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Hepatocellular injury during preservation of human livers with UW and HTK solution
ELSEVIER
Hepatocellular Injury During Preservation of Human Livers With UW and HTK Solution R. Lange, J. Erhard, U. Rauen, H. de Groot, and F.W. Eigler
V
ARIOUS parameters have been suggested for preoperative or perioperative assessment of the function of a liver graft.lm3 However, these parameters are not able to predict satisfactory or good functioning in individual cases.2-4 Postoperative graft function is primarily influenced by the quality of the donor organ and ischemic injury. The extent of preservation injury has mainly been shown in experimental studies.5-7 The results of an earlier retrospective study showed that the measurement of enzyme activities in the effluent of the preserved organ at the end of the cold &hernia time can provide data that are valuable in the assessment of graft prognosis before transplantation.‘x9 To evaluate these retrospective results we collected samples prospectively. We examined 70 human livers that had been procured for transplantation in our centre. MATERIALS AND METHODS Seventy human livers were examined in a 30-month period. The livers were harvested for transplantation at the Essen University transplant centre. The interval of cold ischemic times ranged from 6 to 15 hours (median 10.8). Fifty-one livers were perfused with University of Wisconsin (UW) solution, 19 with histidine-tryptophane-ketoglutarate (HTK) solution. Depending on the preservation solution primarily used, the livers were rinsed again at the beginning of the back table preparation with 500 mL of UW or HTK solution. The perfusion was performed via a balloon catheter placed in the portal vein.‘“,” During perfusion, the infrahepatic vena cava was clamped. The first three lo-mL portions (P l-3) of effluent were collected. These samples were centrifuged. In the supernatant, the enzymes lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) were measured by standardized methods” at 37°C. The back table preparation was done before the hepatectomy of the recipient was finished. Thus, the period between the collection of the effluent samples and the beginning of the warm ischemia (start of transplantation of the graft) was in the range of 60 to 80 minutes. The median warm ischemia time was 60 minutes (range 40 to 70 minutes). All 70 livers were biopsied about 20 minutes after reperfusion (0 biopsy). The specimen was studied for preexisting damage and alterations resulting from the harvesting procedure and preservation. In addition, serum activities of LDH and transaminases were determined 24 hours after reperfusion. To estimate graft function, the parameters of hepatic synthesis (thromboplastin time, partial thrombin time, thrombin time, fibrinogen) and the clinical course were documented for the first 5 postoperative days. Initial nonfunction (INF) was noted in the 0041-1315/97/$17.00 PII SO041 -1345(96)00136-4
Table 1. Enzyme Activities in the Effluent of the Rinse During Back Table Preparation
LDH (U/L) AST (U/L)
uw
HTK
1642 (183-25925) 478 (9-7213)
3112 (223-22500) 880 (45-5505)
UW-preserved livers were rinsed with 500 mL of UW solution, HTK-preserved livers with 500 mL of HTK solution. The samples were collected from the liver veins. The median values and range of the first 30 mL of effluent are given.
event of intensive care treatment with ventilation being required, of increasing circulatory instability, and the need to substitute coagulation factors. Delayed graft function was said to have occurred if the clinical stabilization of the patient was not observed within 48 hours after transplantation and a disorder of coagulation with the need of substitution, a hypodynamic circulation, and an increase of the transaminases (over 2000 U/L) continued for this time.‘.‘” A satisfactory function was defined as a recovery period of 24 to 36 hours with stabilisation of circulation and normalisation of coagulation. The transaminases should not increase over 1000 U/L. RESULTS
The median activities of the enzyme LDH and AST in the effluent of HTK-perfused organs was twice the median of activities of UW-perfused ones (Table 1). The correlation between the ubiquitous cytosolic enzyme LDH as a marker of the “global” injury and AST as a marker of the hepatocellular damage was high (r = .972), which is in line with the fact that hepatocytes represent over 90% of cellular liver volume.” There was no significant correlation between enzyme activities in the effluent and the length of cold ischemia time. However, all four livers with the highest LDH and transaminase activities in the effluent had ischemic preinjury in the donor, two were from donors who had been resuscitated (MK, AI Table 2) and two were from donors with circulatory instability or shock (KN, HF). There were only three further cases involving circulatory instability or resuscitation of the donor. From the Department of General Surgery and Institute of Physiological Chemistry, University Clinic, Essen, Germany. Address reprint requests to Reinhard Lange, MD, Dept of General Surgery, University Clinic, Hufelandstr. 55, D-45147 Essen, Germany.
