A simplified approach to human islet quality assessment.

BACKGROUND: The successful treatment of patients with type 1 diabetes by islet transplantation is affected by a multitude of factors of which infusion of the highest quality tissue is essential. The current standard pretransplant quality assessments lack sensitivity, accuracy, and objectivity in the determination of islet viability and potency. We hypothesized that a multiparametric approach focused on islet cell metabolic state, mitochondrial integrity, and in vitro glucose-stimulated insulin secretion (GSIS) could provide data predictive of in vivo function. The objective of this study was to validate a novel set of islet quality assays and develop a simplified islet quality scoring system for both basic research and clinical applications. METHODS: A series of 42 human islet preparations were screened using standard and novel methods, which included determination of yield, viability by fluorescent microscopy, GSIS, percentage of islet loss in culture, quantification of adenine nucleotides, flow cytometric measurement of viability, apoptosis, and mitochondrial membrane potential (MMP). In vivo functional potency was tested by minimal model transplant in streptozotocin-induced diabetic NOD.scid mice. RESULTS: Functionally potent islet preparations showed significantly greater numbers of cells with polarized MMP, higher ATP-to-ADP ratios, and increased glucose-induced insulin secretion. The MMP, ATP-to-ADP ratio, and GSIS data were combined into a single islet scoring formula that showed more than 86% accuracy in predicting in vivo functional potency. CONCLUSIONS: Our study demonstrates that a multiparametric approach using objective assessments focused on islet cell mitochondrial integrity and in vitro function can provide data predictive of in vivo function.

Assessment of islet function following islet and pancreas transplantation.

Pancreas and islet transplant recipients are monitored using various metabolic and imaging methods. The inaccessibility of the transplanted whole pancreas and of the isolated islets poses specific problems (eg, all assessment techniques are indirect). Although successful pancreas transplantation typically restores normal glucose homeostasis, islet transplantation into the liver does not completely normalize islet hormone secretion and glucose metabolism. Development of better testing strategies, such as direct islet imaging, will significantly advance the field.

Assessment of human islet viability using various mouse models.

To date no in vitro viability test is known to accurately predict in vivo human islet function, making transplantation into various nonimmune animal models mandatory. The diabetic mouse model has been proposed as a standard method for human islet viability assessment. However, the use of streptozotocin for diabetes induction is associated with inconsistency with respect to induction protocols and the significant mortality rate. The purpose of this study was to compare a nondiabetic NOD-scid mouse model to its diabetic counterpart in terms of predicting islet viability. Diabetes was induced in NOD-scid mice using intraperitoneal injection of streptozotocin at concentrations ranging from 100 to 200 mg/kg. Blood glucose levels were monitored for 7 to 10 days, and mice that had levels of >300 mg/dL were used in the experiment. For nondiabetic mice, blood glucose and baseline human C-peptide levels were checked after an overnight fast. Transplantation of 2000 human islet equivalent was done in both models using the same technique. Islet function was determined in the diabetic mice by return to normoglycemia for 2 consecutive days and measurement of fasting human C-peptide on days 7 and 14 posttransplant. Viability was tested in nondiabetic mice after intraperitoneal injection of glucose (2 g/kg) and the measurement of human C-peptide levels using radioimmunoassay. Titration of the streptozotocin dose from 200 to 100 mg/kg showed a significant reduction in mice mortality (40% to 10%) and an increase of diabetes induction (55% to 81%). The 23 human islet isolations tested in both models showed complete consistency of the viability results.

Islet graft assessment in the Edmonton Protocol: implications for predicting long-term clinical outcome.

The success of the Edmonton Protocol for islet transplantation has provided new hope in the treatment of type 1 diabetes. This study reports on the assessment of 83 human islet grafts transplanted using the Edmonton Protocol since 1999. Cellular composition, as assessed by immunohistochemistry, showed a lower islet purity (approximately 40%) than has been reported in previous studies using dithizone staining to quantitate islet equivalents. Furthermore, grafts were found to contain substantial populations of exocrine and ductal tissue. Total cellular insulin transplanted was 8,097.6 +/- 3,164.4 microg/patient, and was significantly lower in bottom gradient layer grafts than top gradient layer or whole/combined grafts (P < 0.0005). A static incubation test for islet function gave a stimulation index of 3-4, although this measure did not correlate with posttransplant metabolic outcome. Furthermore, we confirmed a previously reported trend in which donor age affects islet yield and purity. It is important to note that a significant positive correlation was observed between the number of islet progenitor (ductal-epithelial) cells transplanted and long-term metabolic success as assessed an by intravenous glucose tolerance test at approximately 2 years posttransplant. In summary, careful assessment of islet graft composition is needed in a clinical transplantation program to accurately estimate islet purity and assess the contribution of other cell types present, such as islet progenitor cells.

