Flow cytometry, the laser-based, biophysical technology employed in cell counting, cell sorting, biomarker detection, and protein engineering, is finding increasing applications in the biopharma and healthcare sectors. These include selecting an appropriate therapy, measuring therapeutic responses, evaluating predictive or prognostic biomarkers, and developing assays to aid in diagnosis.

Historically, however, the utility of flow cytometry has been limited by the challenge of standardizing instruments across facilities, companies and geographies. This article provides information for pharmaceutical researchers and flow core staff scientists on a new method of quantitative instrument standardization and set-up using stable, custom-manufactured fluorescent control beads. The use of such beads allows for greater consistency of intra- and inter-platform standardized setup, and provides a satisfactory basis for quantitative instrument standardization.  This new approach allows comparable quantitative results to be generated by multiple flow cytometers at the same or different sites. Topics covered in this article include how to perform quantitative instrument setup and how to address problems that may be encountered when performing quantitative flow cytometry.

The new method of intra and inter laboratory standardization of flow cytometry instruments allows instruments to produce data that shows very little variation from instrument to instrument for the same assays and sample types. The method is based on the use of BD Cytometer Setup and Tracking (CS&T) beads and custom-manufactured BD FC Beads. BD CS&T beads are comprised of three separate beads that are hard-dyed with varying amounts of fluorescent molecules that mimic fluorochromes that are excited by laser wavelengths of 355, 375, 445, 488, 532, 561, 633, 635, 640 nM. The brightest of the three beads was used originally used for the purposes of standardization, as it exhibited the least intrinsic fluorescence. Bright CS&T Bead Targets were set in worksheets that could be moved between instruments, allowing settings to be created and stored. These stored settings were applications settings that updated daily when the CS&T beads were run to cancel out day-to-day variations within individual instruments.

CS&T Bead Standardization Protocol
Instrument characterization data derived from setting baseline targets for each detector with the CS&T beads yields information on the electronic noise (SDen) of each detector, and the maximum linearity of each detector, known as the linearity max channel. This information is used to optimize the dynamic range of each detector. The SDen of each detector is multiplied by 2.5 to yield a standard deviation (SD) value to which unstained lymphocytes can be adjusted so that the contribution of electronic noise to the SD of the negative population is less than 10%.

Lymphocytes stained with a marker that exhibits the highest fluorescent intensity for that detector are run to adjust the positive staining population to below the linearity max channel value for that detector. The resulting photomultiplier voltages (PMTV) are then saved as Application Settings in BD FACSDiVa software, and the CS&T bright bead median fluorescence intensity (MFI) values were saved as target values. As a result, the same settings can be reproduced on any other instrument that needs to be standardized. Sample data are shown in Figure 1.

Problems with Quantitative Flow Assays
Problems were encountered during the transfer of quantitative flow cytometry assays across multiple BD FACSCanto II instruments that had been standardized using CS&T beads. A variation of more than 20% in the mean fluorescent index for fluorochrome-labeled molecules of equivalent soluble fluorochrome (MESF) calibration beads was observed for instruments that had been standardized using CS&T beads. This problem was the most severe for the fluorescein isothiocyanate (FITC) detector and made this detector unusable for quantitative work.

The variation between instruments was much less in the phycoerythrin (PE) and allophycocyanin (APC) detectors with variation ranging between 2% and 11% for the PE detector and 3.5% and 9.2% for the APC detector. These observable differences in MFI were attributed to:

• The use of beads hard-dyed with fluorochrome surrogates rather then the fluorochromes themselves; and
• Variations in the performance of the optical system between instruments (including laser power variability between instruments, filter performance. PMT sensitivity, optical noise, and laser alignment).
• A simple experiment was devised to investigate the utility of various beads to provide the recording and transference of standardization results between instruments. Cells were stained with CD4 conjugates to a cellular “gold standard” reference which removed any setup variation between instruments. The MFI ratio of various beads to the cellular gold standard was then determined. These included:  the BDTM CS&T bright bead; dried covalently linked FITC, PE, and APC fluorescent control beads (BDTM FC Beads) custom manufactured for Quintiles; Bangs Quantum TM FITC beads; SpherotechTM FITC beads; BD QuantibriteTM PE beads; Bangs QuantumTM PE beads; Spherotech PE beads; and Quantum APC beads. Experimental results are illustrated in Figures 2, 3 and 4.