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
400
Transplantation Proceedings,
29, 400-402 (1997)
PRESERVATION
401
WITH UW AND HTK SOLUTIONS
Table 2. LDH Activitv in Back-Table Rinse in Correlation to Morphology and Postoperative Graft Function LDH [U/L]
LDH [U/L] Preservation
CIT (h)
MK KN Al KL PJ PJ HF
HTK uw uw uw uw uw uw
6 14 9 9 9 9 12
13500 26240 13820 6745 5212 4850 13112
HH PG
uw uw
15 6
6650 6242
Liver
Histology
(24h)
Function
5% fatty degeneration 40% fatty degeneration normal normal normal 40% centroacinous necrosis 20% necrosis of parenchyma 40% fatty degeneration normal
8460 3220 4231 1742 1235 2765 2200
Delayed Delayed INF Delayed Delayed INF Delayed
1876 3442
INF INF
Anamnesis of the donor
(PI-3
54 52 58 38 42 56 44
years, years, years, years, years, years, years,
ICE, resuscitation, alcohol abuse ICB, alcohol abuse, adrenaline CCT, resuscitation CCT, resuscitation CCT ICB, adrenaline CCT, noradrenaline
53 years, ICB, diabetes 48 years, resuscitation, AST 420 U/L
The table shows the organs with peculiarities in the effluent (LDH activity more than twice median value) and function (initial nonfunction [INF] or delayed function). Abbreviations: cold ischemia time: CIT; craniocerebral trauma: CCT; intracranial bleeding: ICB.
All livers with abnormalities with regard to histomorphology, LDH activity in the perfusate (LDH > 3300 U/L for UW preserved organs or 6100 U/L for HTK preserved organs), and function (delayed function or primary nonfunction) are detailed in Table 2. The activities of 3300 U/L in UW-preserved organs and 6100 U/L in HTK-preserved organs are double the median of LDH activities of all preserved organs. Nine of 30 livers with LDH values above this limit showed an initial nonfunction after transplantation, and seven livers were subject to delayed functioning. The other 90 livers with LDH activities below the limit did not show any functional disorder (Fig 1). Four of 12 livers with morphologic alterations in the 0 biopsy had an initial nonfunction, but 3 of 58 livers with normal biopsy had an initial nonfunction, too. In three organs that were histologically classified as normal, the function was delayed.
cantly (53%). This is probably due to the high influence of the additional factors that might affect the organ after assessing the effluent samples such as warm ischemia time, circulatory insufficiency, infection, and rejection.3 Unfortunately, a good liver may drift into nonfunction for such reasons or-conversely-a transplantation without any problems may take successfully even with a marginal, severely injured graft4
0
‘I function o k.
DISCUSSION
0 Levels of enzyme activities in effluent samples can show the injury of a preserved liver up to the end of cold ischemia. The preliminary results of the actual study seems to confirm the conclusion of the former retrospective investigation.’ The effluent of HTK-preserved livers showed significantly higher levels of enzyme activities than the effluent of UW-preserved organs. This might be the result of the differing viscosity of the two solutions, leading to better rinsing of the liver with HTK solution,8,9 and does not necessarily indicate superior preservation by UW solution. Not only damage sustained during cold ischemia time, but also preexisting damage such as warm ischemic injury in the donor (livers MK, KN, AI, HF) produced a clear increase in the activity of LDH in the effluent. In all cases of initial nonfunction and delayed function, the effluent samples showed high LDH activities. In contrast, the histologic examination only yielded pathologic results in four of eight livers (grafts KN, PJ, HF, and HH). This might result from the delay in which events lead to morphologic tissue alteration. As shown in Fig 1, high enzyme activities in the effluent samples do not necessarily indicate a malfunction of the graft, yet the danger of dysfunction was increased signifi-
?? ??
delayed function or INF
0
2 x medun I
ii
HTK Fig 1. LDH activities in the effluent in comparison to graft function; the values for UW- (n = 51) and HTK-preserved organs (n = 19) are presented separately. The borderline between normal activity and increased release was set at twice the median of all activities (for LDH: UW 3300 U/L; HTK 6100 U/L). These borderlines are indicated by lines.
