In vitro evaluation of donor liver preservation fluids on human hepatocyte function

Successful liver transplantation depends on adequate preservation of cellular function. We therefore tested the effects of two currently used liver preservation fluids, Euro-Collins (EC) solution and University of Wisconsin (UW) solution, on the viability and some functional activities of hepatocytes isolated from human livers. Cells in primary culture were maintained under hypoxic (95% N₂/5% CO₂) and hypothermic (4°C) conditions for 24 h, either in EC or UW solution. This treatment did not result in significant hepatocyte damage, as judged by phase contrast microscopy, intracellular LDH release, and the MTT mitochondrial test. However, neutral red uptake indicated that lysosomal functions were slightly affected (35% decrease) when compared to control conditions. At the end of the hypoxia/hypothermia period, hepatocyte monolayers were incubated at 37°C under normoxic conditions for 24 h, in order to simulate the reperfusion of a transplanted liver. Three drugs-midazolam, diazepam, zidovudine-were used as diagnostic substrates to check the metabolic abilities of human hepatocytes replaced in normal conditions. Both phase I (hydroxylation, demethylation) and phase II (glucuronidation) metabolic reactions were affected by the hypoxia/hypothermia shock. Indeed, a 30%–50% decrease in these activities was observed as compared to values obtained in control hepatocytes. No difference could, however, be found at the cellular level regarding the solution used for cold storage. These results suggest that the superiority of UW over EC solution, already reported in clinical practice after transplantation of preserved human livers, was not due to a better preservation of the hepatocytes.     

Comparison of histidine-tryptophan-ketoglutarate (HTK) solution versus University of Wisconsin (UW) solution for organ preservation in human liver transplantation

Over a 30-month period, 60 patients (30 in each group) suffering from end-stage liver disease or primary hepatic malignancy and scheduled for liver transplantation were enrolled in a prospective, randomized study to compare two methods of liver preservation: histidinetryptophan-ketoglutarate (HTK) solution versus University of Wisconsin (UW) solution. Entry criteria for both groups were: age (18–65 years), elective surgery (transplantable or urgent category of the recipients), first transplantations and harvesting procedure performed by the same team. The parameters under investigation were the clinical and laboratory data preand post-transplantation, as well as follow-up data such as complications and survival. There were no significant differences in the two groups as far as the evaluation criteria were concerned, even when cold ischemia time was more than 15h (n=7). A slight, yet not significant, increase in late complications of the biliary anastomoses could be seen in the UW group. Hepatocellular injury (SGOT, SGPT, GLDH, lactate) appeared to be more marked in the HTK group. These results suggest that both HTK and UW solutions are appropriate for clinical use in liver transplantation, even if cold ischemia time is more than 15h.     

Comparison of HTK- and UW-solution for liver preservation tested in an orthotopic liver transplantation model in the pig

The aim of this experimental study was to compare the preservation potency of University of Wisconsin (UW) and HTK (Bretschneider) solutions in an orthotopic liver transplantation (OLT) model in pigs. Livers were harvested using an in situ perfusion technique, where organs were flushed with the solution being tested, stored on ice — cold storage (CS) — for 2 or 24 h and then transplanted. Parameters monitored were liver enzymes in serum, hepatic water content, high energy phosphates, nuclear magnetic resonance (NMR) relaxation time T2, light microscopy and bile production. CS for 24 h is an extreme in pig liver preservation and is not compatible with animal survival. Biopsies showed drastic morphological changes and grafts did not produce bile in either group. (Bile production 2 h CS: HTK, 5.6 ± 1.8 ml/h; UW, 4.7 ± 2.3 ml/h) Enzyme release after reperfusion (ASGOT, ?LDH) was higher in long-term preservation. Hepatic tissue water content significantly decreased during CS in UW preserved livers. Edema alter reperfusion (?H₂0: HTK 24 h = + 5.6%, UW 24 h= + 4.8%) and regeneration capacity after reperfusion (UW 2 h = 63%, HTK 2 h = 55%, UW 24 h = 30%, HTK 24 h = 30%) were not significantly different. However, we did not observe major differences in preservation potency between the solutions tested. Differences were correlated, rather, with length 9 time of CS, than with the solution used. Therefore, HTK solution seemed to be a low potassium containing alternative to UW solution.     

