Thomas M. Suszynski

Islet assessment for transplantation.

Purpose of reviewThere is a critical need for meaningful viability and potency assays that characterize islet preparations for release prior to clinical islet cell transplantation. Development, testing, and validation of such assays have been the subject of intense investigation for the last decade. These efforts are reviewed, highlighting the most recent results while focusing on the most promising assays. Recent findingsAssays based on membrane integrity do not reflect true viability when applied to either intact islets or dispersed islet cells. Assays requiring disaggregation of intact islets into individual cells for assessment introduce additional problems of cell damage and loss. Assays evaluating mitochondrial function, specifically mitochondrial membrane potential, bioenergetic status, and cellular oxygen consumption rate, especially when conducted with intact islets, appear most promising in evaluating their quality prior to islet cell transplantation. Prospective, quantitative assays based on measurements of oxygen consumption rate with intact islets have been developed, validated, and their results correlated with transplant outcomes in the diabetic nude mouse bioassay. ConclusionMore sensitive and reliable islet viability and potency tests have been recently developed and tested. Those evaluating mitochondrial function are most promising, correlate with transplant outcomes in mice, and are currently being evaluated in the clinical setting.

Pig Pancreas Anatomy: Implications for Pancreas Procurement, Preservation, and Islet Isolation

Background. Islet transplantation is emerging as a treatment option for selected patients with type 1 diabetes. The limited human islet supply from cadavers and poor islet yield and quality remain substantial impediments to progress in the field. Use of porcine islets holds great promise for large-scale application of islet transplantation. Consistent isolation of porcine islets is dependent on advances in pancreas procurement, pancreas preservation, and islet isolation, requiring detailed knowledge of the porcine pancreatic anatomy. The primary aim of this study was to describe the vascular and ductal anatomy of the porcine pancreas to guide and improve organ preservation and enzyme perfusion. Methods. Pancreata were removed by en bloc viscerectomy from 65 female Landrace pigs. Results. Fifteen percentage of organs exhibited inconsistent vascular branching from the celiac trunk. All organs showed uniform patterns of branching at the superior mesenteric artery. The superior and inferior mesenteric veins merged to become the portal vein in all but one case in which the inferior mesenteric vein drained into the splenic vein. Ninety-seven percent of pancreata had three lobes: duodenal lobe (DL), connecting lobe (CL), and splenic lobe (SL); 39% demonstrated ductal communication between the CL and the other two lobes; 50% had ductal communication only between the CL and duodenal lobe; and 11% presented other types of ductal delineation. Conclusions. Accounting for the variations in vascular and ductal anatomy, as detailed in this study, will facilitate development of protocols for preservation, optimal enzyme administration, and pancreas distention and digestion, and will ultimately lead to substantial improvements in isolation outcomes.

Ten-Year Outcome after Rapid Discontinuation of Prednisone in Adult Primary Kidney Transplantation

BACKGROUND AND OBJECTIVES Rapid discontinuation of prednisone after kidney transplantation potentially allows for minimization of steroid-related side effects. Although intermediate-term data with rapid discontinuation of prednisone have been promising, concern still exists regarding long-term outcomes. The 10-year experience is reported herein. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Between October 1, 1999 and December 31, 2010, 1241 adult primary kidney transplants (791 living donor and 450 deceased donor) were performed using a protocol in which prednisone is discontinued after postoperative day 5. The 10-year actuarial recipient and graft survival rates and prednisone-related side effects were studied. RESULTS Ten-year actuarial patient survival was 71% for living donor transplants and 62% for deceased donor transplants; 10-year graft survival was 61% for living donor transplants and 51% for deceased donor transplants, and was comparable to 10-year Scientific Registry of Transplant Recipients national data. Ten-year death-censored graft survival was 79% for living donor transplants and 80% for deceased donor transplants. Ten-year acute rejection rates were 25% for deceased donor transplants and 31% for living donor transplants; 10-year chronic rejection (interstitial fibrosis/tubular atrophy) rates were 39% for deceased donor transplants and 47% for living donor transplants. For nondiabetic recipients of living donor or deceased donor allografts, the incidence of new-onset diabetes was significantly lower than in historical controls on prednisone (P<0.001). We also found significantly reduced rates of cataracts, avascular necrosis, and cytomegalovirus infection in some subgroups. CONCLUSIONS Prednisone-related side effects can be minimized in a protocol incorporating rapid discontinuation of prednisone for maintenance immunosuppression. Ten-year patient and graft outcomes remain acceptable.

