6 - Characterization of Islet Preparations

Abstract

To move forward in the development of improved methods in Islet transplantation, it is essential to have assays to assess meaningful characteristics of islet preparations. This chapter examines the methods for in vitro characterization of islet preparations. Methods can be divided into three groups: islet purity and composition, quantity of islets, and viability of islets. Light microscopic observation following staining with dithizone (diphenylthiocarbazone, DTZ) is the commonly accepted technique employed currently to assess the purity of islet preparations. Because of its inadequacies, a variety of new techniques have been evolved, such as immunohistochemistry using laser scanning, confocal microscope, and ultrastructural analysis using electron microscope. The methods for measuring the quantity of islets in an islet preparation include DTZ staining large particle flow cytometry and tissue pellet packed cell volume ultrasound scattering. Membrane integrity measurement by visual assessment following staining with fluorescein diacetate and propidium iodide is the most commonly employed method of assessing fractional viability of islet preparations. It also reviews assays currently in use, as well as new methods under study at various laboratories with special attention to the own efforts. In examining these methods, discussion provides attention to utility in terms of adequacy for the intended purpose, accuracy and precision, time required to perform the assay and obtain a result, and cost in terms of materials and equipment.