自制KYL液对大鼠肝脏低温保存后细胞凋亡影响的研究

目的 研究自制的KYL液对大鼠肝脏低温保存后细胞凋亡的影响。方法 采用大鼠肝脏非循环离体灌注模型（noncirculated isolated perfusion of ratliver，IPRL），随机以KYL液和UW液对大鼠肝脏保存0、4、8、16、24、48h，测定灌注流出液氧自由基代谢产物（丙二醛MDA和超氧化物歧化酶SOD）的含量，检测肝细胞内钙离子浓度，检测肝细胞凋亡率和凋亡相关基因表达，观察肝脏组织形态学变化。同时设生理盐水保存阴性对照组，了解器官保存液对大鼠肝脏有无保护作用。结果 KYL液保存的大鼠肝脏肝细胞内钙离子浓度较UW液保存者低，灌注流出液MDA和SOD含量与UW液保存者相近，两者肝细胞凋亡率及凋亡基因表达情况相近，光、电镜观察两者形态学变化基本一致。两组所有指标均较生理盐水保存组好，说明两种液体对大鼠肝脏均有保护作用。结论 自制的KYL液对大鼠肝脏的保存效果在钙拮抗方面略优于UW液，在抑制细胞凋亡方面与UW液相当，而在防止细胞水肿方面较UW液稍差。     

The impact of liver preservation in HTK and UW solution on microcirculation after liver transplantation

Severe microcirculatory disturbances due to endothelial cell damage and leukocyte adherence during reperfusion of transplanted livers are considered to contribute to early graft failure. Since the degree of reperfusion injury after liver transplantation depends on the length of preservation time and the solution used for preservation, the aim of our study was to assess three solutions with respect to microvascular perfusion and leukocyte adhesion. Therefore, rat livers were stored up to 24 h in Euro-Collins (EC), University of Wisconsin (UW), or histidin-tryphtophan-ketoglutarate (HTK) solutions prior to orthotopic transplantation. The livers were studied in situ 60 min postoperatively using intravital fluorescence video microscopy. Using simple syringe flushing (10 ml), sinusoidal perfusion decreased below 50% in EC preserved livers after 8 h preservation, in HTK preserved livers after 16 h preservation, and remained higher than 70% in livers preserved in UW up to 24 h. Permanent adhesion of leukocytes was increased more rapidly in organs after 1, 8, 16, and 24 h preservation in HTK (16%, 15%, 34%, and 49.7% ± 4.7%) compared to those preserved in UW (15%, 18%, 17%; and 32.7% ± 3.3%; P < 0.05). Using a 10-fold volumn of the organ weight of HTK solution during the harvesting procedure, with an 8 min equilibration period, sinusoidal perfusion (39.6 ± 4.7%) and leukocyte adhesion (42.7 ± 3.1%) were not improved after 24 h. In contrast, equilibration with a volumn of approximately 40-times the liver weight improved sinusoidal perfusion (70.8% ± 2.7%; P < 0.01) and leukocyte adhesion (24.9% ± 3.1%; P < 0.01) significantly. Thus, using HTK solution, simple flushing prior to long-term cold storage resulted in microcirculatory disturbances when compared to UW solution. Larger volumns of HTK solution with an additional equilibration period of 8 min, however, reduced leukocyte adhesion and improved sinusoidal perfusion to a similar degree as UW solution.     

自制肝脏保存液在大鼠原位肝移植模型中的应用

目的研制一种新型的专用于肝脏保存的溶液,以期达到价廉、专用、损伤小并能长时间保存的目标。方法自制肝脏保存液;建立大鼠原位肝移植模型;使用自制肝脏保存液(A组)、UW液(B组)和HC-A液(C组)保存大鼠肝脏2、8、24h后行大鼠原位肝移植,于移植后6 h比较各项肝脏功能。结果对于ALT、AST、HA,自制肝脏保存液组及其亚组与UW液组同步升高(P>0.05),与HC-A液组比较,前者的含量均低,差异有统计学意义(P<0.05)。对于LDH、STB,自制肝脏保存液组及其亚组与HC-A液组同步升高(P>0.05),与UW液组比较,前者的含量均高(P<0.05)。结论经大鼠原位肝移植模型证实,自制肝脏保存液与UW液在保护肝脏功能方面作用相当,优于HC-A液。     