Assessment of the severity of hypoglycemia and glycemic lability in type 1 diabetic subjects undergoing islet transplantation.

Currently, the major indications for solitary islet transplantation are recurrent severe hypoglycemia and labile glucose control. Quantifying these problems remains subjective. We have developed a scoring system for both hypoglycemia and glycemic lability, established normative data, and used them in patients who have undergone islet transplantation. A composite hypoglycemic score (HYPO score) was devised based on the frequency, severity, and degree of unawareness of the hypoglycemia. In addition, using 4 weeks of glucose records, a lability index (LI) was calculated based on the change in glucose levels over time and compared with a clinical assessment of glycemic lability. A mean amplitude of glycemic excursions (MAGE) was also calculated based on 2 consecutive days of seven readings each day. These scores were determined in 100 randomly selected subjects with type 1 diabetes from our general clinic to serve as a control group and in patients before and after islet transplantation. The mean age of the control diabetic subjects was 38.4 +/- 1.3 years (+/-SE), with a duration of diabetes of 21.5 +/- 1.1 years. The median HYPO score in the control subjects was 143 (25th to 75th interquartile range: 46-423). The LI in the diabetic control subjects was 223 (25th to 75th interquartile range: 130-329 mmol/l(2)/h.week(-1)). The LI correlated much more closely than the MAGE with the clinical assessment of lability. A HYPO score of > or = 1,047 (90th percentile) or an LI > or = 433 mmol/l(2)/h.week(-1) (90th percentile) indicated serious problems with hypoglycemia or glycemic lability, respectively. The islet transplant patients (n = 51) were 42.1 +/- 1.4 years old, with a duration of diabetes of 25.7 +/- 1.4 years. Islet transplant patients had a mean HYPO score of 1,234 +/- 184 pretransplant, which was significantly higher than that of the control subjects (P < 0.001), which became negligible posttransplantation with the elimination of hypoglycemia. The median LI pretransplant was 497 mmol/l(2)/h.week(-1) (25th to 75th interquartile range: 330-692), significantly higher than that of control subjects (P < 0.001), and fell to 40 (25th to 75th interquartile range: 14-83) within a month after the final transplant. In those who had lost graft function, the LI rose again. The HYPO score and LI provide measures of the extent of problems with hypoglycemia and glycemic lability, respectively, complement the clinical assessment of the problems with glucose control before islet transplantation, and will allow comparison of selection of subjects for transplants between centers.

Factors influencing quantification of in vivo bioluminescence imaging: application to assessment of pancreatic islet transplants.

The aim of this study is to determine and characterize factors influencing in vivo bioluminescence imaging (BLI) and apply them to the specific application of imaging transplanted pancreatic islets. Noninvasive quantitative assessment of transplanted pancreatic islets poses a formidable challenge. Murine pancreatic islets expressing firefly luciferase were transplanted under the renal capsule or into the portal vein of nonobese diabetic-severe combined immunodeficiency mice and the bioluminescence was quantified with a cooled charge coupled device camera and digital photon image analysis. The important, but often neglected, effects of wound healing, mouse positioning, and transplantation site on bioluminescence measurements were investigated by imaging a constant emission, isotropic light-emitting bead (lambda = 600) implanted at the renal or hepatic site. The renal beads emitted nearly four times more light than hepatic beads with a smaller spot size, indicating that light absorption and scatter are greatly influenced by the transplant site and must be accounted for in BLI measurements. Detected luminescence decreased with increasing angle between the mouse surface normal and optical axis. By defining imaging parameters such as postsurgical effects, animal positioning, and light attenuation as a function of transplant site, this study develops BLI as a useful imaging modality for quantitative assessment of islets post-transplantation.

