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Mark Edinger | December 17, 2015

Setting Up for Success

Until recently most flow cytometers were routinely characterized and calibrated using beads that contained surrogate dyes, with standardized quantitative measurements restricted to those few fluorochromes for which Molecules of Equivalent Soluble Fluorochrome (MESF) beads or assay specific setup beads were available.
Until recently most flow cytometers were routinely characterized and calibrated using beads that contained surrogate dyes, with standardized quantitative measurements restricted to those few fluorochromes for which Molecules of Equivalent Soluble Fluorochrome (MESF) beads or assay specific setup beads were available. What was needed were fluorochrome specific calibration beads that would allow all the detectors on any instrument to be setup to provide reproducible quantitative measurements of fluorescence on the same instrument day to day, across instruments, and across multiple instrument platforms.  To this end, working with BDTM, Q2 Solutions has developed a procedure for flow cytometry instrument setup, employing covalently linked lyophilized beads, which reduces the variation between instruments on all detectors, and promises to improve the relevance of many of the key tests performed in the biopharmaceutical and healthcare world. It will allow us to achieve a level of quantitative standardization among instruments that makes flow cytometry data more accurate and reproducible across facilities, geographies, instruments throughout the duration of a study, as a part of daily use.

Flow cytometry has been used for decades to assess key biological hypothesis and to provide insights into physio-pathological mechanisms. However, flow cytometry is now rapidly gaining popularity in the biopharmaceutical sector due to its direct relevance to new therapeutic modalities being investigated, and the ability to readily ascertain the response of the immune system to an individual therapeutic. A flow cytometer can measure identity, function and status at level of the individual cell. Flow cytometry data can thus be used for determining appropriate therapies for a patient, measuring therapeutic responses, and evaluating predictive or prognostic biomarkers. 

However, while there has been an increasing focus on the standardization of flow cytometry by many groups for the last decade or so, the efficacy of flow data has been limited due to the lack of complete quantitative standardization of instruments. Most analytic procedures require standardization for the data be relevant, for if variation exist between different tools, or the same tools on different days, it can compromise the comparative value of those results. Flow cytometry is no exception. Without standardization of these tools, researchers cannot accurately detect changes against baseline measures, conduct tests across varying facilities, or reliably publish results as there is no uniform standard of quality. 

Fluorescent beads are the answer

That all could change if we as an industry adopt quantitative instrument standardization for flow cytometry, using stable, detector matched fluorescent control beads.
Fluorescent control beads, which have a long shelf life and stability, can help achieve standardization of flow cytometry setup because they provide the basis for a long-term and consistent calibration method that allows researchers to reliably set for and measure the quantitative performance of their instruments. This same method also can be used to identify anomalies in performance on a periodic basis, which ensures problems are addressed before study results are negatively impacted. When incorporated into routine practice, calibration with stable fluorescent beads enables greater consistency of intra- and inter-platform performance, which ensures minimal variation from instrument to instrument for the same assays and sample types.

To demonstrate the utility of fluorescent control beads for quantitative setup an experiment was devised whereby the Mean Fluorescence Intensity (MFI) of various fluorescent beads could be compared to a “gold standard”, that gold standard being lymphocytes stained with CD4 conjugated to the fluorochrome best matched to the emission spectra of the fluorescent bead under comparison. The basis of the comparison was the bead to cell MFI ratio.

The results of the bead to cell comparison showed up to 20 percent variation for the fluorescein isothiocyanate (FITC) detector, with the phycoerythrin (PE) and allophycocyanin (APC) detectors showing between 2 and 11 percent variation across five different FACSCanto II instruments standardized with BDTM CS&T Setup and Tracking Beads. It also was found that fluorochrome-specific beads, rather than beads hard-dyed with fluorochrome surrogates, provided a better basis for quantitative setup. 

To be of practical use throughout a long period of time (>1 year), fluorochrome-specific beads must exhibit consistent levels of fluorescent intensity. To achieve this level of stability and consistency, covalently linked fluorochrome beads that are dried, stored with desiccant and refrigerated for long-term storage, are the best candidates for quantitative standardization.

Based on these results, we adopted custom manufactured fluorescent control beads to establish quantitative standardization of all our flow cytometers. New Standard Operating Procedures (SOPs) for the adoption of this approach, were written and distributed globally for routine flow cytometry practice, ensuring an enhanced level of standardization across our seven global flow cytometry facilities. Instruments are tested weekly with these same beads to demonstrate continuity of standardization, with the detection of any drift triggering a standardization of the instrument. 

At Q2 Solutions, we think the use of long-term stabilized fluorescent control beads for quantitative standardization will change the way flow cytometry is practiced in laboratory settings around the world. If this standardization approach is universally applied across the industry, it will provide the basis for comparable quantitative results to be generated by multiple flow cytometers at the same or different sites, especially for assays where determination of the relative levels of antigen expression are of particular interest. This will not only add value for individual researchers, it will generate broad-based value for the entire biopharmaceutical research community.

Hoffman RA, Wang L, Bigos M, Nolan JP. NIST/ISAC standardization study: variability in assignment of intensity values to fluorescence standard beads and in cross calibration of standard beads to hard dyed beads. Cytometry A. 2012;81:785-796.

Yan M, Edinger MG, Zhu L, Crowther E, Sharkey M, Jaimes MC, Rogers T.  A comparison of stable fluorochrome-specific beads and hard-dyed beads for standardized quantitative flow cytometer setup. Poster B221 Cyto/ISAC 2014.

Meinelt E, Reunanen M, Edinger MG, Jaimes MC, Stall A, Sasaki D, Trotter J Standardizing Application Setup Across Multiple Flow Cytometers Using BD FACSDiva™ Version 6 Software BD Biosciences March 2012 Technical Bulletin.

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