SX-1液对肝脏保存效果的实验研究

目的 应用改良后的Kamada二袖套法大鼠原位肝移植模型,检验SX-1液、HC-A液和UW液对肝脏保存的效果.方法 在无菌条件下配制肝脏保存液.建立大鼠原位肝移植模型.使用SX-1液、UW液和HC-A液保存大鼠肝脏2、8、24 h后行大鼠原位肝移植,于移植后6 h比较各项肝脏功能.结果 对于ALT、AST,SX-1液组(2、8、24 h)与UW液组同步升高,分析无统计学意义(P＞0.05),与HC-A液组相比,差异有统计学意义(P＜0.05).对于LDH,SX-1液组(2 h、8 h、24 h)与HC-A液组同步升高,差异无统计学意义(P＞0.05),与UW液组相比,差异有统计学意义(P＜0.05).对于分泌胆汁的肝脏个数,各组别与分泌胆汁的肝脏个数无差别(P＞0.05).本组内各时间点分泌胆汁个数有差别(P＜0.05).随肝脏保存时间增长,分泌胆汁的肝脏个数减少.结论 经大鼠原位肝移植模型证实,SX-1液在肝脏酶学方面与UW液作用相当,超过HC-A液水平.肝移植后6 h肝脏分泌胆汁的个数方面,SX-1液与HC-A液、UW液间无明显差别.     

Protective effects of the lazaroid U74500A and lidoflazine on liver preservation with UW solution

The effect of adding a 21-aminosteroid, U74500A, and a Ca²⁺ antagonist, lidoflazine, alone and together to UW solution was assessed in a rat liver preservation model. Following preservation, the livers were reperfused using a closed circuit, and the release of hepatocellular enzymes (ASAT, ALAT, and LDH) into the perfusate was determined with increasing time. Both drugs reduced the amount of enzymes lost from the liver. The combination of the two drugs was better than either drug alone. These data suggest that both agents may be of value in organ preservation for clinical liver transplantation.     

Myocardial preservation with the UW solution. First European results in clinical heart transplantation

In recent years, there is a growing body of evidence that the University of Wisconsin (UW) solution offers many advantages in organ preservation with regard to preservation quality and time. We, therefore, conducted the first European prospective, randomized, clinical trial comparing myocardial performance after preservation with UW and St. Thomas Hospital (ST) solution. Preliminary results indicated superior heart function after preservation with UW solution.     

Comparison of solutions for preservation of the rabbit liver as tested by isolated perfusion

The University of Wisconsin (UW) solution consists of a relatively complex mixture of agents. In this study we compared simpler preservation solutions, namely, histidine-tryptophan-ketoglutarate glutarate (HTK) and phosphatebuffered sucrose (PBS) with different compositions of UW solution in the isolated perfused rabbit liver model. Livers were stored cold for 24 and 48 h. After 24 h of preservation, the amount of bile produced in UW-preserved livers was significantly greater (P<0.05) than that in HTK-preserved livers. Also, there was less LDH released into the perfusate in UW-preserved livers. There was more edema and lower K+/Na+ rations in HTK-preserved livers than in UW-preserved livers (all data P<0.05). After 48 h of preservation, the differences between livers preserved in UW or HTK solution were less noticeable than at 24 h and bile production was similar. LDH and AST release were greater in HTK-preserved livers than in UW livers, but these differences were not statistically significant. Preservation in PBS for 48 h was worse than in either UW or HTK solution. Substitution of polyethylene glycol (PEG) for hydroxyethyl starch (HES) in 48-h UW-preserved livers was not effective. We conclude that solutions simpler in composition than UW solution may be effective in kidney transplantation but do not appear suitable for successuful liver preservation.     

