Garth L. Warnock

Islet isolation assessment in man and large animals

SummaryRecent progress in islet isolation from the pancreas of large mammals including man, accentuated the need for the development of precise and reproducible techniques to assess islet yield. In this report both quantitative and qualitative criteria for islet isolation assessment were discussed, the main topics being the determination of number, volume, purity, morphologic integrity andin vitro andin vivo function tests of the final islet preparations. It has been recommended that dithizone should be used as a specific stain for immediate detection of islet tissue making it possible to estimate both the total number of islets (dividing them into classes of 50 µ diameter range increments) and the purity of the final preparation. Appropriate morphological assessment should include confirmation of islet identification, assessment of the morphological integrity and of the purity of the islet preparation. The use of fluorometric inclusion and exclusion dyes together have been suggested as a viability assay to simultaneously quantitate the proportion of cells that are intact or damaged. Perifusion of islets with glucose provides a dynamic profile of glucose-mediated insulin release and of the ability of the cells to down regulate insulin secretion after the glycemic challenge is interrupted. Although perifusion data provides a useful guide to islet viability the quantity and kinetics of insulin release do not necessarily predict islet performance after implantation. Therefore, the ultimate test of islet viability is their function after transplantation into a diabetic recipient. For this reason,in vivo models of transplantation of an aliquot of the final islet preparation into diabetic nude (athymic) rodents have been suggested. We hope that these general guidelines will be of assistance to standardize the assessment of islet isolations, making it possible to better interpret and compare procedures from different centers.

Variables in organ donors that affect the recovery of human islets of Langerhans.

In an attempt to reduce the variability in the yields of human islets isolations and to identify donor factors that were potentially deleterious, we retrospectively reviewed 153 human islets isolations in our center over a 3-year period. Isolations were performed using controlled collagenase perfusion via the duct, automated dissociation, and Ficoll purification. Factors leading to successful isolations (recovery of >100,000 islet equivalents at a purity >50%) were analyzed retrospectively using univariate and multivariate analysis. Critical factors in the multiorgan cadaveric donors that were identified using univariate analysis included donor age (P<0.01), body mass index (BMI)(P<0.01), cause of death (P<0.01), and prolonged hypotensive episodes (systolic blood pressure <90 mmHg or mean arterial pressure <60 mmHg for > 15 min) requiring high vasopressors (>15 microgram/kg/min dopamine or >5 microgram/kg/min Levophed) (P>0.01). Independent analysis of 19 donor variables using multivariate logistic stepwise regression showed six factors were statistically significant. Odds ratio (OR) showed that donor age (OR 1.1, P<0.01), local procurement team (OR 10.9, P<0.01), and high BMI (OR 1.4, P<0.01) had a positive correlation with islet recovery. In contrast, hyperglycemia (all blood glucose >10 mmol/L) (OR 0.63, P<0.01), frequency and duration of cardiac arrest (OR 0.7, P<0.01), and increased duration of cold storage before islet isolation (OR 0.83, P<0.01) had negative correlation. Using these combinations of factors, the prediction of success was 85% accurate. By donor age, success was 13% for 2.5- to 18-year-old donors (n=23), 37% for 19- to 28-year-old donors (n=30), 65% for 29- to 50-year-old donors (n=70), and 83% for 51- to 65-year-old (n=29) donors. However, when vitro function was assessed by perifusion, the insulin secretory capabilities of islets isolated from the >50-year-old donor group was significantly reduced as compared with the 2.5- to 18-year-old group (P<0.02). Multiple regression analysis using postdigestion and postpurification islet recovery as outcome variables identified BMI, procurement team, pancreas weight, and collagenase digestion time factors tht can affect the recovery of human islets. Locally procured pancreases and donors with elevated minimum blood glucose levels were identified as factors that affect the insulin secretory capabilities of the isolated islets. This review of parameters suggests an improved approach to the prediction of successful islet isolation from human pancreases. Selection of suitable pancreases for processing may improve consistency in human islet isolation and thereby decrease costs.

Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion.

