Failure mode and effect analysis in human islet isolation: from the theoretical to the practical risk.

This study aimed to assess the global mapping risk of human islet isolation, using a failure mode and effect analysis (FMEA), and highlight the impact of quality assurance procedures on the risk level of criticality. Risks were scored using the risk priority number (RPN) scoring method. The risk level of criticality was made based on RPN and led to risk classification (low to critical). A raw risk analysis and a risk control analysis (with control means and quality assurance performance) were undertaken. The process of human islet isolation was divided into 11 steps, and 230 risks were identified. Analysis of the highest RPN of each of the 11 steps showed that the 4 highest risks were related to the pancreas digestion and islet purification stages. After implementation of reduction measures and controls, critical and severe risks were reduced by 3-fold and by 2-fold, respectively, so that 90% of risks could be considered as low to moderate. FMEA has proven to be a powerful approach for the identification of weaknesses in the islet isolation processes. The results demonstrated the importance of staff qualification and continuous training and supported the contribution of the quality assurance system to risk reduction.

Impact of ischemia time on islet isolation success and posttransplantation outcomes: A retrospective study of 452 pancreas isolations.

Many variables impact islet isolation, including pancreas ischemia time. The ischemia time upper limit that should be respected to avoid a negative impact on the isolation outcome is not well defined. We have performed a retrospective analysis of all islet isolations in our center between 2008 and 2018. Total ischemia time, cold ischemia time, and organ removal time were analyzed. Isolation success was defined as an islet yield ≥200 000 IEQ. Of the 452 pancreases included, 288 (64%) were successfully isolated. Probability of isolation success showed a significant decrease after 8 hours of total ischemia time, 7 hours of cold ischemia time, and 80 minutes of organ removal time. Although we observed an impact of ischemia time on islet yield, a probability of isolation success of 50% was still present even when total ischemia time exceeds 12 hours. Posttransplantation clinical outcomes were assessed in 32 recipients and no significant difference was found regardless of ischemia time. These data indicate that although shorter ischemia times are associated with better islet isolation outcomes, total ischemia time >12 hours can provide excellent results in appropriately selected donors.

Combined Pancreatic Islet-Lung-Liver Transplantation in a Pediatric Patient with Cystic Fibrosis-Related Diabetes.

BACKGROUND: Cystic fibrosis-related diabetes (CFRD) is the most frequent extrapulmonary complication of cystic fibrosis (CF). METHODS: We report the first combined pancreatic islet-lung-liver transplantation in a 14-year-old adolescent. CFTR was analyzed by Sanger sequencing. Further genes were analyzed by high-throughput sequencing. RESULTS: The patient was diagnosed with CF at the age of 14 months. Nine years later, after diagnosis of CFRD, the patient's BMI and lung function began to decline. Bilateral lung transplantation with simultaneous liver transplantation was performed at the age of 14.5 years. The first islet transplantation (IT) was carried out 10 days later. Six months later, C-peptide secretion after arginine stimulation showed peak values of 371 pmol/L (vs. 569 pmol/L before IT) and insulin doses had slightly increased (1.40 vs. 1.11 units/kg/day before IT). A second IT was performed at the age of 15 years, a third at 16 years. Two years after the first IT, arginine-stimulated C-peptide secretion increased to 2,956 pmol/L and insulin doses could be reduced to 0.82 units/kg/day. HbA1c decreased from 7.3% (57.4 mmol/mol) to 5.9% (41.0 mmol/mol). CONCLUSION: IT following lung and liver transplantation, with injection of islets into a transplanted organ, is feasible. It improves C-peptide secretion, decreases insulin needs, and lowers HbA1c.

Successful pregnancy and delivery after simultaneous islet-kidney transplantation.

Allogeneic islet of Langerhans transplantation is a recognized beta-cell replacement therapy for patients affected by type 1 diabetes mellitus. Type 1 diabetes mellitus is a condition associated with an increased risk of adverse outcomes for pregnant women and fetuses. We report the case of a 29-year-old woman with type 1 diabetes mellitus, who underwent successful allogeneic islet transplantation with simultaneous kidney transplantation. She achieved durable insulin independence after 2 islet infusions. Pregnancy was desired and planned 2 years after the last islet infusion. Multidisciplinary monitoring of pregnancy was carried out and the immunosuppressive regimen was adapted. Euglycemia was maintained throughout pregnancy without the need for exogenous insulin. After an uneventful pregnancy, she delivered on term an otherwise healthy male child with imperforate anus that was immediately surgically corrected. In conclusion, allogeneic islet transplantation is a suitable treatment for women of childbearing age with complicated type 1 diabetes mellitus, allowing physiologic glycemic control during pregnancy with a low risk of graft loss. This target can be achieved only by a tight multidisciplinary follow-up, including immunosuppressive therapy adaptation and adequate diabetes and obstetrical monitoring.