High-Na + low-K + UW cold storage solution reduces reperfusion injuries of the rat liver graft

The isolated perfused rat liver model was used to assess graft viability after 24 h of cold preservation. Two solutions were compared for liver preservation: Belzer's original UW solution (high-K ⁺ UW) and a solution containing the same components but with inverted concentrations of sodium and potassium (high-Na ⁺ UW). During the 120 min of normothermic reperfusion, livers preserved in the high-Na ⁺ UW solution released lower levels of creatine kinase-BB isoenzyme, transaminases (ALT and AST), and potassium than those preserved in the high-K ⁺ UW solution. Bile flow and biliary excretion of indocyanine green increased when livers were preserved in the high-Na ⁺ UW solution. We found no statistical differences for oxygen consumption and tissue ATP concentration. The results of this study support the concept that a high-Na ⁺ UW solution is a more effective means of preserving rat livers, at least after 24 h of cold-storage and 120 min of reperfusion in the isolated perfused model, than the original high-K ⁺ UW solution. Liver preservation in the high-Na ⁺ UW solution reduces damage to sinusoidal endothelial and hepatocellular cells. The use of an extracellular-like Belzer cold storage solution eliminates potassium-related problems in cold preservation and subsequent normothermic reperfusion while keeping all the qualities of the original UW solution. Received: 26 August 1997 Received after revision: 12 November 1997 Accepted: 28 November 1997     

Postoperative recovery of mitochondrial function of the human liver graft procured and preserved with University of Wisconsin (UW) solution

Changes in arterial blood ketone body ratio (KBR) were investigated in 47 human liver transplantations. Of the 20 grafts preserved with University of Wisconsin (UW) solution, 10 had a cold preservation period of less than 10 h (UWS group) and 10 of more than 10 h (UWL group). In 27 other cases, grafts were preserved with EuroCollins (EC) solution for less than 10 h (EC group). In the EC group, KBR increased over 0.7 within 6 h after reperfusion of the graft in 17 cases (63%) and within 24 h in 7 cases (26%). In the 3 other cases, KBR failed to recover, and these patients underwent retransplantation. In the UW group, KBR recovered within 6 h in 13 cases (65%) and within 24 h in 7 cases (35%). There were no significant differences between the UWS and UWL groups. It is shown that the mitochondrial function of liver grafts preserved with UW solution can be well maintained even after extended preservation periods of more than 10 h.     

A comparison of histadine lactobionate solution with University of Wisconsin solution for rat liver and heart preservation

We developed a new solution mainly composed of Na-lactobionate and histidine (HL) and compared the effectiveness of this solution with that of University of Wisconsin (UW) solution using orthotopic liver and heterotopic heart transplantation in rats. The new solution has a higher sodium content and a lower potassium content (Na, 90 mEq/l; K, 45 mEq/l) than UW. Hydroxyethyl starch, adenosine, dexamethasone and insulin are not included. Buffering capacity is increased by adding histidine (90 mm/l) together with KH₂PO₄ (20 mm/l). Rat liver was perserved in either UW or HL solution hypothermically for 24 h and then transplanted orthotopically into the recipient rat. The heart was preserved in either solution for 18 h and transplanted heterotopically into the recipient rat. The 1-week survival rate for rats receiving livers preserved in UW for 24 h at 4°C was 29% (5/17). In contrast, the new solution (HL) gave a 78% (11/14) survival rate (P < 0.01). The 1-week heart graft survival rate, using UW solution was 50% (3/6), following 18-h cold preservation, whereas all hearts (7/7) continued to beat for over a week using new HL solution (P < 0.05). These results demonstrated that the new HL solution, with a substantial buffering capacity, was superior to UW solution in rat liver and heart preservation.     

Advantages of Celsior solution in graft preservation from non-heart-beating donors in a canine liver transplantation model.