402
In conclusion, the effluent samples were a sensible marker for preexisting damage or for damage acquired during cold ischemia. Thus, effluent samples offer a helpful parameter for the decision on whether to transplant a marginal organ. The test can be performed during back table preparation before the recipient hepatectomy is performed so the result of the analysis is available in time to make a decision. This is an advantage to the alternative procedure of measurement during warm ischemia time.14 REFERENCES
1. Erhard J, Lange R, Gersing E, et al: Langenbecks Arch Chir 378:233, 1993 2. Fukusazawa K, Schwartz ME, Acarli K, et al: J Am C Surg 178541, 1994 3. Strasberg S, Howard T, Molmenti E, et al: Hepatology 20:829, 1994 4. Doyle HR, Marino IR, Jabbour N, et al: Transplantation 57:1028, 1994
LANGE, ERHARD, RAUEN ET AL
5. Aminalai A, Kehrer G, Grossmann F, et al: Langenbecks Arch Chir 377:81, 1992 6. Caldwell-Kenkel JC, Currin RT, Tanaka Y, et al: Hepatology 13:83, 1991 7. Shimada M, Yanaga K, Kishikawa K, et al: Transplant Int 6:4, 1993 8. Rauen U, Erhard J, Klhnhenrich Chir 379:241, 1994
P, et al: Langenbecks Arch
9. Lange R, Erhard J, Rauen U, et al: Transplantation
62:1996
10. Erhard J, Lange R, Scherer R, et al: Transplant Int 7:177, 1994 11. Gubernatis G, Pichlmayr R, Lamesch P, et al: Langenbecks Arch Chir 375:66, 1990 12. Bergmeyer HU: Methoden Weinheim: Verlag Chemie. 1974
der enzymatischen
Analyse.
13. Blouin A, Bolender RP, Weibel ER: J Cell Biol 72:441, 1977 14. Devlin J, Dunne JB, Sherwood RA, et al: Transplantation 60:627, 1995
Hepatocellular Injury During Preservation of Human Livers With UW and HTK Solution R. Lange, J. Erhard, U. Rauen, H. de Groot, and F.W. Eigler
V
ARIOUS parameters have been suggested for preoperative or perioperative assessment of the function of a liver graft.lm3 However, these parameters are not able to predict satisfactory or good functioning in individual cases.2-4 Postoperative graft function is primarily influenced by the quality of the donor organ and ischemic injury. The extent of preservation injury has mainly been shown in experimental studies.5-7 The results of an earlier retrospective study showed that the measurement of enzyme activities in the effluent of the preserved organ at the end of the cold &hernia time can provide data that are valuable in the assessment of graft prognosis before transplantation.‘x9 To evaluate these retrospective results we collected samples prospectively. We examined 70 human livers that had been procured for transplantation in our centre. MATERIALS AND METHODS Seventy human livers were examined in a 30-month period. The livers were harvested for transplantation at the Essen University transplant centre. The interval of cold ischemic times ranged from 6 to 15 hours (median 10.8). Fifty-one livers were perfused with University of Wisconsin (UW) solution, 19 with histidine-tryptophane-ketoglutarate (HTK) solution. Depending on the preservation solution primarily used, the livers were rinsed again at the beginning of the back table preparation with 500 mL of UW or HTK solution. The perfusion was performed via a balloon catheter placed in the portal vein.‘“,” During perfusion, the infrahepatic vena cava was clamped. The first three lo-mL portions (P l-3) of effluent were collected. These samples were centrifuged. In the supernatant, the enzymes lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) were measured by standardized methods” at 37°C. The back table preparation was done before the hepatectomy of the recipient was finished. Thus, the period between the collection of the effluent samples and the beginning of the warm ischemia (start of transplantation of the graft) was in the range of 60 to 80 minutes. The median warm ischemia time was 60 minutes (range 40 to 70 minutes). All 70 livers were biopsied about 20 minutes after reperfusion (0 biopsy). The specimen was studied for preexisting damage and alterations resulting from the harvesting procedure and preservation. In addition, serum activities of LDH and transaminases were determined 24 hours after reperfusion. To estimate graft function, the parameters of hepatic synthesis (thromboplastin time, partial thrombin time, thrombin time, fibrinogen) and the clinical course were documented for the first 5 postoperative days. Initial nonfunction (INF) was noted in the 0041-1315/97/$17.00 PII SO041 -1345(96)00136-4
Table 1. Enzyme Activities in the Effluent of the Rinse During Back Table Preparation
LDH (U/L) AST (U/L)
uw
HTK
1642 (183-25925) 478 (9-7213)
3112 (223-22500) 880 (45-5505)
UW-preserved livers were rinsed with 500 mL of UW solution, HTK-preserved livers with 500 mL of HTK solution. The samples were collected from the liver veins. The median values and range of the first 30 mL of effluent are given.
