Methodology for evaluating and comparing flow cytometers: A multisite study of 23 instruments

Parks, David R.; Moore, Wayne A.; Brinkman, Ryan R.; Chen, Yong; Condello, Danilo; Khettabi, Faysal El; Nolan, John P.; Perfetto, Stephen P.; Redelman, Doug; Špidlen, Josef; Dyke, Jonathan Van; Wang, Lili; Wood, James C. S.

doi:10.1002/cyto.a.23605

Cited by 17 publications

(20 citation statements)

References 10 publications

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“…The feasibility of a multi-instrument interlaboratory setup was documented by EuroFlow (Canto II, LSR II, and Cyan ADP) (37), COG (Canto and LSR II instrument) (31), and Harmonemia (Canto II and Navios instrument) (42). Methods for the objective comparison of the capability of instruments to perform fluorescence measurements are being developed and will be necessary for making assumptions about the "similar" performance of diverse cytometers (66). However, the differences can be more prominent in channels where a larger difference in filter wavelength specification exists.…”

Section: Boxmentioning

confidence: 99%

“…It remains to be seen how similar the patterns can be between instruments working on a different photon detection principle (PMT detector, semiconductor detector arrays "avalanche diode"). Methods for the objective comparison of the capability of instruments to perform fluorescence measurements are being developed and will be necessary for making assumptions about the "similar" performance of diverse cytometers (66).…”

Section: Boxmentioning

confidence: 99%

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Reproducibility of Flow Cytometry Through Standardization: Opportunities and Challenges

Kalina

2019

Cytometry Pt A

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There is an agreement in the field that interlaboratory reproducibility of flow cytometry measurements as well as the whole studies might be improved by a consensual use of methodological approach. Typically, a consensus is made on a crucial markers needed in the immunostaining panel, sometimes on the particular fluorochrome conjugates and rarely on a complete set of methods for sample preparation. The term "standardization" is used to describe the complete set of methodical steps, while "harmonization" is used for partial agreement on the method. Standardization can provide a platform for improved reproducibility of cytometry results over prolonged periods of time, across different sites and across different instruments. For the purpose of structured discussion, several desired aims are described: common interpretation of the immunophenotype definition of a target subset, accurate quantification, reproducible pattern of a multicolor immunophenotype, and reproducible intensity of all measured parameters. An overview of how standardization was approached by several large consortia is provided: EuroFlow, The ONE Study, Human Immunology Project Consortium (HIPC), and several other groups. Their particular aims and the tools adopted to reach those aims are noted. How those standardization efforts were adopted in the field and how the resulting outcome was evaluated is reviewed. Multiple challenges in the instrument hardware design, instrument setup tools, reagent design, and quality features need to be addressed to achieve optimal standardization. Furthermore, the aims of different studies vary, and thus, the reasonable requirements for standardization differ. A framework of reference for the reasonable outcomes of different approaches is offered. Finally, it is argued that complete standardization is important not only for the reproducibility of measurements but also for education, for quality assessment and for algorithmic data analysis. The different standardized approaches can and in fact should serve as benchmarking reference tools for the development of future flow cytometry studies.

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Section: Boxmentioning

confidence: 99%

Section: Boxmentioning

confidence: 99%

Reproducibility of Flow Cytometry Through Standardization: Opportunities and Challenges

Kalina

2019

Cytometry Pt A

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“…In such cases, there are alternate mechanisms that can be used to establish standardization (28)(29)(30)(31). For specific longitudinal studies, such as immune monitoring of patients receiving sequential immunotherapy treatments, the use of a dedicated instrument is strongly recommended, as most facilities have not standardized multiple instruments to each other.…”

Section: Instrument Designmentioning

confidence: 99%

“…The use of application settings may not be available on all platforms. In such cases, there are alternate mechanisms that can be used to establish standardization (28)(29)(30)(31). One example of an alternative method, which in our experience has been successful, is setting target median fluorescence intensities (MFI) values for eight-peak beads.…”

Section: Instrument Designmentioning

confidence: 99%

Rigor and Reproducibility of Cytometry Practices for Immuno‐Oncology: A multifaceted challenge

et al. 2019

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The rapid advancement of immunotherapy strategies has created a need for technologies that can reliably and reproducibly identify rare populations, detect subtle changes in modulatory signals, and assess antigenic expression patterns that are time-sensitive. Accomplishing these tasks requires careful planning and the employment of tools that provide greater sensitivity and specificity without demanding extensive time. Flow Cytometry has earned its place as a preferred analysis platform. This technology offers a flexible path to the interrogation of protein expression patterns and detection of functional properties in cell populations of interest. Mass Cytometry is a newcomer technology that has generated significant interest in the field. By incorporating mass spectrometry analysis to the traditional principles of flow cytometry, this innovative tool promises to significantly expand the ability to detect multiple proteins on a single cell. The use of these technologies in a manner that is consistent and reproducible through multiple sample sets demands careful attention to experiment design, reagent selection, and instrumentation. Whether applying flow or mass cytometry, reaching successful, reliable results involves many factors. Sample preparation, antibody titrations, and appropriate controls are major biological considerations that impact cytometric analysis. Additionally, instrument voltages, lasers, and run quality assessments are essential for ensuring comparability and reproducibility between analyses. In this article, we aim to discuss the critical aspects that impact flow cytometry, and will touch on important considerations for mass cytometry as well. Focusing on their relevance to immunotherapy studies, we will address the importance of appropriate sample processing and will discuss how selection of suitable panels, controls, and antibodies must follow a carefully designed plan. We will also comment on how educated use of instrumentation plays a significant role in the reliability and reproducibility of results.Through this work, we hope to contribute to the effort toward establishing higher standards for rigor and reproducibility of cytometry practices by researchers, operators, and general cytometry users employing cytometry-based assays in their work.

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“…Advances in the technology of modern cytometers now allow for some conventional commercial instruments to detect biological particles down to the 100 nm diameter range with minor to no modifications to default instrument configurations (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). However, several key challenges remain for small particle FCM and these include: variations in instrument configurations and detection capabilities across platforms and facilities, widely differing sample processing and labeling methods, and a lack of consensus for data reporting (15).…”

mentioning

confidence: 99%

Engineered Retroviruses as Fluorescent Biological Reference Particles for Small Particle Flow Cytometry

Tang

Fritzsche

Renner

et al. 2019

Preprint

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There has been renewed interest in the use of flow cytometry for single particle phenotypic analysis of particles in the nanometer size-range such as viruses, organelles, bacteria and extracellular vesicles (EVs). However, many of these particles are smaller than 200 nm in diameter, which places them at the limit of detection for many commercial flow cytometers. The use of reference particles of diameter, fluorescence, and light-scattering properties akin to those of the small biological particles being studied is therefore imperative for accurate and reproducible data acquisition and reporting across different instruments and analytical technologies. We show here that an engineered murine leukemia virus (MLV) can act as a fluorescence reference particle for other small particles such as retroviruses and EVs. More specifically, we show that engineered MLV is a highly monodisperse enveloped particle that can act as a surrogate to demonstrate the various effects of antibody labeling on the physical properties of small biological particles in a similar diameter range.

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Methodology for evaluating and comparing flow cytometers: A multisite study of 23 instruments

Cited by 17 publications

References 10 publications

Reproducibility of Flow Cytometry Through Standardization: Opportunities and Challenges

Reproducibility of Flow Cytometry Through Standardization: Opportunities and Challenges

Rigor and Reproducibility of Cytometry Practices for Immuno‐Oncology: A multifaceted challenge

Engineered Retroviruses as Fluorescent Biological Reference Particles for Small Particle Flow Cytometry

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