BACKGROUND: The optimal method for preserving livers from non-heart-beating donors (NHBD) is still unknown. We compared the Celsior solution, a new extracellular-type, low-potassium, low-viscosity preservation solution, with the University of Wisconsin (UW) solution in a canine orthotopic liver transplantation from NHBD. MATERIALS AND METHODS: Fourteen adult mongrel dogs, weighing 9 to 17 kg, were divided into two groups: the Celsior or the UW group (n = 7 each). The thoracic descending aorta and supradiaphragmatic inferior vena cava were cross-clamped for 20 min to induce warm ischemia as a NHBD model. The liver was flushed with the respective cold preservation solution and then stored at 4 degrees C for 4 h. The grafts were transplanted using the piggy-back technique under portal decompression by leaving the native right lobe as a temporary shunt. RESULTS: The duration of liver flushing out (min) was shorter (P < 0.05), and the serum glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, lactate dehydrogenase, lactate, and alpha-glutathione S-transferase concentrations 2 and 6 h after reperfusion of the graft (RPF) were lower (P < 0.05) in the Celsior group than in the UW group. Hepatic tissue blood flow (HTBF) did not deteriorate as much (P < 0.05) in the Celsior group. The serum endothelin-1 level was lower (P < 0.05) in the Celsior group 2 h after RPF. Histopathology of liver specimens revealed portal congestion and hepatocyte necrosis with neutrophil infiltration in the UW group, while these findings were mild in the Celsior group. CONCLUSIONS: The Celsior solution improves vascular endothelial injury in livers from NHBDs and may have advantages in graft flush and preservation of grafts from NHBDs.     

An underlying mechanism for improved liver preservation with a combined histidine-lactobionate-raffinose flush solution

In previous experimental liver transplant studies, it was possible to extend cold ischaemic time (CIT) by using a flush/storage solution combining histidine, lactobionate and raffinose (HLR). In this study, energy metabolism, glycolytic substrate (glucose) and anaerobic end-product (lactate) were examined in rat liver over 24 h of cold storage to determine the mechanism of action of the HLR solution. In livers subjected to simple flush and storage with the HLR solution. levels of ATP and ADP were considerably higher than livers stored with modified UW throughout 24 h of storage; at 4 h of storage, ATP and ADP levels were 1.1 and 3.1 mol/g for HLR solution versus 0.18 and 0.81 mol/g for UW solution. Total adenylate contents (TA=ATP+ADP+AMP) also remained 1–2 mol/g higher in HLR-treated livers than those preserved in UW; TA values ranged from 3.8 to 5.7 mol/g. Glucose increased to 20–35 mol/g by 10–24 h of storage (similar to the UW group). Lactate rose to almost twice that in livers stored in UW; total lactate accumulation was approximately 10.0 mol/g. This study demonstrated that the combined HLR solution is able to prolong the maximum safe CIT by increasing anaerobic metabolism and consequently preserving liver energetics. The second part of the experiment examined the effect of continuous perfusion (with/without O₂) over the 1st h of cold ischaemia. Under current methods of liver flushing and excision, the 1st h of cold storage may be the critical time of metabolic adjustment since most of the pH and ATP changes occur during this period. Therefore, we tested the hypothesis that the combination of a simple flush with an additional brief 1-h perfusion period prior to storage would enhance the maintenance of hepatic energetics. There was no beneficial effect of 1 h of perfusion without O₂ compared to simple HLR flush and storage. However, perfusion with O₂ resulted in prolonged maintenance of high energy adenylates and total adenylates; at 10 h of storage ATP was 1.0, ADP 3.3, and TA 5.7 mol/g. However, any improvement in ultimate viability following long-term storage of the livers in these two groups needs to be tested in an animal transplant model.     

UW器官保存液在胰腺移植中的研究进展

近年来,糖尿病发病率在全球呈快速增长的趋势,我国患病率已达到3%.多数Ⅰ型和部分Ⅱ型糖尿病患者需要采用外源性胰岛素注射控制血糖,患者不但要承受胰岛素注射带来的痛苦,而且还不可避免地要发生远期的并发症.因此,重建内源性胰岛分泌系统是近年来人们关注的热点.大量实验表明,胰腺移植不但可以中止胰岛素依赖性糖尿病并发症的发展,还可以转归已有的并发症,明显改善患者的预后.与其他器官移植一样,移植器官的获取及保存是移植成功的前提,由于胰腺组织的特殊性,不同的器官保存液对于保存胰腺效果不一.本文将对University of Wisconsin(UW)保存液在胰腺移植中作用作一论述.     