Effective intraductal delivery of the enzyme collagenase into the pancreas is crucial to the subsequent ability to isolate viable islets. Most clinical islet transplant centers load the enzyme into the pancreas by retrograde injection using a syringe following cannulation of the pancreatic duct. An alternative approach is to perfuse the pancreas via the pancreatic duct with collagenase solution using a recirculating perfusion device system. This provides control over perfusion pressures and collagenase temperature. This study reports on our evaluation of the delivery of Liberase™-HI into the pancreas of 14 consecutive adult multiorgan cadaveric donors. Alternate glands were procured and processed using an identical protocol with the exception of collagenase delivery. The first group of pancreases was loaded using the perfusion technique where cold (4°C) Liberase™-HI was perfused at 80 mmHg for 5 min after which the pressure was increased to 180 mmHg. The collagenase solution was then slowly warmed to 35°C, transferred to the dissociation chamber and mechanically dissociated, and then purified using discontinuous gradients of Ficoll. Pancreases in the second group were loaded with collagenase (28–32°C) using the syringe technique before mechanical dissociation and purification. There were no significant differences in pancreas cold ischemia, donor age, body mass index, maximum blood glucose, or serum amylase of the donors between the two groups. Mean collagenase digestion time in the digestion chamber was not different between the two groups; however, the amount of undigested tissue remaining after dissociation was significantly higher in the syringe-loaded group (15.3 ± 2.6 g vs. 4.6 ±2.1 g, mean ± SEM, p < 0.05). Postdigestion recovery of islets was 471 ± 83 × 103 IE in the perfusion group compared with 391 ± 57 × 103 IE for the syringe-loaded group. Postpurification recovery was higher in the perfused group (379 ± 45 vs. 251 ± 28 × 103 IE, p < 0.05, two-tailed paired t-test). No difference in in vitro islet viability was observed between the two groups following glucose perifusion with the calculated stimulation index of 4.6 ± 0.6 for the perfusion group and 4.2 ± 0.7 for the syringe-loaded group. Controlled perfusion via the pancreatic duct allows the effective delivery of the enzyme achieving maximal distension to all regions of the pancreas leading to an increased recovery of the islets with no detrimental effect on subsequent in vitro islet function.

Long-term follow-up after transplantation of insulin-producing pancreatic islets into patients with Type 1 (insulin-dependent) diabetes mellitus

SummaryPurified human islets and a kidney from the same donor were transplanted into four patients with Type 1 (insulin-dependent) diabetes mellitus. Two of the patients received additional islets that were isolated from multiple donors, cryopreserved, and stored in a tissue bank. The islets were embolized into the liver via the portal vein. Immunosuppression was induced with antilymphocyte globulin and maintained with azathioprine, prednisone and cyclosporine. In the first two patients, fasting serum C-peptide rose to levels of 0.5–2.0 ng/ml during the first 4–8 weeks and mixed meal feeding elicited increases to 2–3 ng/ml. C-peptide secretion persisted for 8 months, but at progressively lower levels and insulin therapy could not be withdrawn. In the next two patients who received cryopreserved islets in addition to fresh islets, serum C-peptide levels (fasting/post-meal) rose to 4–7 ng/ml and serum glucose was more stable, allowing withdrawal of insulin therapy after 69 days in one patient, and reduced insulin doses in the other. The insulin-independent patient has maintained normal fasting glucose, glycosylated haemoglobin, and oral glucose tolerance at 1 year following cessation of daily insulin therapy. Episodes of renal graft rejection occurred in three patients, including the insulin-independent patient. High-dose steroid therapy reversed the rejection in all instances, with apparent preservation of C-peptide secretion. These data show that transplantation of purified freshly-prepared and cryopreserved islets into Type 1 diabetic patients results in prolonged insulin secretion, and that sufficient function could be provided in one patient to sustain euglycaemia in the absence of insulin therapy at 1 year of follow-up.