The variance in the ratio reveals any error the bead may generate in the setting up for stained cellular samples.

Results: Reproducible Quantitative Set-Up
Fluorochrome specific beads rather than beads hard-dyed with fluorochrome surrogates provide a better basis for quantitative setup. To be of practical use over a long period of time, fluorochrome specific beads must be exhibit stabile levels of fluorescent intensity. Covalently linked fluorochrome beads that are dried and refrigerated for long-term storage are the best candidates for quantitative standardization for this reason.

Fluorescent control beads (BD FC Beads) were custom ordered from BD Biosciences for this purpose. The existing qualitative standardization of all flow cytometers at Quintiles was modified to utilize these new beads. New SOPs were written and distributed globally for routine flow cytometry practice. Data from standardized instruments are shown in Figure 5.

FCB Bead Standardization Protocol
Instrument characterization data derived from setting a baseline targets for each detector with the BDTM CS&T beads yields information on the SDen and maximum linearity of each detector. This information is used to optimize the dynamic range of each detector. The SDen of each detector is multiplied by 2.5 to yield an SD value to which unstained lymphocytes can be adjusted so that the contribution of electronic noise to the SD of the negative population is less than10%.

Lymphocytes stained with a marker that exhibits the highest fluorescent intensity for that detector are run to adjust the positive staining population to below the maximum linearity channel value for that detector. The resulting PMT voltages are then saved as Application Settings in BD FACSDiVa software, and the BD FC bead mean fluorescence intensity (MFI) values are saved as target values for each detector so that the same settings can be reproduced on any other instrument to be standardized.

BD FACSDiVA worksheets incorporating the targets and target ranges are emailed globally to all sites, as well as BD FC Bead lot information. A new baseline and a daily performance check are run on each instrument with the current global lot of CS&T beads. A new experiment is created in BD FACSDiVa and the FCB target worksheet is loaded. Detector PMT voltages are adjusted to the predetermined FCB target values for each detector. The resulting PMT voltages are then saved as Application Settings in BD FACSDiVa software. The Application Settings are used to create a master compensation matrix using a Master Compensation Panel (globally distributed). The master compensation matrix is linked to tube specific settings for all existing assay panels in a Master Experiment (globally distributed). Assays panels are then exported for daily use.

Daily Practice
A daily instrument characterization and update is performed by running a daily performance check with CS&T beads. Characterization data is compared to performance specifications developed from baseline values derived from all global cytometers. A daily folder is created in BD FACSDIVa. A new experiment is created in the daily folder, and updated Applications Settings are applied to the cytometer settings of the experiment. Assay panels to be run are added to the experiment, and a carousel or plate is loaded for automated acquisition. The data is acquired and immediately backed up centrally. Data is downloaded to dedicated data analysts for analysis and reporting.

Conclusion
Key learnings from these standardization exercises included:

• Fluorochrome-labeled particles are required to accurately and consistently set up and quantitatively standardize flow cytometers. Demonstrated setup accuracy as low as 5% is attainable, which is essential for quantitative and MFI-based assay analysis;
• Stabilized fluorochrome specific particles are an essential tool for reliable setup of one or multiple flow cytometry platforms in a single or multiple laboratories, and provide the basis of the calibration needed to achieve comparable results;
• Hard-dyed beads are satisfactory for setting up instruments for a given platform with reasonable accuracy, and for tracking of daily performance, but are not an adequate calibrator for quantitative standardization; and
• Consistent setup using fluorochrome-labeled particles allows users to compare quantitative assay results easily across instruments. 

References

1. 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. Available at http://www.ncbi.nlm.nih.gov/pubmed/22915363 [accessed March 22, 2015]
2. 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. Available at http://cytoconference.org/CMSPages/GetFile.aspx?nodeguid=8a4fabe3-9eec-409e-9513-ccecdca2e3b4&lang=en-US [accessed March 22, 2015]

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Mark Edinger is global scientific director at Quintiles.