The Use Of A Novel Stereological Sampling Approach To Estimate Islet, Duct And Acinar Total Cell Number And Compartment Volume In Nonhuman Primate Pancreas

Abstract

Design‐based stereology is used to obtain quantitative estimates of biological structures from sections with known precision and unlike model‐based stereology, is free of any assumptions concerning tissue structure. Practical solutions for incorporation of stereological estimates of total number in regulatory toxicology studies should provide appropriate sections for both the estimation of cell or particle number and routine histopathology evaluation in standard paraffin format from the same animal. Development of novel sampling principles, stereological software and advancements in computer hardware technology make it possible to fulfill both these criteria. A novel form of sampling based on non‐uniform statistical sampling principles known as the Proportionator has been recently developed that provides precise estimates of non‐uniformly distributed structures efficiently. In this test system, the pancreas was used because of the inhomogeneous distribution of cell types with in the organ. Coupled with the Fractionator sampling principles, estimates of total acinar, duct and islet cell number and compartment volume can be efficiently quantified. Using a systematic uniform sampling strategy, entire pancreas from 6 nonhuman primates were sampled and processed to paraffin blocks. Disectors were generated and immunostained to delineate the islet and duct cells. Virtual images of disectors were captured and imported into the Visiopharm Integrator System (VIS) and sampled using the Autodisector/Proportionator software. Matched fields of view (FOV) were superimposed with either an unbiased counting frame (test probe for cell number) or a set of test points (test probe for compartment volume), and counts were performed. Mean total cell estimates were 656.78 × 106 (acinar), 267.7 × 106 (duct) and 49.47 × 106 (islet). Mean total compartment volumes were 1575.46 mm3 (acinar), 477.04 mm3 (duct), 35.91 mm3 (islet). Application of the Proportionator Fractionator sampling principles allowed for a more efficient, less labor intensive approach to quantitate organ sub‐structures and heterogeneously distributed cell populations. This study was conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and was reviewed by the ethical review process at the institution where the work was performed.