Evaluation of Sort Recovery via Rmax

Cell sorting performance can be evaluated in regard to the purity and recovery of the sorted fractions. The purity provides checks on sample quality, acquisition settings, gating strategy, and the sort decisions made by the instrument, but alone it is not sufficient to evaluate sorting performance. Recovery, defined here as the number of target particles sorted relative to the number of original target particles to be sorted, is a key metric of sort fitness and performance but is often neglected due to difficulties in its measurement. Both purity and recovery require re-sampling of the sorted fraction, but unlike determining purity, calculating recovery calls for the absolute counting of particles in the sorted fraction that comes with large errors, and may not be feasible for rare populations or precious samples. Here, we describe a recently developed metric and method for calculating sort recovery called Rmax, representing the maximum expected recovery for a particular set of instrument settings. Rmax calculation avoids re-sampling of the total sorted fraction and absolute counting, being instead based on the ratios of target and non-target populations in the original pre-sort sample and in the waste stream or center stream catch. The Rmax method is ideal to evaluate and troubleshoot the optimum drop-charge delay of the sorter or any instrument-related failures that will affect sort performance. It can be used as a daily quality control check but can be particularly useful to assess instrument fitness before single-cell or rare population sorts. Because the sorted fraction is not perturbed, we can calculate Rmax during the sort run. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Evaluating sorter setup with Rmax Basic Protocol 2: Finding the maximum Rmax: scanning over the drop charge delay Alternate Protocol: Finding the maximum Rmax for cells: scanning over the drop charge delay Basic Protocol 3: Estimating sorted cell number with Rmax.