Rmax: A systematic approach to evaluate instrument sort performance using center stream catch

Andrew Riddell,Rui Gardner,Alexis Perez-Gonzalez,Telma Lopes,Lola Martinez

doi:10.1016/j.ymeth.2015.02.017

Abstract

Sorting performance can be evaluated with regard to Purity, Yield and/or Recovery of the sorted fraction. Purity is a check on the quality of the sample and the sort decisions made by the instrument. Recovery and Yield definitions vary with some authors regarding both as how efficient the instrument is at sorting the target particles from the original sample, others distinguishing Recovery from Yield, where the former is used to describe the accuracy of the instrument’s sort count. Yield and Recovery are often neglected, mostly due to difficulties in their measurement. Purity of the sort product is often cited alone but is not sufficient to evaluate sorting performance. All of these three performance metrics require re-sampling of the sorted fraction. But, unlike Purity, calculating Yield and/or Recovery calls for the absolute counting of particles in the sorted fraction, which may not be feasible, particularly when dealing with rare populations and precious samples. In addition, the counting process itself involves large errors.Here we describe a new metric for evaluating instrument sort Recovery, defined as the number of particles sorted relative to the number of original particles to be sorted. This calculation requires only measuring the ratios of target and non-target populations in the original pre-sort sample and in the waste stream or center stream catch (CSC), avoiding re-sampling the sorted fraction and absolute counting. We called this new metric Rmax, since it corresponds to the maximum expected Recovery for a particular set of instrument parameters. Rmax 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 performance before single-cell sorting experiments. Because we do not perturb the sort fraction we can calculate Rmax during the sort process, being especially valuable to check instrument performance during rare population sorts.

Highlights

Even when comparing the number of sort decisions made by the instrument with the final number of cells in the sorted tube, it remains very hard to ascertain whether loss occurred due to instrument failure or cell death and/or cell adherence to the tube walls
Current methods to measure Recovery rely on counting the end-sort product, which is dependent on both instrument performance and sample loss, preventing researchers from understanding whether loss of Recovery is due to instrument or sample, or both
Rmax can be used as quality control (QC) tool for sorters, by assessing the maximum Recovery of the instrument using an ‘‘ideal’’ sample in ‘‘ideal’’ conditions, and can be useful in single-cell sorting experiments

Summary

Introduction

Particles for analysis and sorting are injected into the core of the nozzle and confined to a single file through hydrodynamic forces. The jet’s core, particles will ideally move one at a time through a focused laser beam spot, allowing light scatter and fluorescent measurements to be taken from each particle. Classification of particles can be made, and because the break-off point (BOP) of the jet is stable in time, drops containing desired target particles can be selectively charged, deflected by an electrostatic field, and collected in a container. Coincidence events occur when two or more particles arrive together at the laser interrogation point or within the boundaries of a drop. Drop coincidence events are controlled differently according to the selected sort mode.

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Discussion

Conclusion