Pancreas preservation fluid microbial contamination is associated with poor islet isolation outcomes - a multi-centre cohort study.

The microbiological safety of islet preparations is paramount. Preservation medium contamination is frequent, and its impact on islet yield and function remains unclear. Microbiological samples collected during islet isolations from 2006 to 2016 were analyzed and correlated to isolation and allo- and autotransplantation outcomes. Microbial contamination of preservation medium was found in 64.4% of processed donor pancreases (291/452). We identified 464 microorganisms including Staphylococcus (253/464, 54.5%), Streptococcus (31/464, 6.7%), and Candida species (25/464, 5.4%). Microbial contamination was associated with longer warm and cold ischemia times and lower numbers of postpurification islet equivalents, purity, transplant rate, and stimulation index (all P < 0.05). Six percent of the preparations accepted for transplantation showed microbial contamination after isolation (12/200); 9 of 12 were Candida species. Six patients were transplanted with a sample with late microbial growth discovered after the infusion. Insulin independence rate was not affected. This risk of transplanting a contaminated islets preparation was reduced by half following the implementation of an additional sampling after 24 h of islet culture. Pancreas preservation fluid microbial contamination is associated with lower transplant rate and poorer in vitro function, but not with changes in graft survival. Culture medium testing 1 day after isolation reduces the risk of incidental transplantation with contaminated islets.

Alloimmune Monitoring After Islet Transplantation: A Prospective Multicenter Assessment of 25 Recipients.

Islet transplantation is an effective treatment for selected patients with type 1 diabetes. However, an accurate test still lacks for the early detection of graft rejection. Blood samples were prospectively collected in four university centers (Geneva, Grenoble, Montpellier, and Strasbourg). Peripheral blood mononuclear cells were stimulated with donor splenocytes in the presence of interleukin-2. After 24 h of incubation, interferon- (IFN-) ELISpot analysis was performed. After a total of 5 days of incubation, cell proliferation was assessed by fluorescence-activated cell sorting (FACS) analysis for Ki-67. Immunological events were correlated with adverse metabolic events determined by loss of 1 point of -score and/or an increased insulin intake 10%. Twenty-five patients were analyzed; 14 were recipients of islets alone, and 11 combined with kidney. Overall, 76% (19/25) reached insulin independence at one point during a mean follow-up of 30.7 months. IFN- ELISpot showed no detectable correlation with adverse metabolic events [area under the curve (AUC)=0.57]. Similarly, cell proliferation analysis showed no detectable correlation with adverse metabolic events (CD3+/CD4+ AUC=0.54; CD3+/CD8+ AUC=0.55; CD3/CD56+ AUC=0.50). CD3/CD56+ cell proliferation was significantly higher in patients with combined kidney transplantation versus islet alone (6 months, p=0.010; 12 months, p=0.016; and 24 months, p=0.018). Donor antigen-stimulated IFN- production and cell proliferation do not predict adverse metabolic events after islet transplantation. This suggests that the volume of transplanted islets is too small to produce a detectable systemic immune response and/or that alloimmune rejection is not the sole reason for the loss of islet graft function.

Low risk of anti-human leukocyte antigen antibody sensitization after combined kidney and islet transplantation.

Anti-human leukocyte antigen (HLA) antibody could lead to humoral rejection and a decrease in graft survival after kidney transplantation. A recent report has suggested that islet transplantation alone is associated with a high rate of sensitization. The withdrawal of the immunosuppressive therapy because of the progressive nonfunction of the islets could explain the high rate of sensitization. Because the specific risk of immunization of multiple islet infusions remains unknown, we studied the immunization rate in our cohort of multiple islet infusions transplant recipients. De novo anti-HLA antibodies were analyzed in 37 patients after islets alone (n=8), islet-after-kidney (n=13), and simultaneous islet-kidney (n=16) transplantation by solid phase assays over time. The rate of immunization was 10.8% that is comparable with the risk of immunization after kidney transplantation alone. Multiple islet infusions do not represent a specific risk for the development of anti-HLA antibodies after combined kidney-islets transplantation.

Impairment of renal function after islet transplant alone or islet-after-kidney transplantation using a sirolimus/tacrolimus-based immunosuppressive regimen.

The immunosuppressive (IS) regimen based on sirolimus/low-dose tacrolimus is considered a major determinant of success of the Edmonton protocol. This regimen is generally considered safe or even protective for the kidney. Herein, we analyzed the impact of the sirolimus/low-dose tacrolimus combination on kidney function. The medical charts of islet transplant recipients with at least 6 months follow up were reviewed. There were five islet-after-kidney and five islet transplantation alone patients. Serum creatinin, albuminuria, metabolic control markers and graft function were analyzed. Impairment of kidney function was observed in six of 10 patients. Neither metabolic markers nor IS drugs levels were significantly associated with the decrease of kidney function. Although a specific etiology was not identified, some subsets of patients presented a higher risk for decline of kidney function. Low creatinin clearance, albuminuria and long-established kidney graft were associated with poorer outcome.

