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AutoSpill: A method for calculating spillover coefficients to compensate or unmix high-parameter flow cytometry data

View ORCID ProfileCarlos P. Roca, Oliver T. Burton, Teresa Prezzemolo, Carly E. Whyte, Richard Halpert, View ORCID ProfileŁukasz Kreft, James Collier, View ORCID ProfileAlexander Botzki, View ORCID ProfileJosef Spidlen, View ORCID ProfileStéphanie Humblet-Baron, View ORCID ProfileAdrian Liston
doi: https://doi.org/10.1101/2020.06.29.177196
Carlos P. Roca
1VIB Center for Brain and Disease Research, 3000 Leuven, Belgium
2KU Leuven – University of Leuven, Department of Microbiology and Immunology, 3000 Leuven, Belgium
3Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, United Kingdom
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  • For correspondence: carlosproca@gmail.com adrian.liston@babraham.ac.uk
Oliver T. Burton
3Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, United Kingdom
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Teresa Prezzemolo
1VIB Center for Brain and Disease Research, 3000 Leuven, Belgium
2KU Leuven – University of Leuven, Department of Microbiology and Immunology, 3000 Leuven, Belgium
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Carly E. Whyte
3Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, United Kingdom
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Richard Halpert
4BD Life Sciences–FlowJo, Ashland, OR 97520, USA
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Łukasz Kreft
5VIB Bioinformatics Core, Rijvisschestraat 120, 9052 Ghent, Belgium
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James Collier
5VIB Bioinformatics Core, Rijvisschestraat 120, 9052 Ghent, Belgium
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Alexander Botzki
5VIB Bioinformatics Core, Rijvisschestraat 120, 9052 Ghent, Belgium
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  • ORCID record for Alexander Botzki
Josef Spidlen
4BD Life Sciences–FlowJo, Ashland, OR 97520, USA
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Stéphanie Humblet-Baron
1VIB Center for Brain and Disease Research, 3000 Leuven, Belgium
2KU Leuven – University of Leuven, Department of Microbiology and Immunology, 3000 Leuven, Belgium
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Adrian Liston
1VIB Center for Brain and Disease Research, 3000 Leuven, Belgium
2KU Leuven – University of Leuven, Department of Microbiology and Immunology, 3000 Leuven, Belgium
3Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, United Kingdom
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  • For correspondence: carlosproca@gmail.com adrian.liston@babraham.ac.uk
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Abstract

Compensating in classical flow cytometry or unmixing in spectral systems is an unavoidable challenge in the data analysis of fluorescence-based flow cytometry. In both cases, spillover coefficients are estimated for each fluorophore using single-color controls. This approach 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, a novel approach for calculating spillover coefficients or spectral signatures of fluorophores. 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, but it differs in two key aspects from current methods: (1) it is much less demanding in the preparation of controls, as it does not require the presence of well-defined positive and negative populations, and (2) it does not require manual tuning of the spillover matrix, as the algorithm iteratively computes the tuning, producing an optimal compensation matrix. Another algorithm, AutoSpread, complements this approach, providing a robust estimate of the Spillover Spreading Matrix (SSM), while avoiding the need for well-defined positive and negative populations. Together, AutoSpill and AutoSpread provide a superior solution to the problem of fluorophore spillover, allowing simpler and more robust workflows in high-parameter flow cytometry.

Competing Interest Statement

The VIB and the Babraham Institute received funding from BD Bioscience in return for pre-publication access to and consultancy on the AutoSpill algorithm, in order to be incorporated into FlowJo v.10.7. RH and JS are affiliated with FlowJo, a wholly owned subsidiary of Becton, Dickinson and Company. The other authors declare no competing financial interests.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted August 10, 2020.
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AutoSpill: A method for calculating spillover coefficients to compensate or unmix high-parameter flow cytometry data
Carlos P. Roca, Oliver T. Burton, Teresa Prezzemolo, Carly E. Whyte, Richard Halpert, Łukasz Kreft, James Collier, Alexander Botzki, Josef Spidlen, Stéphanie Humblet-Baron, Adrian Liston
bioRxiv 2020.06.29.177196; doi: https://doi.org/10.1101/2020.06.29.177196
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AutoSpill: A method for calculating spillover coefficients to compensate or unmix high-parameter flow cytometry data
Carlos P. Roca, Oliver T. Burton, Teresa Prezzemolo, Carly E. Whyte, Richard Halpert, Łukasz Kreft, James Collier, Alexander Botzki, Josef Spidlen, Stéphanie Humblet-Baron, Adrian Liston
bioRxiv 2020.06.29.177196; doi: https://doi.org/10.1101/2020.06.29.177196

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