影响致敏受者肾移植术后人/肾存活的相关因素

目的：探讨影响致敏受者肾移植术后人／肾存活的相关因素。方法：对82例致敏受者尸体肾移植患者可能影响移植肾存活相关的20个因素47个水平，进行Logrank单因素分析和Cox模型多因素回归分析。结果：82例的1、2和3年的人存活率分别为98％、96％和94％；移植肾存活率分别为90％、87％和83％。总的移植肾半生存期为4．7年。单因素分析，群体反应抗体水平和动态变化、供体特异性抗体、急性排斥、慢性排斥、冷缺血时间、早期肾功能、血肌酐水平和免疫诱导剂等9个因素；多因素分析，急性排斥、供体特异性抗体和群体反应抗体类型等3个因素；单因素和多因素同时分析，急性排斥和供体特异性抗体2个因素，对致敏受者移植肾短期和长期的存活率有重要的影响(P＜0．05)。结论：高质量的供肾、群体反应抗体动态监测、尽力避免和有效处理术前和术后相关危险因素，对提高致敏受者肾移植的人／肾存活率有重要的作用。     

Long-term survival and prognostic factors in the surgical treatment for intrahepatic cholangiocarcinoma

Background/Purpose. We retrospectively investigated the clinicopathologic features and outcome of 51 patients who underwent hepatectomy for intrahepatic cholangiocellular carcinoma (ICC) between 1991 and 2000, and we also analyzed the potential prognostic factors for long-term survival. Methods. There were 27 men and 24 women, with a mean age of 63.7 years. The surgical procedures were extended right or left hepatectomy (15 cases), right or left hepatectomy (19 cases), bisegmentectomy (3 cases), segmentectomy (7 cases), and subsegmentectomy (7 cases). The macroscopic findings of the excised tumor showed the mass-forming (MF) type (31 cases), the periductal-infiltrating (PI) type (13 cases), and the intraductal growth (IG) type (7 cases). Results. The patients with the MF type had a significantly higher incidence of lymph node metastasis (44.8%), as compared to those with the PI or IG type (15.0%). Two patients who died of hepatic failure during their hospital stay were excluded from this survival study. The cumulative 1-, 3-, and 5-year survival rates in 49 patients who underwent liver resection were 68.2%, 44.1%, and 32.4%, respectively. The patients with the IG type had the best outcome, followed by those with the PI type and MF type. The survival rates with or without lymph node metastasis were 9.0% and 60.6% at 3 years, and 9.0% and 42.9% at 5 years, respectively (P 0.05). The 1-, 2-, and 3-year survival rates in the MF-type patients with lymph node metastasis were 25.4%, 16.9%, and 0%, respectively. Eight patients (15.7%) survived for more than 5 years after operation. The gross appearance of these tumors was the PI type in 5 patients, the IG type in 2, and the IG + MF type in 1. Except for one case with the PI-type tumor, lymph node metastasis was not observed. All of the 5-year survivors underwent curative resection and none of them had any positive surgical margin. Conclusion. Analysis of the clinicopathologic factors influencing the survival after surgical treatment showed that the macroscopic type, surgical curability, lymph node metastasis, tumor size, and cancer-free margin were the most predictive.     

Analysis of risk factors following pediatric liver transplantation

Abstract Several recipient, donor and operation factors as well as postoperative complications related to patient survival after liver transplantation (LT) in children were studied by univariate and multivariate analyses. In a 13‐year period, 103 patients under 15 years of age underwent 120 LT; the mean age was 63 months and 36% were under 2 years of age. Indications for LT were cholestatic disease in 68 (56%), metabolic diseases in 18 (14%), fulminant hepatic failure in 8 (7.5%), cirrhosis in 7 (5.8%), and retransplants in 17 (14%). Whole liver was transplanted in 79% of cases and partial liver in 21 %. Actuarial survival at 1, 5, and 10 years was 70 %, 61 %, and 57 %, respectively. United Network of Organ Sharing (UNOS) I recipients (RR = 2.7), primary non‐function (PNF) (RR = 13.9), and hepatic artery thombosis (HAT) (RR = 3.8) were independent factors for lower patient survival in multivariate analysis. Thus, in our experience, postoperative mortality as a consequence of the patient's condition before transplantation, or complications such as PNF or HAT, are the major causes of decreased survival in pediatric LT.