Human islet isolation outcomes from pancreata preserved with Histidine-Tryptophan Ketoglutarate versus University of Wisconsin solution

This study was designed to compare Histadine-Tryptophan-Ketogluterate (HTK) with University of Wisconsin (UW) solution. Pancreata from extended criteria donors were flushed and transported with HTK (n=41) or UW (n=45). Isolation outcomes were determined by islet yields, viability and in vitro and in vivo function. Final yields were similar between two groups (HTK: 383,085 vs. UW: 328,514 EIN, P=0.14). In the HTK group, 63.4% (26/41) of isolations resulted in a yield of over 300,000, and in the UW group this was achieved in 46.7% (21/45; P=0.12). Viability results were similar (HTK: 82.9 vs. UW: 82.7%, P=0.93). Stimulation index in the HTK and UW groups were comparable (5.28 vs. 4.91, P=0.62). Ten out of 41 islet preparations in HTK and 4 of 45 in UW group were suitable for clinical transplantation (P=0.05). Our study shows HTK is equivalent to UW solution in the preservation of pancreata for islet isolation.     

University of Wisconsin solution versus Celsior solution in clinical pancreas transplantation

INTRODUCTION: This study compared the safety and efficacy of University of Wisconsin solution (UW) and Celsior solution (C) in pancreas transplantation (PTx). METHODS: A retrospective review of 154 PTx performed over a 61-month period included 77 grafts preserved with UW and 77 with C. The two groups were comparable for both donor and recipient characteristics. RESULTS: After a mean cold ischemia time of 624 minutes (range 360 to 945 minutes) for UW versus 672 minutes (range 415 to 1005 minutes) for C (P = NS), no primary endocrine nonfunction occurred. Delayed endocrine function was diagnosed in two grafts in the UW group (2.6%) versus none in the C group (P = NS). After a minimum follow-up of 4 months (mean 26.5 +/- 15.2 months), 22 recipients (UW = 11 vs C = 11; P = NS) required relaparotomy. Overall, 18 pancreata were lost due to either patient death with functioning graft (UW = 4 vs C = 1; P = NS) or graft loss due to other reasons (UW = 8 vs C = 5; P = NS). Actuarial 1- and 5-year patient survival rates were 93.5% and 86.8% for UW compared with 98.7% and 98.7% for C (P = .04). Actuarial graft survival rates at the same times were 88.3% and 75.0% for UW compared with 90.4% and 90.4% for C (P = NS). CONCLUSIONS: Within the range of cold ischemia times reported in this study, UW and C show similar safety and efficacy profiles for PTx.     

Ameliorating injury during preservation and isolation of human islets using the two-layer method with perfluorocarbon and UW solution

This study assessed the effects of a two-layer method (TLM), using perfluorocarbon and UW solution, on the quality of human pancreata following storage and islet yield/function after isolation. In part A, TLM was applied immediately after procurement and the energetic profile was compared to a group treated with UW solution only (control) throughout 24-h storage. In part B, cadaveric human pancreata were procured and subjected to a TLM after cold storage in UW solution (TLM group) or UW solution (control group). Energetics, lipid peroxidation, and islet recovery/function were assessed after preservation at 4 degrees C. In part A, after 9-h storage, the energetic profile (ATP, ATP/ADP, energy charge) for the TLM group was superior to controls. In part B, TLM treatment resulted in consistently greater ATP, ATP/ADP, and energy charge values than with storage in UW solution alone (p < 0.05). UW treatment resulted in 40% greater peroxidative damage than in the TLM group (p < 0.05). Islet recovery and functional viability were 30-40% higher following TLM treatment (p < 0.05). These data support the hypothesis that islet viability and yields can be significantly improved using a brief period of TLM treatment following conventional UW storage; reduced energetic and oxidative stress are implicated as potential mechanisms.     

