Performance comparison of the Spectral Signature, Spillover Spreading Matrix, and Data Unmixing Quality between the spectral Cytek Aurora and the spectral BD FACS Symphony A5 SE by a 35-Color Human Broad Immunophenotyping Panel

Spectral flow cytometry allows the collection of the entire spectral signature across all lasers. This aids in the overall workflow including improvements in data collection, finetuned visualization of autofluorescence and panel design. Moreover, by the process of spectral unmixing we can take the sum of the fluorescence and mathematically separate out each individual color, allowing us to further discriminate between the variations within the spectral signature of a molecule and use novel fluorochrome combinations that were historically not used in the same panel. In recent years, many cytometry manufacturers have developed their own instrumentation to perform such multicolor spectral cytometry experiments, most notably Cytek with their Aurora system as well as more recently by BD Biosciences with their FACS Symphony A5 SE (Spectrally Enabled). In this study, we independently compared the performance between the Cytek Aurora and the FACS Symphony A5 SE. First, we assessed human peripheral blood mononuclear cells (PBMCs) individually stained with anti-human CD4 antibodies (35 different colors, clone SK3) to compare the stain index for each fluorescent channel as well as the overall spillover spreading matrix, which visualizes the interactions between pairs of fluorochromes and estimates the spread. Secondly, based on instrument optical configurations, stain index, and spillover spread matrix, we designed a 35-color broad human immunophenotyping panel to stain lysed whole blood from 3 donors and compared the data quality and resolution of previously-described and anticipated immune cell populations between the instruments and have determined an accurate comparative analysis of both instruments.

Content

Author and article information

Journal

Title: ScienceOpen Posters

Publisher: ScienceOpen

Publication date (Electronic preprint): 26 June 2022

Affiliations

[1 ] Tumor Microenvironment Thematic Research Center, Bristol Myers Squibb, Redwood City, CA

Author notes

[* ]Email: Gabriel.DeNiro@ 123456bms.com .

Author information

Gabriel DeNiro https://orcid.org/0000-0002-4633-4773

Article

DOI: 10.14293/S2199-1006.1.SOR-.PPHND5B.v1

SO-VID: 52546518-0f4f-4ae8-805b-7d62d7c60f98

License:

This work has been published open access under Creative Commons Attribution License CC BY 4.0 , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Conditions, terms of use and publishing policy can be found at www.scienceopen.com .

History

Date received : 26 June 2022

Data availability: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

ScienceOpen disciplines: Immunology

Keywords: Spectral Flow,Flow Cytometry,35 color,Spectral Signiture,Aurora,FACS Symphony,SSM,BD,Cytek,Immunophenotyping

References

Maecker Holden T., Frey Tom, Nomura Laurel E., Trotter Joe. Selecting fluorochrome conjugates for maximum sensitivity. Cytometry. Vol. 62A(2):169–173. 2004. Wiley. [Cross Ref]
Seong Yekyung, Nguyen Denny X, Wu Yian, Thakur Archana, Harding Fiona, Nguyen Tuan Andrew. Novel <scp>PE</scp> and <scp>APC</scp> tandems: Additional <scp>near‐infrared</scp> fluorochromes for use in spectral flow cytometry. Cytometry Part A. 2022. Wiley. [Cross Ref]
Nguyen Richard, Perfetto Stephen, Mahnke Yolanda D., Chattopadhyay Pratip, Roederer Mario. Quantifying spillover spreading for comparing instrument performance and aiding in multicolor panel design. Cytometry Part A. Vol. 83A(3):306–315. 2013. Wiley. [Cross Ref]
Ferrer‐Font Laura, Pellefigues Christophe, Mayer Johannes U., Small Sam J., Jaimes Maria C., Price Kylie M.. Panel Design and Optimization for High‐Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry. Current Protocols in Cytometry. Vol. 92(1)2020. Wiley. [Cross Ref]
Lawrence William G., Varadi Gyula, Entine Gerald, Podniesinski Edward, Wallace Paul K.. Enhanced red and near infrared detection in flow cytometry using avalanche photodiodes. Cytometry Part A. Vol. 73A(8):767–776. 2008. Wiley. [Cross Ref]

Comments

Comment on this article

[1] Maecker Holden T., Frey Tom, Nomura Laurel E., Trotter Joe. Selecting fluorochrome conjugates for maximum sensitivity. Cytometry. Vol. 62A(2):169–173. 2004. Wiley. [Cross Ref]

[2] Seong Yekyung, Nguyen Denny X, Wu Yian, Thakur Archana, Harding Fiona, Nguyen Tuan Andrew. Novel <scp>PE</scp> and <scp>APC</scp> tandems: Additional <scp>near‐infrared</scp> fluorochromes for use in spectral flow cytometry. Cytometry Part A. 2022. Wiley. [Cross Ref]

[3] Nguyen Richard, Perfetto Stephen, Mahnke Yolanda D., Chattopadhyay Pratip, Roederer Mario. Quantifying spillover spreading for comparing instrument performance and aiding in multicolor panel design. Cytometry Part A. Vol. 83A(3):306–315. 2013. Wiley. [Cross Ref]

[4] Ferrer‐Font Laura, Pellefigues Christophe, Mayer Johannes U., Small Sam J., Jaimes Maria C., Price Kylie M.. Panel Design and Optimization for High‐Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry. Current Protocols in Cytometry. Vol. 92(1)2020. Wiley. [Cross Ref]

[5] Lawrence William G., Varadi Gyula, Entine Gerald, Podniesinski Edward, Wallace Paul K.. Enhanced red and near infrared detection in flow cytometry using avalanche photodiodes. Cytometry Part A. Vol. 73A(8):767–776. 2008. Wiley. [Cross Ref]