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Flow Cytometer Performance Characterization, Standardization, and Control

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Single Cell Analysis

Part of the book series: Series in BioEngineering ((SERBIOENG))

Abstract

Flow cytometry is a widely used technique for the analysis of single cells and particles. It is an essential tool for immunological research, drug and device development, clinical trials, disease diagnosis, and therapy monitoring. However, measurements made on different instrument platforms are often inconsistent, leading to variable results for the same sample on different instruments and impeding advances in biomedical research. This chapter describes methodologies to obtain key parameters for characterizing flow cytometer performance, including precision, sensitivity, background, electronic noise, and linearity. Further, various fluorescent beads, hard dyed and surface labeled, are illustrated for use in quality control, calibration, and standardization of flow cytometers. To compare instrument characteristics, fluorescence intensity units have to be standardized to mean equivalent soluble fluorochrome (MESF) or equivalent reference fluorophore (ERF) units that are traceable to the existing primary fluorophore solution standards. With suitable biological controls or orthogonal method, users will be able to quantitatively measure DNA and RNA content per cell or biomarker expression in antibodies bound per cell. Comparable, reproducible, and quantitative measurements using flow cytometers can be accomplished only upon instrument standardization through performance characterization and calibration, and use of proper biological controls.

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Acknowledgements

The authors would like to thank Adolfas Gaigalas of NIST for his pioneering work and continued engagement in developing traceable fluorescence standards. They would also like to thank David Parks of Stanford for reviewing and clarifying the chapter section on the TEST-FILL method.

Disclaimer

Certain commercial equipment, instruments, and materials are identified in this paper to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment are necessarily the best available for the purpose.

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Authors and Affiliations

  1. Biosystems and Biomaterials Division, National Institute of Standards and Technology, 100 Bureau Drive, Galthersburg, MD, 20899, USA

    Lili Wang

  2. Livermore, CA, USA

    Robert A. Hoffman

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  1. Lili Wang
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Correspondence to Lili Wang .

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Editors and Affiliations

  1. Purdue University Cytometry Laboratories, Department of Biomedical Engineering, Purdue University, West Lafayette, Indiana, USA

    J. Paul Robinson

  2. Chair of Pathology and Immunology, Department of Medical and Surgical Sciences for Children and Adults, University of Modena and Reggio Emilia School of Medicine, Modena, Italy

    Andrea Cossarizza

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Wang, L., Hoffman, R.A. (2017). Flow Cytometer Performance Characterization, Standardization, and Control. In: Robinson, J., Cossarizza, A. (eds) Single Cell Analysis. Series in BioEngineering. Springer, Singapore. https://doi.org/10.1007/978-981-10-4499-1_8

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  • DOI: https://doi.org/10.1007/978-981-10-4499-1_8

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  • Online ISBN: 978-981-10-4499-1

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