OXYGEN PERSUFFLATION INCREASES PANCREATIC ATP LEVELS AND VIABLE ISLET YIELD FOLLOWING 24 HOURS PRESERVATION COMPARED WITH THE TWO-LAYER METHOD (TLM): 1196

W.E. Scott III1, E.S. Avgoustiniatos2, J. Ferrer-Fabrega2, B.P. Weegman2, V.A. Kircner2, T. Anazawa3, M.D. Rizzari4, L.S. Kidder2, S.A. Stein2, S. Matsumoto2, J.J. Stone2, T.M. Suszynski5, T.C. Aasheim2, B.E. Hammer6, A.N. Balamurugan2, T.D. O’Brien7, M.P. Murtaugh8, L.A. Tempelman9, D.E. Sutherland2, B.J. Hering2, K.K. Papas2 1, , St. Paul/MN/UNITED STATES OF AMERICA, 2Surgery, University of Minnesota, Minneapolis/UNITED STATES OF AMERICA, 3Organ Regenerative Surgery, Fukushima Medical University, Fukushima/ Fukushima/JAPAN, 4Department Of Surgery, University of Minnesota, Minneapolis/Minnesota/UNITED STATES OF AMERICA, 5Department Of Surgery, University of Minnesota, Minneapolis/MN/UNITED STATES OF AMERICA, 6Radiology, University of Minnestoa, Minneapolis/MN/UNITED STATES OF AMERICA, 7Veterinary Population Medicine, University of Minnestoa, St. Paul/MN/UNITED STATES OF AMERICA, 8Veterinary Biosciences, University of Minnestoa, St. Paul/MN/UNITED STATES OF AMERICA, 9, Giner Inc., Newton/MA/UNITED STATES OF AMERICA

Pancreas Oxygen Persufflation Increases ATP Levels as Shown by Nuclear Magnetic Resonance

BACKGROUND Islet transplantation is a promising treatment for type 1 diabetes. Due to a shortage of suitable human pancreata, high cost, and the large dose of islets presently required for long-term diabetes reversal; it is important to maximize viable islet yield. Traditional methods of pancreas preservation have been identified as suboptimal due to insufficient oxygenation. Enhanced oxygen delivery is a key area of improvement. In this paper, we explored improved oxygen delivery by persufflation (PSF), ie, vascular gas perfusion. METHODS Human pancreata were obtained from brain-dead donors. Porcine pancreata were procured by en bloc viscerectomy from heparinized donation after cardiac death donors and were either preserved by either two-layer method (TLM) or PSF. Following procurement, organs were transported to a 1.5-T magnetic resonance (MR) system for (31)P nuclear magnetic resonance spectroscopy to investigate their bioenergetic status by measuring the ratio of adenosine triphosphate to inorganic phosphate (ATP:P(i)) and for assessing PSF homogeneity by MRI. RESULTS Human and porcine pancreata can be effectively preserved by PSF. MRI showed that pancreatic tissue was homogeneously filled with gas. TLM can effectively raise ATP:P(i) levels in rat pancreata but not in larger porcine pancreata. ATP:P(i) levels were almost undetectable in porcine organs preserved with TLM. When human or porcine organs were preserved by PSF, ATP:P(i) was elevated to levels similar to those observed in rat pancreata. CONCLUSION The methods developed for human and porcine pancreas PSF homogeneously deliver oxygen throughout the organ. This elevates ATP levels during preservation and may improve islet isolation outcomes while enabling the use of marginal donors, thus expanding the usable donor pool.

Persufflation (or Gaseous Oxygen Perfusion) as a Method of Organ Preservation

Improved preservation techniques have the potential to improve transplant outcomes by better maintaining donor organ quality and by making more organs available for allotransplantation. Persufflation, (PSF, gaseous oxygen perfusion) is potentially one such technique that has been studied for over a century in a variety of tissues, but has yet to gain wide acceptance for a number of reasons. A principal barrier is the perception that ex vivo PSF will cause in vivo embolization post-transplant. This review summarizes the extensive published work on heart, liver, kidney, small intestine and pancreas PSF, discusses the differences between anterograde and retrograde PSF, and between PSF and other conventional methods of organ preservation (static cold storage, hypothermic machine perfusion). Prospective implications of PSF within the broader field of organ transplantation, and in the specific application with pancreatic islet isolation and transplant are also discussed. Finally, key issues that need to be addressed before PSF becomes a more widely utilized preservation strategy are summarized and discussed.

The ATP/DNA ratio is a better indicator of islet cell viability than the ADP/ATP ratio.

Real-time, accurate assessment of islet viability is critical for avoiding transplantation of nontherapeutic preparations. Measurements of the intracellular ADP/ATP ratio have been recently proposed as useful prospective estimates of islet cell viability and potency. However, dead cells may be rapidly depleted of both ATP and ADP, which would render the ratio incapable of accounting for dead cells. Since the DNA of dead cells is expected to remain stable over prolonged periods of time (days), we hypothesized that use of the ATP/DNA ratio would take into account dead cells and may be a better indicator of islet cell viability than the ADP/ATP ratio. We tested this hypothesis using mixtures of healthy and lethally heat-treated (HT) rat insulinoma cells and human islets. Measurements of ATP/DNA and ADP/ATP from the known mixtures of healthy and HT cells and islets were used to evaluate how well these parameters correlated with viability. The results indicated that ATP and ADP were rapidly (within 1 hour) depleted in HT cells. The fraction of HT cells in a mixture correlated linearly with the ATP/DNA ratio, whereas the ADP/ADP ratio was highly scattered, remaining effectively unchanged. Despite similar limitations in both ADP/ADP and ATP/DNA ratios, in that ATP levels may fluctuate significantly and reversibly with metabolic stress, the results indicated that ATP/DNA was a better measure of islet viability than the ADP/ATP ratio.