Normoglycaemia after transplantation of freshly isolated and cryopreserved pancreatic islets in Type 1 (insulin-dependent) diabetes mellitus

SummaryPurified islets of Langerhans and a kidney were transplanted into a 36-year-old patient who suffeded from renal failure secondary to a 25 year history of Type 1 (insulin-dependent) diabetes mellitus. The islet graft contained 243 000 fresh islets (mean islet diameter 150 μm) that were syngeneic with the kidney fraft and 368 000 cryopreserved islets that had been collected from four other donors. The total of 10 000 islets/kg body weight was infused into the liver via the umbilical vein. Immunosupperession was induced with antilymphocyte globulin and maintained with prednisone, cyclosporine and azathioprine. Serum C-peptide levels (ng/ml) during fasting and after standard mixed metal feeding (Sustacal) were <0.12 preoperatively. Postoperatively, insulin secretion was restored: fasting C-peptide rose during the first 4 weeks to levels of 4 to 5 and Sustacal elicited a further rise to 6 to 7. Transplant renal function was stable. Dialy fasting glucose (mmol/l, mean±SD) was 5.6±1 and 5.3±0.6 during the first and second months respectively and post-Sustacal glucose was 5.7+-0.8. Exogenous insulin therapy was progressively withdrawn and stopped duting the ninth week. Thereafter, fasting glucose was 4.7+-0.5, 24 h mean glucose was 6.6+-0.5, and normoglycaemia was maintained after Sustacal. These data show that this mass of freshly isolated and cryopreserved islets from multiple donors provided sustained function (3 months) that reversed insulin-dependence in an immunosuppressed Type 1 diabetic patient treated with simultaneous islet-kedney transplantation.

Does volume of clinical experience affect performance of clinical clerks on surgery exit examinations

BACKGROUNDnControversy persists over the educational value of student clerkship clinical activities.nnnMETHODSnStudents (109) from the class of 1995 recorded their clinical experiences in a logbook during their surgical clerkship at one of four affiliated teaching hospitals. The influence of clinical experience on examination scores and on correlations between prerotation and postrotation examination performance was determined.nnnRESULTSnBetween sites, marked variation in clinical experience was observed but postrotation scores were similar. High-volume experience in emergency admissions and feedback was associated with better objective structured clinical examination (OSCE) performance, but high-volume outpatient clinic experience was associated with less satisfactory OSCE performance. Correlations between prerotation examination performance and the OSCE was increased by feedback on emergency and elective admissions, in a positive and negative direction, respectively.nnnCONCLUSIONSnThese data show that surgical clerks clinical skills were enhanced by an increased volume of some but not all clinical experiences and that feedback does not necessarily enhance performance. These data suggest that both the volume of clinical experience and the quality of feedback should be carefully monitored by surgical clerkship directors.