Clinical application of the two-layer (University of Wisconsin solution/perfluorochemical plus O2) method of pancreas preservation before transplantation

BACKGROUND: The two-layer method [University of Wisconson solution (UW)/perfluorochemical plus O2] for pancreas preservation has been demonstrated to be superior to simple UW storage alone in the canine model. For the first time, we applied the two-layer method to clinical whole-pancreas transplantation. METHODS: Pancreases were placed in the two-layer method in 10 cases and UW alone in 44 cases before transplant. The mean cold ischemic time was 16.5 hr in the two-layer group versus 18.1 hr in the UW group (P=NS). We compared the condition of graft at the time of reperfusion, and then 3 months posttransplant graft function and complications. RESULTS: At the time of reperfusion, no grafts in the two-layer group were edematous, compared with 10(23.3%) of 43 in the UW group (P=0.18). Seven (70%) of 10 grafts in the two-layer group obtained the best overall quality score, compared with 24(57.1%) of 42 in the UW group (P=0.72). Nine (90%) of 10 recipients in the two-layer group became insulin-independent during hospitalization, compared with 31(70.5%) of 44 in the UW group (P=0.26). Time to insulin independence was no different between the two groups. No pancreas grafts preserved by the two-layer method suffered acute rejection. Conclusions. The two-layer preservation method is feasible in human clinical transplantation. It was at least equivalent and may be superior to UW alone in both morphologic and functional assessment of the transplanted pancreas.     

The effect of two-layer (University of Wisconsin solution/perfluorochemical) preservation method on clinical grade pancreata prior to islet isolation and transplantation

BACKGROUND: Research-grade pancreata preserved by the two-layer method (TLM) compared to organs stored with University of Wisconsin (UW) solution prior to islet isolation result in significantly better islet yields. However, it is unknown whether the TLM improves islet yields from pancreata that meet the criteria for the selection of clinical-grade organs. METHODS: Six clinical-grade pancreata were preserved for 4.8 +/- 0.5 hour in UW and three clinical-grade pancreata were preserved by the TLM for 11.7 +/- 2.0 hour. The local team procured all pancreata. All donors were hemodynamically stable without norepinephrine usage and length of hospitalization was less than 96 hour. Causes of death were either head trauma or cerebrovascular accident. Islets were isolated and evaluated according to the Edmonton protocol. RESULTS: The TLM as compared to UW resulted in a significant increase in islet yields (3415 +/- 227 vs 2006 +/- 337 IE/g pancreas, P <.03). The quality of islets as assessed by visual score was significantly better in the TLM group (8.7 +/- 0.2 vs 7.3 +/- 0.3, P <.02) but other parameters (viability, survival rate after culture, insulin content, stimulation index) were similar between the two groups. We transplanted all three islet preparations in the TLM group but only two of six preparations from the UW group. CONCLUSION: Compared to UW, exposure of pancreata to the TLM resulted in greater islet yields and extended preservation times with clinical grade pancreas. Pancreata should be preserved by the TLM prior to islet isolation even for donors that meet clinical grade organ selection criteria. The Human Islet Transplantation in Seattle (HITS) Consortium is supported in part by a grant from the Juvenile Diabetes Research Foundation International. The HITS consortium is an islet transplant program involving the University of Washington, Pacific Northwest Research Institute, the Puget Sound Blood Center, Fred Hutchinson Cancer Research Center, Swedish Hospital, and the Virginia Mason Research Center.     

Ultrastructural analyses of pancreatic grafts preserved by the two-layer cold-storage method and by simple cold storage in University of Wisconsin solution

The two-layer cold storage method (TLM) using University of Wisconsin (UW) solution supplies sufficient oxygen to pancreatic grafts during preservation and extends pancreas preservation time to up to 96 h in the canine model. Simple cold storage in UW (UWM) on the other hand, preserves canine pancreas grafts for up to 72 h by preventing cell swelling, mainly because of its high osmotic pressure. The aim of this study is to analyze morphologically dog pancreatic grafts preserved by these two methods with their different mechanisms. Immediately after preservation of canine pancreata by TLM for 72 h and 96 h (group 1 and group 3, respectively), and by UWM for 72 h and 96 h (group 2 and group 4, respectively), tissue ATP levels were determined using high-performance liquid chromatography (HPLC), and detailed morphological analyses of intragraft components were performed using light- and electron microscopy. The mean areas of one mitochondrion and rough endoplasmic reticulum (RER) vacuolization were calculated by computer-graphic analyses using NIH image 1.62 f soft. The tissue ATP levels were significantly higher in groups 1 and 3 than groups 2 and 4 ( P < 0.05). Light microscopy demonstrated no marked difference among the 4 groups. By electron microscopy however, mitochondrial swelling and RER vacuolization were observed in acinar cells to various extents in the 4 groups. They were significantly more evident in group 2 than group 1 ( P < 0.05), and in group 4 than group 3 ( P < 0.05). In conclusion, TLM demonstrated excellent protection of intracellular organelles, mitochondria, and RER, up to 72-96 h. Well-maintained graft ATP levels in TLM groups may result in maintaining the integrity of intracellular organelle membranes as well as cellular membranes.     

