Considerations for the control of background fluorescence in clinical flow cytometry
Open accessAbstract
Accurate measurement of antigen‐positive cells by flow cytometry can be hampered by background fluorescence of antigen‐negative cells and other particles (e.g., debris). This article focuses on three major causes of background (autofluorescence, spectral overlap, and undesirable antibody binding) by reviewing individual aspects of flow cytometric measurements that contribute to these causes. The appropriate use of controls facilitates a thorough understanding of these contributing factors as well as the development of robust cell labeling protocols intended for routine flow cytometric analysis. We present a set of recommendations that enables the user to develop an optimized cell labeling protocol that minimizes background and maximizes the ability to reliably distinguish between a positive and a negative population of cells. These recommendations are also intended to augment existing guidelines designed to aid in the formulation of a consensus regarding the utility of flow cytometry for the analysis of clinical samples. © 2009 Clinical Cytometry Society
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An isotype control antibody is not useful for determining or excluding non-specific binding in flow cytometry.
“Although some continue to use an isotype control Ab to determine/exclude non-specific binding, the isotype control is not useful in this context [2].”
From Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition) (2021) · cites this paper as [2]
The 'Understand and Minimize Undesirable Antibody Binding' section says isotype controls are not recommended for determining nonspecific binding in quantitative cell-counting assays. But the 'ISOTYPE CONTROLS' section says a perfectly matched control can help determine undesirable binding and that isotype controls may have utility in some qualitative assays. The claim's unqualified statement that they are not useful is therefore broader than the paper's conclusion. (LLM, conf=0.88)
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References 58 · Cited by 255
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- Authors
- Hulspas, R., O'Gorman, M. R. G., Wood, B. L., Gratama, J. W., & Sutherland, D. R.
- Affiliations
- University of Washington (US)
- Erasmus MC Cancer Institute (NL)
- University Health Network (CA)
- Corresponding author
- Hulspas, Ruud
- Venue
- Cytometry Part B: Clinical Cytometry
- Publisher
- Wiley
- Published
- 2009-07-02
- Volume
- 76B
- Issue
- 6
- Pages
- 355-364
- ISSN
- 1552-4949 (ISSN-L)
- 1552-4957
- OpenAlex
- W1997315475
- Cited by
- 255
- References
- 58
- Citations by year
- 2026: 10 · 2025: 17 · 2024: 25 · 2023: 18 · 2022: 17 · 2021: 21 · 2020: 25 · 2019: 20 · 2018: 19 · 2017: 17 · 2016: 18 · 2015: 9 · 2014: 6 · 2013: 10 · 2012: 11
- FWCI
- 0.80
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Cytometry Part B Clinical Cytometry (publishedVersion)
- PubMed (publishedVersion)
- EUR Research Repository (Erasmus University Rotterdam) (submittedVersion)
- Topics
- Health Sciences › Medicine › Physiology › Clinical Laboratory Practices and Quality Control
- Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Keywords
- background fluorescence, autofluorescence, spectral overlap, antibody binding, clinical flow cytometry, debris
- MeSH terms
- Antibodies, Antibody Specificity, Flow Cytometry, Fluorescence, Fluorescent Dyes, Humans, Image Processing, Computer-Assisted, Quality Control, Staining and Labeling, Clinical Laboratory Techniques
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