Transplantation of discordant xenogeneic islets using repeated therapy with anti-CD154.

BACKGROUND: Costimulatory blockade has been shown to allow long-term survival of xenogeneic islets. The aim of the present study was to analyze the possibility of xenogeneic islet retransplantation using costimulatory blockade. METHODS: Streptozotocin-induced diabetic C57/BL6 mice were transplanted with 1000 human islet equivalents. After 14 days, mice were nephrectomized (graftectomy) and retransplanted with human leukocyte antigen (HLA)-mismatched human islets under contralateral kidney capsule. Four groups were performed: I: all transplants (Tx) without MR1; II: first Tx without MR1, second Tx with MR1; III: first Tx with MR1, second Tx without MR1; and IV: all Tx with MR1. Recipient serums were analyzed by cross-match for serum-mediated cytotoxicity against human lymphocytes and islets. RESULTS: In group I, the second graft rejection was accelerated (graft survival, 5 +/- 3 days) compared with the first graft without MR1 (13 +/- 7 days). In groups II and III, second graft survivals were 16 +/-1 3 and 62 +/- 15 days, respectively. In group IV, second graft function was maintained for >100 days. Pretransplant cross-matches were all negative. Post-second Tx cross-matches were positive in groups I and II and negative in group IV. In group III, post-second Tx cross-matches were negative only for cells with HLA molecules present in the first donor. CONCLUSIONS: MR1 was unable to induce tolerance after sensitization. MR1 given at the first Tx only allowed prolonged survival of the second Tx, but rejection still occurred with development of antibodies against molecules not present on first donor cells, indicating that costimulatory blockade does not induce linked-suppression against species-specific antigens of xenografts but can induce donor-specific unresponsiveness. MR1 given for all sequential transplantation allowed long-term regraft survival and prevented occurrence of antidonor antibodies.

Logistics and transplant coordination activity in the GRAGIL Swiss-French multicenter network of islet transplantation.

BACKGROUND: Since the Edmonton trial in 2000, increasing numbers of transplant centers have been implementing islet transplantation programs. Some institutions have elected to associate in multicenter networks, such as the Swiss-French GRAGIL (Groupe Rhin-Rhône-Alpes-Genève pour la Transplantation d'Ilots de Langerhans) consortium. METHODS: All pancreata offers to the University of Geneva Cell Isolation and Transplantation Center from within the network in 2002 and 2003 were reviewed. Islet preparations were attributed to the most suitable recipient on a centrally managed waiting list. All shipments were performed by ambulance in less than 5 hr. RESULTS: Over the period of study, 260 pancreata were offered, from a total of 1,304 cadaveric donors in the four allocation regions (20%). Fifty-two patients were on the waiting list at any time during this 2-year period. The percentage of organs offered varied in the range of 0.5% to 42%, depending on region of origin, with a correlation with number of patients on the waiting list in each region. Of these, 104 (40%) were accepted for processing. Ninety-two pancreata were actually processed, resulting in 42 islet preparations being transplanted. The number of international equivalents of transplanted preparations was 378,500+/-16,000 versus 165,400+/-15,400 (P<0.0001) for nontransplanted preparations. Total cold ischemia time was 6+/-0.3 hr for transplanted preparations versus 6.7+/-0.4 hr for nontransplanted preparations (not significant). CONCLUSIONS.: A high rate of pancreas offers, successful isolation, and islet transplantation can be achieved in multicenter networks such as GRAGIL. Such an approach can expand both the donor pool and the recipient population.

Positron-emission tomography imaging of early events after transplantation of islets of Langerhans.

The aim of our study was to assess cell trafficking and early events after intraportal islet transplantation. Sprague-Dawley rat islets were incubated for various times, with various concentrations of 2-[F]fluoro-2deoxy-D-glucose (FDG), and in presence of various glucose concentrations. FDG-labeled syngeneic islets or FDG alone were injected in rats. Radioactivity was measured in the liver and in various organs by positron-emission tomography for 6 hours. FDG uptake increased with incubation time or FDG concentration and decreased in presence of glucose. In vivo, all islets implanted in the liver, with an uptake 4.4 times higher than controls (44.2% vs. 10.1%, P=0.02). Radioactivity in the liver decreased at the same rate after injection of labeled-islets and FDG alone. Ex vivo labeling of islets and imaging of posttransplant early events were feasible. Islets engrafted exclusively in the liver. No islet loss could be demonstrated 6 hours after transplantation.