Assessment of some porcine strains as donors of islets of Langerhans.

Mass isolation of viable porcine islets is a difficult task because of their fragility, and because of donor variability with respect to strain, age, sex, feeding, and methods of slaughtering. Not all strains are equally suitable for islet separation. The aim of this study was to evaluate porcine pancreata as an alternative source of islets for clinical transplantation. Pancreata were digested from pig strains available in Poland: 248 market weight slaughterhouse pigs and 42 pigs, belonging to the Polish Large White (WBP, 14 sows and 3 males), Polish White Pendant-Ears (PBZ; 16 sows), Pietrain (8 sows), and Yorkshire (1 sow) races. Prepurification data of recoverable islets/g and islet equivalents/g were considered as representative for the number of recoverable islets. Acceptable results namely, islet and/or islet-equivalent (IE) number of at least 1000/g, were obtained from only 56 of 248 slaughterhouse pigs, namely 2073 +/- 137.4 SE (median 1767/g) islets with values of IE of 2994 +/- 303 SE (median 1874/g). Our data support Krickhahn et al suggesting that only pancreata with an average islet size exceeding 199 microm should be digested and that only from 1 of 3 to 5 porcine pancreata is an adequate amount of islets generated.

In vitro and in vivo viability assessment of unpurified pancreatic islet tissue.

The viability of porcine collagenase-prepared islet preparations (n = 16) was classified by 31P-NMR spectroscopy, staining by neutral red and trypan blue, and in vitro insulin secretion following glucose challenge. Vital islets exhibited a phosphate diester/phosphate monoester (PDE/PME) ratio of 0.5-0.9, a staining score of 18-30 and an insulin secretion responding well to glucose challenge. Damaged islets performed at a PDE/PME of 0.2-0.49 and a staining score of 9-17 and necrotic islets had 0.0-0.49 and a staining score of 9-17 and necrotic islets had 0.0-0.19 and 0-8, respectively. The islets of the latter two groups did not adequately respond to glucose. The in vivo function following autotransplantation of these islets into the spleen was investigated in five recipients of more than 3000/kg vital islets of which 4 expressed daily normoglycemia (< 200 mg%), normalized intravenous glucose tolerance (K = -2.21), and a prolonged survival (mean +/- SD) of 167 +/- 12 days compared to five recipients of > 3000/kg damaged islets (K = -0.814) (P = 0.0017) and a survival of 86 +/- 21 days (P = 0.0096). It is suggested that 31P-NMR spectroscopy is a valuable and practical method to predict islet graft viability prior to transplantation in order to assure good graft function in the recipient.

Assessment of long-term (1 year) graft survival and metabolic and hormonal changes after intrasplenic canine pancreatic microfragment transplantation.

Few data have been published so far on the long-term metabolic and endocrine consequences of intrasplenic islet autografts in dogs and the available information mainly deals with glucose response and, more rarely, insulin secretion during intravenous glucose tolerance tests. The effect of islet transplantation on glucagon levels has never been reported. In this study we measured glucose, insulin, total glucagon, and pancreatic glucagon in dogs before and after (up to 1 year) intrasplenic islet autografts. Pancreata were retrieved from 21 adult dogs and the islets were isolated by collagenase digestion. The endocrine tissue was transplanted into the spleen by direct pulp injection. Autografts resulted in normoglycaemia in 19 out of 21 dogs (90%). Among the successfully transplanted dogs, the percentage of functioning grafts was 71% at 1 year. The metabolic and hormonal results of the follow-up showed that fasting glucose and insulin concentrations did not differ significantly before and after transplantation. However, glucose tolerance and insulin secretion on intravenous glucose tolerance testing were significantly reduced in transplanted animals. Fasting total and pancreatic glucagon, and their integrated releases during the test did not change significantly after islet transplantation. These results demonstrate that: 1) long-term function of intrasplenic canine islet autografts can be achieved in high percentages; 2) autotransplanted animals have normal or near normal fasting glucose and insulin levels, but reduced glucose disappearance and insulin secretion on intravenous glucose tolerance tests; 3) normal glucagon secretion is a feature of successful islet grafts.