Cryopreservation of insulin-producing tissue in rats and dogs

Transplantation of frozen-thawed islets of Langerhans normalized urine volume, urine glucose, plasma glucose, and weight gain in diabetic rats. However, these animals had abnormal intravenous glucose tolerance tests (ivGTT) at 3 mo post-transplant. Modified cryopreservation protocols were extended to canine islet-containing tissue: a higher temperature was used during equilibration in the 2 M dimethyl sulfoxide. Recipients of frozen-thawed isografts had prolonged normoglycemia with plasma glucose <105 mg/100 ml for up to 18 mo. During ivGTT at 6 mo post-transplant, K values (decline in glucose concentration, %/min) were similar in dogs autotransplanted with fresh (K=1.22±0.37) or cryopreserved tissue (K=1.8±0.4). Because of the similar compactness of the pancreas in humans and dogs, this canine model should be useful for formulating an optimal cryopreservation technique for human islets. La transplantation dîlots de Langerhans conservés par congélation normalise la diurèse, la glycosurie, la glycémie et la croissance pondérale des rats diabétiques. Cependant ces animaux ont une courbe dhyperglycémie provoquée par voie veineuse (HGPV) anormale 3 mois après la greffe. Des modifications ont été apportées aux procédés de conservation pour leur adaptation aux îlots de chien: une température plus élevée est utilisée pour léquilibration dans le diméthylsulfoxide 2M. Les receveurs de greffons isogéniques conservés congelés ont une normoglycémie prolongée avec une glycémie inférieure à 105 mg/100 ml pendant un temps allant jusquà 18 mois. Les tests dhyperglycémie provoquée intraveineuse 6 mois après la greffe donnent des valeurs de K (baisse de la glycémie, en % par min.) similaires chez les chiens autotransplantés avec des îlots frais (K=1.22±0.37) ou conservés congelés (K=1.8±0.4). Le tissu pancréatique du chien étant aussi compact que celui de lhomme, ce modèle canin devrait aider à la mise au point dune méthode optimale pour la conservation des îlots humains. El transplante de islotes de Langerhans congelados-descongelados resulta en la normalización del volumen urinario, de la glucosa urinaria, de la glucosa plasmática y de la ganancia de peso en ratas diabéticas. Sin embargo, estos animales exhiben resultados anormales en las pruebas intravenosas de tolerancia a la glucosa 3 meses después del transplante. Protocolos de criopreservación modificados han sido utilizados con tejido insular canino, usando una mayor temperatura durante el equilibrio en el 2M dimetilsulfóxido. Los animales recipientes de isotransplantes congelados-descongelados exhibieron normoglicemia prolongada, con valores de glucosa plasmática <105 mg/100 ml hasta por 18 meses. En las pruebas intravenosas de tolerancia a la glucosa realizadas 6 meses después del transplante, los valores K (declinación de la concentración de glucosa, %/min) fueron similares autotrans-plantados con tejido fresco (K=1.22±0.37) o con tejido criopreservado (K=1.8±0.4). En razón de la similitud en la densidad del páncreas humano y del páncreas canino, este modelo canino puede ser útil en la formulación de la técnica óptima de criopreservación de islotes humanos.Transplantation of frozen-thawed islets of Langerhans normalized urine volume, urine glucose, plasma glucose, and weight gain in diabetic rats. However, these animals had abnormal intravenous glucose tolerance tests (ivGTT) at 3 mo post-transplant. Modified cryopreservation protocols were extended to canine islet-containing tissue: a higher temperature was used during equilibration in the 2 M dimethyl sulfoxide. Recipients of frozen-thawed isografts had prolonged normoglycemia with plasma glucose <105 mg/100 ml for up to 18 mo. During ivGTT at 6 mo post-transplant, K values (decline in glucose concentration, %/min) were similar in dogs autotransplanted with fresh (K=1.22±0.37) or cryopreserved tissue (K=1.8±0.4). Because of the similar compactness of the pancreas in humans and dogs, this canine model should be useful for formulating an optimal cryopreservation technique for human islets.RésuméLa transplantation dîlots de Langerhans conservés par congélation normalise la diurèse, la glycosurie, la glycémie et la croissance pondérale des rats diabétiques. Cependant ces animaux ont une courbe dhyperglycémie provoquée par voie veineuse (HGPV) anormale 3 mois après la greffe. Des modifications ont été apportées aux procédés de conservation pour leur adaptation aux îlots de chien: une température plus élevée est utilisée pour léquilibration dans le diméthylsulfoxide 2M. Les receveurs de greffons isogéniques conservés congelés ont une normoglycémie prolongée avec une glycémie inférieure à 105 mg/100 ml pendant un temps allant jusquà 18 mois. Les tests dhyperglycémie provoquée intraveineuse 6 mois après la greffe donnent des valeurs de K (baisse de la glycémie, en % par min.) similaires chez les chiens autotransplantés avec des îlots frais (K=1.22±0.37) ou conservés congelés (K=1.8±0.4). Le tissu pancréatique du chien étant aussi compact que celui de lhomme, ce modèle canin devrait aider à la mise au point dune méthode optimale pour la conservation des îlots humains.ResumenEl transplante de islotes de Langerhans congelados-descongelados resulta en la normalización del volumen urinario, de la glucosa urinaria, de la glucosa plasmática y de la ganancia de peso en ratas diabéticas. Sin embargo, estos animales exhiben resultados anormales en las pruebas intravenosas de tolerancia a la glucosa 3 meses después del transplante. Protocolos de criopreservación modificados han sido utilizados con tejido insular canino, usando una mayor temperatura durante el equilibrio en el 2M dimetilsulfóxido. Los animales recipientes de isotransplantes congelados-descongelados exhibieron normoglicemia prolongada, con valores de glucosa plasmática <105 mg/100 ml hasta por 18 meses. En las pruebas intravenosas de tolerancia a la glucosa realizadas 6 meses después del transplante, los valores K (declinación de la concentración de glucosa, %/min) fueron similares autotrans-plantados con tejido fresco (K=1.22±0.37) o con tejido criopreservado (K=1.8±0.4). En razón de la similitud en la densidad del páncreas humano y del páncreas canino, este modelo canino puede ser útil en la formulación de la técnica óptima de criopreservación de islotes humanos.

