Standardizing Flow Cytometry Assays

With flow cytometry increasingly being used in laboratory developed tests (LDTs) and throughout the development of biological therapeutics, there is a growing need to move toward greater standardization. This article discusses some inherent challenges of standardizing flow cytometry assays and suggests ways of addressing sources of variation. It also provides links to open-access resources and publications from experts in the field.

Standardization has many different levels

Standardization in flow cytometry can have many meanings. Emily Cartwright, Ph.D., Product Marketing Specialist at Bio-Techne, notes that it could refer to the standardization of instrumentation, markers used to define cellular subsets, gating strategies for data analysis or, more generally, the requirements for reporting flow cytometry data. “Standardization of flow cytometry assays would allow experimental results to be compared directly to results acquired on a different cytometer, on a different day, and across different institutions and study centers,” she explains. “It could also lead to a role for flow cytometry in clinical diagnostics.”

Critically, at the present time, flow cytometry is not completely standardized despite exhaustive efforts by the scientific community. Dr. Maria C. Jaimes, Vice President, Applications at Cytek Biosciences, reports that because standardization in the context of flow cytometry requires reporting results in calibrated rather than in arbitrary units, and also means obtaining the same readout whenever exactly the same particle is run, the very nature of the technique makes this difficult to achieve. “When it comes to a biological assay that uses cells stained with fluorochromes, there are numerous sources of variation, including the particle itself,” she says. “Although the National Institute of Standards and Technology launched an initiative to develop fluorochrome-labeled particles for use as standards, there has so far been little traction from users and manufacturers.”

Addressing sources of variation

Every application comes with its own challenges for standardization, yet the uniqueness of flow cytometry presents inherent difficulties not associated with other immunoassays. First, with so many different flow cytometers available, instrument performance represents a major source of variation. “While it is possible to standardize instrument setup using calibration beads for monitoring mean fluorescence intensity across detectors over time, this is specific to the instrument in question,” notes Jaimes. “Standardizing performance between instruments is currently more difficult unless data output can be calibrated in standardized units due to the sheer range of hardware components available and the variation within each component.” For example, laser power can vary greatly between flow cytometers. “Fluorochromes will be brighter when exposed to more photons, so any variation in the number of excitation events will impact fluorescence intensity,” explains Cartwright. “This can make the same sample appear differently on two different instruments.”

Sample preparation technique can also cause variation. Mike Blundell, Product Manager for Flow Cytometry Reagents at Bio-Rad, comments that while any application used for analyzing live cells can be hard to standardize, sample preparation for flow cytometry can especially present problems due to the requirement for a single-cell suspension. “The method used for preparing a single-cell suspension can be critical,” he says. “Dead cells will bind antibodies non-specifically and have higher autofluorescence, meaning the resolution of detection may be reduced. One way of minimizing variability is to establish a standard operating procedure for sample preparation that preserves the quality of your cells. Once conditions have been optimized, adhering to the protocol helps ensure that any observed differences between samples are real and not simply an artefact of sample handling.”

Following sample preparation, immunostaining introduces yet further variability. “There is no universal standard for conjugated antibodies for flow cytometry and different vendors can have different conjugation protocols, resulting in different fluorescence to protein ratios,” says Cartwright. “Using the same fluorochrome from a different vendor can consequently lead to variation in fluorescence intensity.” Castle Funatake, Ph.D., Senior Manager, Research and Development, Protein and Cell Analysis, at Thermo Fisher Scientific, recommends that to minimize variability over time, and especially for large, long-term studies, a sufficiently large stock of fluorochrome-labeled antibodies should be sourced and any expiration dating noted. “If using a single lot of a specific fluorochrome-conjugated antibody throughout the study is not possible, then a side-by-side comparison of the different lots should be performed to ensure consistency,” she suggests. “Alternatively, multiple lots could be combined and titrated as a ‘mixed lot’ but this assumes that the performance variation between lots was minimal to start and that there were no significant differences in expiration date.”

Analyzing flow cytometry data is largely subjective since the inherent variability of biological samples limits the use of automatic gating tools. Yet, by standardizing the data files of any flow cytometer, the Flow Cytometry Standard (FCS) goes some way toward addressing user bias. “The FCS ensures that all of the information needed to describe the data is contained within the data file and that the file format generated by any instrument can be analyzed by any other instrument or third-party software,” explains Funatake. Guidelines around the minimum information for a flow cytometry experiment (MIFlowCyt) needed for publishing have also been established. “Parameters required for reporting include the instrument used for acquisition, the clone and fluorochrome for each antibody marker, the gating strategy for defining cell subsets, and the software used for analysis,” says Cartwright. “Requiring reporting of other parameters, such as antibody incubation temperature and time, will help increase experimental reproducibility.”

Next steps

Attempts to standardize flow cytometry are many and varied. Blundell highlights the work of the EuroFlow consortium, which has led to the development of a range of open-access protocols and SOPs that have been standardized and validated in multi-center trials, as well as that of the Human ImmunoPhenotyping Consortium, which aims to promote standardization of flow cytometry immunophenotyping in clinical studies. Funatake adds that these are complemented by the guidelines published by Cossarizza et al in 2017, which are based on the combined knowledge of experts in flow cytometry, immunology, and biotechnology and provide recommendations for standardizing assays and protocols successfully. “Most recently, the Clinical and Laboratory Standards Institute published H62—Validation of Assays Performed by Flow Cytometry, which is applicable to flow cytometry performed in any setting,” she says. While flow cytometry still has a long way to go before it becomes fully standardized, things are definitely moving in the right direction.

Useful resources

CYTO University—a portal hosted by the International Society for Advancement of Cytometry (ISAC) that includes webinars, e-learning courses, and other flow cytometry resources.

EuroFlow consortium—a range of protocols and SOPs that have been standardized and validated in multi-center trials.

Flow Cytometry Expert for the Non-Expert—a book providing a brief history of flow cytometry and its use in research, as well as protocols and guidance on experimental setup 

Open Flow initiative—a collaboration between The Francis Crick Institute, Memorial Sloan Kettering Cancer Center, and the Whitehead Institute for Biomedical Research that regularly hosts free webinars that are also available on YouTube.

Purdue Cytometry Discussion List—a platform where users can engage in discussions about technology, experimental design, troubleshooting, and data interpretation, and can search an archive for topics of interest.

Cossarizza, A., et al. (2019). Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). European journal of immunology, 49(10), 1457–1973.

Finak, G., et al. (2016). Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium. Scientific reports, 6, 20686.

Spidlen, J., Breuer, K., & Brinkman, R. (2012). Preparing a Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) compliant manuscript using the International Society for Advancement of Cytometry (ISAC) FCS file repository (FlowRepository.org). Current protocols in cytometry, Chapter 10