrahepatic resistance and cellular injury (Fig. 5A-C). EC had a negative correlation with RPCs which almost reached significance (r = -0.52, p = 0.055), indicating that liver grafts with more RPCs had a lower metabolic energy status during perfusion (Fig. 5D).