ASSESSMENT OF ISLET ISOLATION EFFICACY IN DOGS

The outcome of islet isolation is considered uncertain because of the large variability of islet and insulin yield, but comparison of the is olated and native islet PoPulation has not been attempted. We therefor e addressed the efficacy of collagenase digestion, and density gradien t purification of islets from the splenic dog pancreas (n = 31) by mor phometry of the islet volume and size distribution, and by extraction of insulin and amylase, in samples from the pancreas, the digest, and gradient fractions. In contrast to a approximately 90% recovery of pan creatic insulin and amylase after digestion, islet yield amounted to 5 0% of the islet content of the pancreas. After density separation, isl ets were mainly found in the purified fractions, while half of the rec overed insulin was located in the acinar fraction of the gradients-ind icating a substantial proportion of islets entrapped in acinar fragmen ts. The islet and insulin content of the pancreas correlated well with islet and insulin yield after digestion (r = 0.7, p < .0001). The ins ulin content of digest suspensions did neither correlate with islet no r insulin recovery in the purified fraction of the gradients (r = 0.4) as opposed to the islet content of digest suspensions, which correlat ed with both (r = 0.7, p < .0001). After density separation near 100% purity was obtained, and no loss of insulin from isolated islets was d emonstrated by extraction and microscopy. Size distributions of native and isolated islets demonstrated no fragmentation. We conclude that t he variability of isolation outcome may be attributed to a large exten t to the variability of the native endocrine pancreas. Isolation effic acy was best documented by morphometry, because insulin extraction did not discriminate between free and entrapped islets. However, assessme nt by both morphometry and extraction allowed the quantitation of entr apped islets, and demonstrated preservation of beta-cell granulation. Similar studies should facilitate the analysis of other factors affect ing islet isolation in man.