AutoSpill is a principled framework that simplifies the analysis of multichromatic flow cytometry data

Carlos P Roca,Václav Gergelits,Josef Spidlen,James Collier,Richard Halpert,Carly E Whyte,Łukasz Kreft,Adrian Liston,Stéphanie Humblet-Baron,Oliver T Burton,Alexander Botzki,Teresa Prezzemolo

doi:10.1038/s41467-021-23126-8

Abstract

Compensating in flow cytometry is an unavoidable challenge in the data analysis of fluorescence-based flow cytometry. Even the advent of spectral cytometry cannot circumvent the spillover problem, with spectral unmixing an intrinsic part of such systems. The calculation of spillover coefficients from single-color controls has remained essentially unchanged since its inception, and is increasingly limited in its ability to deal with high-parameter flow cytometry. Here, we present AutoSpill, an alternative method for calculating spillover coefficients. The approach combines automated gating of cells, calculation of an initial spillover matrix based on robust linear regression, and iterative refinement to reduce error. Moreover, autofluorescence can be compensated out, by processing it as an endogenous dye in an unstained control. AutoSpill uses single-color controls and is compatible with common flow cytometry software. AutoSpill allows simpler and more robust workflows, while reducing the magnitude of compensation errors in high-parameter flow cytometry.

Highlights

Compensating in flow cytometry is an unavoidable challenge in the data analysis of fluorescence-based flow cytometry
Flow cytometry has been a revolutionary force in single-cell analysis
Initial limitations were overcome through ingenious technical developments: the number of fluorescent parameters were expanded through the development of new dyes and lasers, intracellular staining protocols were optimized for the detection of intracellular proteins, RNAflow techniques allowed measurement at the RNA level[25], and numerous non-antibody-based dyes were able to detect processes from redox potential[26] to organelle content and status[27]

Summary

Introduction

Compensating in flow cytometry is an unavoidable challenge in the data analysis of fluorescence-based flow cytometry. The calculation of spillover coefficients from single-color controls has remained essentially unchanged since its inception, and is increasingly limited in its ability to deal with high-parameter flow cytometry. The development from single-color flow cytometry to ultra high-parameter flow cytometry has allowed an enormous growth in the data collected per cell. A key limitation with highparameter flow cytometry, is the spectral overlap of fluorescent dyes[7] This results in the spillover of fluorescence to detectors different from the detector assigned to each dye (in classical flow cytometry). Removing this unwanted spillover, i.e. State-of-the-art flow cytometers, with ~30 channels, make compensation increasingly difficult as the number of channels grows, due to the unavoidable overlap between emission spectra of fluorescent dyes. The difficulty of experimental design has followed the growth in fluorophore options, to the point where the development, refinement, and validation of ultra-high parameter panels can take months to years of expert input[4,8,9,10]

Methods

Results

Conclusion