event of intensive care treatment with ventilation being required, of increasing circulatory instability, and the need to substitute coagulation factors. Delayed graft function was said to have occurred if the clinical stabilization of the patient was not observed within 48 hours after transplantation and a disorder of coagulation with the need of substitution, a hypodynamic circulation, and an increase of the transaminases (over 2000 U/L) continued for this time.‘.‘” A satisfactory function was defined as a recovery period of 24 to 36 hours with stabilisation of circulation and normalisation of coagulation. The transaminases should not increase over 1000 U/L. RESULTS
The median activities of the enzyme LDH and AST in the effluent of HTK-perfused organs was twice the median of activities of UW-perfused ones (Table 1). The correlation between the ubiquitous cytosolic enzyme LDH as a marker of the “global” injury and AST as a marker of the hepatocellular damage was high (r = .972), which is in line with the fact that hepatocytes represent over 90% of cellular liver volume.” There was no significant correlation between enzyme activities in the effluent and the length of cold ischemia time. However, all four livers with the highest LDH and transaminase activities in the effluent had ischemic preinjury in the donor, two were from donors who had been resuscitated (MK, AI Table 2) and two were from donors with circulatory instability or shock (KN, HF). There were only three further cases involving circulatory instability or resuscitation of the donor. From the Department of General Surgery and Institute of Physiological Chemistry, University Clinic, Essen, Germany. Address reprint requests to Reinhard Lange, MD, Dept of General Surgery, University Clinic, Hufelandstr. 55, D-45147 Essen, Germany.
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
400
Transplantation Proceedings,
29, 400-402 (1997)
PRESERVATION
401
WITH UW AND HTK SOLUTIONS
Table 2. LDH Activitv in Back-Table Rinse in Correlation to Morphology and Postoperative Graft Function LDH [U/L]
LDH [U/L] Preservation
CIT (h)
MK KN Al KL PJ PJ HF
HTK uw uw uw uw uw uw
6 14 9 9 9 9 12
13500 26240 13820 6745 5212 4850 13112
HH PG
uw uw
15 6
6650 6242
Liver
Histology
(24h)
Function
5% fatty degeneration 40% fatty degeneration normal normal normal 40% centroacinous necrosis 20% necrosis of parenchyma 40% fatty degeneration normal
8460 3220 4231 1742 1235 2765 2200
Delayed Delayed INF Delayed Delayed INF Delayed
1876 3442
INF INF
Anamnesis of the donor
(PI-3
54 52 58 38 42 56 44
years, years, years, years, years, years, years,
ICE, resuscitation, alcohol abuse ICB, alcohol abuse, adrenaline CCT, resuscitation CCT, resuscitation CCT ICB, adrenaline CCT, noradrenaline
53 years, ICB, diabetes 48 years, resuscitation, AST 420 U/L
The table shows the organs with peculiarities in the effluent (LDH activity more than twice median value) and function (initial nonfunction [INF] or delayed function). Abbreviations: cold ischemia time: CIT; craniocerebral trauma: CCT; intracranial bleeding: ICB.
