Validation of Large Particle Flow Cytometry for the Analysis and Sorting of Intact Pancreatic Islets

Abstract

Accurate quantification of total islet yield is an essential step prior to transplantation and for research. The standard method of manually determining an islet equivalent (IEQ) count is subjective and prone to error. We evaluated Complex Object Parametric Analyzer and Sorter (COPAS) large particle flow cytometry for the determination of islet equivalent counts and purities of islet preparations. Initial validation of the sensitivity and accuracy of the COPAS flow cytometer was performed by analysis and sorting of uniform polystyrene microspheres with sizes similar to islets. Human and Rhesus monkey islets were stained with the zinc-specific fluorescent dye Newport Green to discriminate islet from nonislet tissue. Islet sizes were extrapolated from standard curves obtained using microspheres from which individual islet volumes were calculated. IEQ counts on six islet preparations were performed by the standard manual method and compared with results obtained by automated COPAS flow cytometry. The COPAS flow cytometer was highly accurate in the detection and measurement of both polystyrene microspheres and islets. IEQ counts determined by COPAS flow cytometry were consistent with manual counts although subject to error when assessing preparations with significant numbers of islets embedded within acinar tissue. Size-specific islet sorting with retention of morphology and dithizone staining was also shown using the COPAS flow cytometer. COPAS large particle flow cytometry provides a novel automated approach for quantification of intact islets and determination of islet equivalent yield. In addition, the ability to analyze and sort islets based upon user defined criterion opens unique avenues for experimentation.