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

Continuous Quadrupole Magnetic Separation of Islets during Digestion Improves Purified Porcine Islet Viability

Islet transplantation (ITx) is an emerging and promising therapy for patients with uncontrolled type 1 diabetes. The islet isolation and purification processes require exposure to extended cold ischemia, warm-enzymatic digestion, mechanical agitation, and use of damaging chemicals for density gradient separation (DG), all of which reduce viable islet yield. In this paper, we describe initial proof-of-concept studies exploring quadrupole magnetic separation (QMS) of islets as an alternative to DG to reduce exposure to these harsh conditions. Three porcine pancreata were split into two parts, the splenic lobe (SPL) and the combined connecting/duodenal lobes (CDL), for paired digestions and purifications. Islets in the SPL were preferentially labeled using magnetic microparticles (MMPs) that lodge within the islet microvasculature when infused into the pancreas and were continuously separated from the exocrine tissue by QMS during the collection phase of the digestion process. Unlabeled islets from the CDL were purified by conventional DG. Islets purified by QMS exhibited significantly improved viability (measured by oxygen consumption rate per DNA, p < 0.03 ) and better morphology relative to control islets. Islet purification by QMS can reduce the detrimental effects of prolonged exposure to toxic enzymes and density gradient solutions and substantially improve islet viability after isolation.Schulze Foundation; Schulze Diabetes Institute, NIH [R44DK072647-03, R01DK068717]; Carol Olson Memorial Diabetes Research Fund; Iacocca Family Foundation; Schott Family Foundation; Kettering Family FoundationThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Summary of cases where <i>OCR dose</i> correctly predicted clinical transplant outcome, whereas <i>IE dose</i> did not.

<p>Summary of cases where <i>OCR dose</i> correctly predicted clinical transplant outcome, whereas <i>IE dose</i> did not.</p

Overlap and correlation of islet characterization methods with clinical transplant outcome.

<p>Data from our study illustrating that membrane integrity staining (based on FDA/PI), oxygen consumption rate (OCR) normalized to DNA content (OCR/DNA), and OCR/DNA normalized to the islet size index (ISI) (OCR/DNA/ISI) are not correlated with the clinical outcome [insulin independence vs. dependence] at 6–12 months following islet autotransplant (IAT). However, both <i>islet equivalent</i> (IE) <i>dose</i> and the <i>OCR dose</i> were correlated with post-IAT outcome. The gray region indicates the range of <i>IE</i> and <i>OCR doses</i> that is associated with uncertain IAT outcome. Note that the width of the gray region is much narrower with the <i>OCR dose</i>. The black dotted line represents the calculated cut-off point for clinical outcome (<i>IE dose</i>: 5,794 and <i>OCR dose</i>: 6.23). The second column of graphs represents receiver operating characteristic (ROC) curves for each of the five islet product characteristics from this data set. The area-under-the-curve (AUC) has been calculated for each islet product characteristic and these values are shown above each ROC curve.</p

Previously published data showing overlap and correlation of islet equivalent dose with clinical transplant outcome.

<p>Data from Anazawa <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134428#pone.0134428.ref010" target="_blank">10</a>] illustrating that the <i>islet equivalent</i> (<i>IE</i>) <i>dose</i> correlates with the clinical outcome [insulin independence vs. dependence] at 6–12 months following islet autotransplant (IAT). However, the gray region indicates a wide range of <i>IE dose</i> (IE/kg of recipient) that is associated with an uncertain IAT outcome. The second graph shows the receiver operating characteristic (ROC) curve for this previously published data set with the area-under-the-curve (AUC) above.</p

Comparison of demographic, isolation, and quality assessment variables between insulin dependent and independent clinical transplant outcome 6–12 months post-transplant.

<p>Data are means ± standard error or percentage.</p><p>^asignificant difference (p< 0.05) between groups as calculated by a Mann Whitney non-parametric test.</p><p>Comparison of demographic, isolation, and quality assessment variables between insulin dependent and independent clinical transplant outcome 6–12 months post-transplant.</p