Comparison of histidine-tryptophan-ketoglutarate solution and University of Wisconsin solution for organ preservation in clinical pancreas transplantation

BACKGROUND: University of Wisconsin (UW) solution is currently the standard preservation solution used for abdominal organ transplantation. This study assesses the efficacy of histidine-tryptophan-ketoglutarate (HTK) compared with UW in pancreas transplantation. METHODS: Between October 2002 and August 2003, 20 pancreas transplants were performed. Patients were divided into two groups: UW (n=10) and HTK (n=10). Donor and recipient demographics were similar in both groups. The mean cold ischemia time for both groups was 11 +/-3 hr. RESULTS: There was an anticipated difference between total preservative volumes used (HTK: 4.5 +/- 1.2 L vs. UW: 3.4 +/-0.8 L; P =0.03). Patient and graft survivals to date were 100% in both groups. Serum fasting blood glucose, peak amylase, and serial amylase levels remained comparable at all intervals posttransplantation. CONCLUSIONS: Within this range of cold ischemia time, UW and HTK demonstrate similar efficacy in pancreas preservation.     

Influence of pancreas preservation on human islet isolation outcomes: impact of the two-layer method

BACKGROUND: Human pancreas preservation for islet transplantation holds additional challenges and considerations compared with whole pancreas transplantation. The purpose of this study was to clarify the limitations of the University of Wisconsin (UW) solution and the potentials of the two-layer method (TLM) for pancreas preservation before human islet isolation. METHODS: We retrospectively evaluated human islet isolation records between January 2001 and February 2003. One hundred forty-two human pancreata were procured from cadaveric donors and preserved by means of the UW solution (n=112) or TLM (n=30). Human islet isolations were performed using a standard protocol and assessed by islet recovery and in vitro function of islets. RESULTS: Eight to ten hours of cold ischemia in the UW solution is a critical point for successful islet isolations. It is difficult to recover a sufficient number of viable islets for transplantation from human pancreata with more than 10 hours of cold storage in the UW solution. The overall islet recovery in the TLM group was significantly higher than in the UW group. With 10 to 16 hours of cold storage, the success rates of islet isolations remained at 62% in the TLM group but decreased to 22% in the UW group. Transplanted islets in the TLM group worked well in the recipients. CONCLUSIONS: There are time limitations for using the UW solution for pancreas preservation before human islet isolation. The TLM is a potential method to prolong the optimal cold storage time for successful islet isolations.     

Functional and histological comparison of rat liver preserved in University of Wisconsin solution compared with tissue preserved in a novel solution

An isolated perfused rat liver model was used to investigate biochemical and histologic changes during 2 hours of reperfusion after 24 hours of cold storage to compare Leeds solution (LS) with University of Wisconsin solution (UW). Compared with livers stored in UW, those perfused with LS showed significantly higher bile flow and lower enzyme production (P < .05 by 1-way analysis of variance). For example, after 120 minutes, alanine aminotransferase results were: LS 38.9 U/L vs UW 66.8 U/L and bile flows were LS 10.3 μg/15 min/g liver vs UW 9.2 μg/15 min/g liver. Histologically the reticulin breakdown was greater and its reformation slower in UW-preserved livers. Liver tissue was viable in both groups, as shown by the increased glycogen content after reperfusion in both groups, but seen at a higher rate among LS, perfused livers. In conclusion, LS compared favorably with UW to prevent ischemic damage and so could offer an alternative perfusion medium to UW.     