All livers with abnormalities with regard to histomorphology, LDH activity in the perfusate (LDH > 3300 U/L for UW preserved organs or 6100 U/L for HTK preserved organs), and function (delayed function or primary nonfunction) are detailed in Table 2. The activities of 3300 U/L in UW-preserved organs and 6100 U/L in HTK-preserved organs are double the median of LDH activities of all preserved organs. Nine of 30 livers with LDH values above this limit showed an initial nonfunction after transplantation, and seven livers were subject to delayed functioning. The other 90 livers with LDH activities below the limit did not show any functional disorder (Fig 1). Four of 12 livers with morphologic alterations in the 0 biopsy had an initial nonfunction, but 3 of 58 livers with normal biopsy had an initial nonfunction, too. In three organs that were histologically classified as normal, the function was delayed.
cantly (53%). This is probably due to the high influence of the additional factors that might affect the organ after assessing the effluent samples such as warm ischemia time, circulatory insufficiency, infection, and rejection.3 Unfortunately, a good liver may drift into nonfunction for such reasons or-conversely-a transplantation without any problems may take successfully even with a marginal, severely injured graft4
0
‘I function o k.
DISCUSSION
0 Levels of enzyme activities in effluent samples can show the injury of a preserved liver up to the end of cold ischemia. The preliminary results of the actual study seems to confirm the conclusion of the former retrospective investigation.’ The effluent of HTK-preserved livers showed significantly higher levels of enzyme activities than the effluent of UW-preserved organs. This might be the result of the differing viscosity of the two solutions, leading to better rinsing of the liver with HTK solution,8,9 and does not necessarily indicate superior preservation by UW solution. Not only damage sustained during cold ischemia time, but also preexisting damage such as warm ischemic injury in the donor (livers MK, KN, AI, HF) produced a clear increase in the activity of LDH in the effluent. In all cases of initial nonfunction and delayed function, the effluent samples showed high LDH activities. In contrast, the histologic examination only yielded pathologic results in four of eight livers (grafts KN, PJ, HF, and HH). This might result from the delay in which events lead to morphologic tissue alteration. As shown in Fig 1, high enzyme activities in the effluent samples do not necessarily indicate a malfunction of the graft, yet the danger of dysfunction was increased signifi-
?? ??
delayed function or INF
0
2 x medun I
ii
HTK Fig 1. LDH activities in the effluent in comparison to graft function; the values for UW- (n = 51) and HTK-preserved organs (n = 19) are presented separately. The borderline between normal activity and increased release was set at twice the median of all activities (for LDH: UW 3300 U/L; HTK 6100 U/L). These borderlines are indicated by lines.
402
In conclusion, the effluent samples were a sensible marker for preexisting damage or for damage acquired during cold ischemia. Thus, effluent samples offer a helpful parameter for the decision on whether to transplant a marginal organ. The test can be performed during back table preparation before the recipient hepatectomy is performed so the result of the analysis is available in time to make a decision. This is an advantage to the alternative procedure of measurement during warm ischemia time.14 REFERENCES
1. Erhard J, Lange R, Gersing E, et al: Langenbecks Arch Chir 378:233, 1993 2. Fukusazawa K, Schwartz ME, Acarli K, et al: J Am C Surg 178541, 1994 3. Strasberg S, Howard T, Molmenti E, et al: Hepatology 20:829, 1994 4. Doyle HR, Marino IR, Jabbour N, et al: Transplantation 57:1028, 1994
LANGE, ERHARD, RAUEN ET AL
5. Aminalai A, Kehrer G, Grossmann F, et al: Langenbecks Arch Chir 377:81, 1992 6. Caldwell-Kenkel JC, Currin RT, Tanaka Y, et al: Hepatology 13:83, 1991 7. Shimada M, Yanaga K, Kishikawa K, et al: Transplant Int 6:4, 1993 8. Rauen U, Erhard J, Klhnhenrich Chir 379:241, 1994
P, et al: Langenbecks Arch
9. Lange R, Erhard J, Rauen U, et al: Transplantation
62:1996
10. Erhard J, Lange R, Scherer R, et al: Transplant Int 7:177, 1994 11. Gubernatis G, Pichlmayr R, Lamesch P, et al: Langenbecks Arch Chir 375:66, 1990 12. Bergmeyer HU: Methoden Weinheim: Verlag Chemie. 1974
der enzymatischen
Analyse.
13. Blouin A, Bolender RP, Weibel ER: J Cell Biol 72:441, 1977 14. Devlin J, Dunne JB, Sherwood RA, et al: Transplantation 60:627, 1995