Bacteremia due to transplantation of contaminated cryopreserved pancreatic islets.

Objective To report two cases of pancreatic islet transplantation-related septicemia, and the results of an investigative protocol to identify potential sources of contamination. Design Case series. Setting University hospital clinical investigational islet transplantation program. Results The last two of our first seven islet transplantation recipients developed Enterobacter cloacae septicemia within hours of islet infusion. Both had received thawed cryopreserved islet infusions. No source of infection apart from islets could be identified. Pancreas harvesting and islet isolation protocols provided multiple opportunities for contamination. Environmental cultures during a mock islet isolation procedure failed to identify a source of Enterobacter. Previously cryopreserved islet lots were thawed and submitted for culture, 14/47 grew micro-organisms including E. cloacae in four instances. Following revision of protocols for aseptic handling of islets during processing and cryopreservation 55 consecutive pancreata undergoing processing were evaluated; 7 grew micro-organisms on arrival and in 3 cases these persisted through to cryopreservation. Conclusion Two of seven islet transplantation recipients developed septicemia, likely related to infusion of contaminated cryopreserved islets. Using existing technology, for isolating islets from donor pancreata, recipients will remain at risk for this complication. Prevention should entail strict adherence to aseptic technique, and, possibly, use of surveillance microbial cultures during the islet isolation process.

Viable Purified Islets of Langerhans From Collagenase-Perfused Human Pancreas

Human islets of Langerhans were isolated with the principles of collagenase perfusion via the pancreatic duct and gentle dissociation of tissue. The number of islets released was 161 × 103, distributed as 76 × 103large (>100-μm) and 85 × 103 small (<100-μm) islets. Recovery after Ficoll-gradient purification was 61% for the large islets and 42% for the small islets. The final islet volume was 240 μl, with purity of 70–90% (large islets) and.20–40% (small islets). Perifusion with glucose elicited a biphasic release of insulin, with the response rising sixfold from basal secretion. Implantation of pure islets under the kidney capsule of normal or streptozocin-induced diabetic nude mice resulted in human C-peptide secretion and partial or complete reversal of hyperglycemia, confirmed by histological recovery. The data show that these methods provide large quantities of viable purified human islets.

Natural human antibody-mediated destruction of porcine neonatal islet cell grafts

Abstract: Porcine pancreata may be considered a potential source of islets for transplantation into diabetic recipients; however, whether porcine islet grafts will be susceptible to damage by natural antibody‐mediated hyperacute rejection remains unknown. In this study, we performed Western blots to determine whether membrane proteins present on porcine neonatal islet cells (NIC) are recognized by xenoreactive antibodies present in human sera. Western blots of freshly isolated porcine NICs with AB sera detected the presence of 14 antigens (MW 24–164 kDa) and 4 antigens (MW 101–150 kDa) to which antiserum against human IgM and IgG bound, respectively. The most prominent antigens with IgM reactivity had MWs of 36, 63, and 120 kDa, whereas for IgG, the most intensely reactive antigen had a MW of 120 kDa. When membrane fractions prepared from purified porcine aortic endothelial cells and LLC‐PK1 cells were analyzed, the major antigens had molecular weights comparable to those seen for NICs. After culturing the NICs for 5 days, the number of detected xenoreactive antigens binding IgM or IgG decreased and the antigens present at 36, 63, and 120 kDa with IgM reactivity were shown to have a decreased intensity of binding. Incubation of cultured porcine NICs for 18 hr in the presence of human AB serum containing complement resulted in a 55% loss of cellular insulin content (P < 0.0001), a 45% reduction in recoverable DNA (P < 0.0001), and a marked reduction in insulin secretory response to an in vitro glucose challenge. Recovery and viability of porcine NICs was not affected when incubated with AB serum depleted of anti‐Gal antibodies with Synsorb 90. These results demonstrate that natural human antibodies of both IgM and IgG subtypes bind to antigens present on Department of Laboratory Medicine and complement reduces islet cell survival and functional viability. Adsorbing serum with the αGal(1–3)βGal(1–4)βGlc carbohydrate removes natural human antibody‐mediated destruction of porcine neonatal islet cell grafts.