A prospective randomized multicenter trial comparing histidine–tryptophane–ketoglutarate versus University of Wisconsin perfusion solution in clinical pancreas transplantation

We aimed to evaluate early pancreas transplant graft function after histidine–tryptophan–ketoglutarate (HTK) versus University of Wisconsin (UW) perfusion. Prospective randomized multicenter study including 68 pancreas transplantations stratified according to preservation fluid used (27 HTK vs. 41 UW). Primary endpoint was pancreas graft survival at 6 months. Serum α-amylase, lipase, C-peptide, HbA1C and exogenous insulin requirement were compared at several time points. Mean pancreas cold ischemia time was 10.8 ± 3.7 (HTK) vs. 11.8 ± 3.4 h (UW) ( P  = 0.247). Simultaneous pancreas–kidney transplantation was performed in 95.6% of the patients, pancreas transplantation alone in 2.9%, and pancreas after kidney transplantation in 1.5%. Six months graft survival was 85.2% (HTK) vs. 90.2% (UW) ( P  = 0.703). Serum amylase and lipase values did not differ between both the groups during the observation period. C-peptide levels were elevated in both the groups without significant differences at each time point. Higher exogenous insulin requirement early after transplantation in the UW group had resolved at 3 months. Six month patient survival was 96.3% (HTK) vs. 100% (UW) ( P  = 0.397). With a mean cold ischemia time of 10 h in this study, HTK and UW solutions appear to be equally suitable for perfusion and organ preservation in clinical pancreas transplantation.     

Augmentation of tissue ATP level during digestion using preoxygenated perfluorocarbon results in high islet yield-new strategy for islet isolation

BACKGROUND: Recently, preservation using oxygenated perfluorocarbon (the two-layer method) has shown beneficial effects on islet yield and viability. In this paper, we apply this concept on isolation processes to examine the effectiveness of oxygenation. METHODS: Rat pancreata were digested using four different methods: (groups 1A, 1B, 2A, and 2B) with or without oxygenated perfluorocarbon in groups 1 and 2, respectively. Adenosine was added into the collagenase solution in subgroup A whereas it is not added in subgroup B. RESULTS: Tissue oxygen tension in group 1 was about 0 during digestion; whereas it rapidly reached about 300 mm Hg and was maintained in group 2. Tissue ATP level just after laparotomy (control) was 4.2 +/- 0.7 micromol/g dry weight. The ATP levels after digestion were 0.12 +/- 0.03 in group 1A (P < 0.01 vs control); 0.70 +/- 0.10 in group 1B (P < 0.01 vs control); 0.30 +/- 0.18 in group 2A (P < 0.01 vs control); and 2.90 +/- 0.80 in group 2B (P = 0.19 vs control). Islet yields (IEQ/pancreas) were 1600 +/- 400 in group 1B; 1400 +/- 400 in group 1B; 1300 +/- 400 in group 2A; and 2400 +/- 100 in group 2B. The amount in group 2B was significantly greater than that in the other three groups. CONCLUSIONS: Oxygen provision by preoxygenated perfluorocarbon itself showed no beneficial effect on islet yield. However, if oxygen provision was associated with adenosine administration into the pancreas, tissue ATP levels after digestion were well maintained, and a greater number of islets were retrieved.     

UW与HTK保存液在肝脏移植术中临床效果对比的系统评价和meta分析

目的比较肝脏移植术中两种常用的器官保存液(UW液与HTK液)的临床效果。方法全面检索PubMed、Embase、Cochrane Library、中国期刊全文数据库、中国生物医学文献数据库、万方、维普等中英文数据库,纳入对比UW(UW液组)与HTK(HTK液组)两种保存液对移植肝脏保存效果的研究,提取资料并评价后用RevMan 5.3软件进行分析。结果最终纳入16篇文献共35 024例受者,meta分析结果显示,与UW液组比较,HTK液组的术后胆管并发症发生率[RR=1.30,95%CI(1.07,1.58),P=0.008]和术后7 d内天门冬氨酸氨基转移酶峰值[MD=112.45,95%CI(93.34,131.56),P<0.01]均较低,而术后移植肝原发性无功能发生率[RR=1.07,95%CI(0.52,2.18),P=0.86]、术后不同时间点移植肝和受者存活率(P>0.05)、术后再移植率[RR=0.83,95%CI(0.48,1.45),P=0.51]、急性排斥反应发生率[RR=1.27,95%CI(0.96,1.68),P=0.33]、7 d内丙氨酸氨基转移酶峰值[MD=31.79,95%CI(–161.84,225.42),P=0.75]、总胆红素水平[MD=19.42,95%CI(–10.83,49.67),P=0.21]、凝血酶原时间[MD=1.75,95%CI(0.01,3.49),P=0.838]等指标比较差异均无统计学意义。结论 HTK保存液对移植肝的保存安全且有效,具有与UW保存液相似的效果,关于二者对肝移植术后受者和移植肝远期存活率的影响仍需要大样本、高质量的随机对照试验研究来系统评价。     