Consideration of donor age and human leukocyte antigen matching in the setting of multiple potential living kidney donors.

Background. Defining living donor (LD)-related risk factors affecting kidney transplant outcome will allow better donor selection and more educated informed consent when there is more than one potential donor. We studied risk factors in a large cohort at a single institution. Methods. We reviewed 1632 recipients who underwent LD kidney transplantation at the University of Minnesota between January 1, 1990, and October 1, 2009. Using Cox regression, we studied the effect of donor and recipient risk factors on patient and graft survival. We specifically examined the effect of donor age and human leukocyte antigen (HLA) matching because these are variables that may help clinical decision making when multiple potential donors exist. Results. Mean donor age was 40.6 years for all transplants; 180 (11%) donors were 55 years or older, and 24 (1.5%) donors were older than 65 years. Mean number of HLA mismatches (per transplant) was 2.9 (29.2% of recipients had one to two HLA mismatches, 39.8% had three to four HLA mismatches, and 25% had five to six HLA mismatches). Donor age more than 65 years, five to six HLA mismatches, delayed graft function, and acute rejection were independent predictors of decreased patient and graft survival. When controlling for recipient age, donor age more than 65 years remained a risk factor for worse outcome. Conclusions. Our data suggest that advanced donor age (>65 years) and degree of HLA mismatch (≥5) are independent donor-related risk factors associated with worse outcome. When multiple potential LDs exist, it may be ideal to attempt to use a donor younger than 65 years and with less than five HLA mismatches.

Islet Oxygen Consumption Rate (OCR) Dose Predicts Insulin Independence in Clinical Islet Autotransplantation

Background Reliable in vitro islet quality assessment assays that can be performed routinely, prospectively, and are able to predict clinical transplant outcomes are needed. In this paper we present data on the utility of an assay based on cellular oxygen consumption rate (OCR) in predicting clinical islet autotransplant (IAT) insulin independence (II). IAT is an attractive model for evaluating characterization assays regarding their utility in predicting II due to an absence of confounding factors such as immune rejection and immunosuppressant toxicity. Methods Membrane integrity staining (FDA/PI), OCR normalized to DNA (OCR/DNA), islet equivalent (IE) and OCR (viable IE) normalized to recipient body weight (IE dose and OCR dose), and OCR/DNA normalized to islet size index (ISI) were used to characterize autoislet preparations (n = 35). Correlation between pre-IAT islet product characteristics and II was determined using receiver operating characteristic analysis. Results Preparations that resulted in II had significantly higher OCR dose and IE dose (p<0.001). These islet characterization methods were highly correlated with II at 6–12 months post-IAT (area-under-the-curve (AUC) = 0.94 for IE dose and 0.96 for OCR dose). FDA/PI (AUC = 0.49) and OCR/DNA (AUC = 0.58) did not correlate with II. OCR/DNA/ISI may have some utility in predicting outcome (AUC = 0.72). Conclusions Commonly used assays to determine whether a clinical islet preparation is of high quality prior to transplantation are greatly lacking in sensitivity and specificity. While IE dose is highly predictive, it does not take into account islet cell quality. OCR dose, which takes into consideration both islet cell quality and quantity, may enable a more accurate and prospective evaluation of clinical islet preparations.

Islet size index as a predictor of outcomes in clinical islet autotransplantation.

Background The islet size distribution in a preparation may contribute to islet transplant outcomes. At the same islet equivalent (IE) dose, larger islets may exhibit poorer therapeutic value and this may be because of oxygen diffusion limitations that worsen in proportion to islet size. Methods To test this hypothesis, we studied the impact of islet size index (ISI) and other islet product characteristics on outcomes after islet autotransplant (IAT) in recipients receiving a marginal islet dose (2000–4999 IEs per kg body weight) from January 1, 2009 to June 11, 2012, at the University of Minnesota (n=58). ISI was defined as the number of IE divided by the number of islet particles (IPs) in a preparation; an ISI less than 1 indicates a mean islet diameter that is less than 150 &mgr;m. The primary post-IAT outcome was 6-month insulin use status. Results Logistic regression analysis indicate that IPs/kg (P=0.001), IEs/kg (P=0.019), total IPs transplanted (P=0.040), and ISI (P=0.074) were most strongly correlated with the primary outcome. The ISI (mean±standard error) was lower for recipients achieving insulin independence at 6 months (0.71±0.05) versus those partially (0.83±0.05) or completely (1.00±0.07) insulin dependent. The combination of islet dose (expressed as units IPs/kg) and ISI exhibited a sensitivity of 75% and specificity of 74% in predicting insulin independence in this population of patients. Conclusion Islet autotransplant recipients of a marginal islet doses were more likely to achieve insulin independence when transplanted with a greater number of smaller islets.