Comparison of histidine-tryptophan-ketoglutarate solution and University of Wisconsin solution in adult liver transplantation

BACKGROUND: A safe and effective preservation solution is a precondition for successful orthotopic liver transplantation (OLT). This study compared University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate (HTK) solutions in OLT. PATIENTS AND METHODS: We retrospectively reviewed the medical records of 137 primary cadaveric. OLT performed between January 2003 and December 2006 at our institution. Sixty-eight grafts were harvested using UW and 69 using HTK. Recipients were managed similarly in regard to operative techniques and immunosuppression. We collected donor data including serum transaminases, serum sodium, ICU stay and assessed macroscopic liver quality. Recipient serum transaminases were collected on postoperative days 1, 7, 14, and 30. We compared biliary and vascular complications, as well as patient and graft survivals. RESULTS: Mean serum bilirubin levels were slightly higher in the HTK group at 1,7,14, and 30 days after transplantation, whereas transaminases were higher in the UW group. Primary nonfunction occurred in 1 patient in each group. Retransplantation was performed in 5 patients in the UW and in 9 patients in the HTK group. Biliary complication rates were similar in the UW and HTK groups (22% and 17%, respectively). Six arterial complications occurred in the HTK (8.7%) and 2 in the UW group (2.9%; P < .05). Mean follow-up was 25 months. Graft survival at 1, 12, and 36 months was 90%, 78%, and 75% versus 90%, 71%, and 71% in the UW versus HTK groups, respectively. One-, 12-, and 36-month patient survival rates were 93%, 78%, and 75% versus 93%, 78%, and 78% in the UW versus HTK groups, respectively. CONCLUSIONS: There were no significant differences in graft and patient survivals between the 2 groups. Whereas the biliary complication rates were comparable in both groups, the arterial complications were clearly higher in the UW group (8.7% vs 2.9%; P < .05%). UW and HTK solutions seemed to be equally safe and effective in the preservation of liver grafts. The high incidence of arterial complications in the UW group requires further prospective studies.     

A meta-analysis for comparison of the two-layer and university of Wisconsin pancreas preservation methods in islet transplantation

Conflicting results have been reported on the effectiveness of the two-layer method (TLM) compared with the University of Wisconsin (UW) method for preserving pancreata. The objective of this study was to compile the evidence for or against any difference in human islet yield and viability between these two. PubMed (January 2000 to May 2008) and Cochran Library searches were performed and 17 studies were included for the meta-analysis. Data on donor characteristics, preservation time, and outcomes were abstracted. Studies were subgrouped based on how TLM was used (UW + TLM or TLM alone), on mean cold ischemic time (CIT) (>20 h or <20 h), and on whether special chemical was used (yes or no). Meta-analysis of all studies and subgroups was performed and the pooled standardized mean differences (SMD) with 95% confidence intervals (CI) were reported. Overall, the use of TLM significantly increased islet yield [SMD, 0.74 (0.44-1.04)] and viability [SMD, 0.63 (0.14-1.12)]. The beneficial effects of TLM on islet yield were more evident when TLM was used following UW storage or when prolonged CIT was used. TLM used alone, shorter CIT, and no chemical use all resulted in similar islet viability between TLM and UW groups. Beneficial effects of TLM on islet viability were demonstrated only when TLM was used following UW storage, or with prolonged CIT, or with chemical use. In conclusion, the TLM was beneficial for prolonged pancreas preservation before human islet isolation; however, benefit of the TLM for short-term preservation was not clear.