Expanding the Flow Cytometry Toolkit with Next-generation Polymer Dyes

Figure 1: Absorption and emission spectra (λ_exc = 405 nm) of SuperNova v428 in phosphate-buffered saline.¹

by Brice Ezzouaouy, Senior Product Manager, Beckman Coulter Life Sciences

Since the emergence of SARS-CoV-2, scientists have discovered more about the role of the immune system and cytokine-associated processes responsible for systemic immune reactions that are typical in patients with COVID-19.¹ Flow cytometry is used to understand those processes at a single-cell level. It is the standard method used in immunology to characterize multiple phenotypic and functional parameters of single cells, including cytokine analysis.

Despite advances in flow cytometry instrumentation, reagents and analytical software, several challenges remain. Conjugated antibodies with improved brightness, stability and specificity are needed. They also need to have narrow excitation and emission spectra, so that different fluorochromes can be used in combination. That would enable scientists to analyze multiple parameters from one sample simultaneously. Low sensitivity also is a problem. Fluorescent conjugates can detect highly abundant markers accurately. But the biopharmaceutical industry also needs conjugated antibodies that are sensitive enough to precisely detect and quantify low-abundance markers. A further complication encountered with antibody-based methods, including flow cytometry, is that of non-specific binding of antibodies to Fc receptors expressed by multiple immune cell types.²

Brighter Options to Detect Dimly Expressed Markers

Manufacturers continue to innovate and produce new types of fluorochrome conjugates. Beckman Coulter Life Sciences, for example, recently launched a polymer dye conjugate called SuperNova v428 that is optimally excited by a violet laser at a wavelength of 405 nm (Figure 1).³ It has an excitation maximum of 414 nm and its emission wavelength peaks at 428 nm and is detectable using a 450/50 bandpass filter or equivalent. It is one of the brightest dyes excitable by a violet laser. SuperNova v428 dye is 10 times brighter than Pacific Blue dye. Because of this, it can better discriminate between negative and positive cell populations with significantly less noise.¹ Additionally, SuperNova v428 core polymer dye can be linked to acceptor dyes to create tandem polymer dyes, which share the same absorbance properties as the core, but absorbed light is transmitted through the polymer chain to the acceptor dye, which re-emits light at a higher wavelength.

Thanks to optimal fluorescence resonance energy transfer (FRET) efficiency, tandems maintain optimal brightness and provide additional options for flow cytometrists to assess dimly expressed markers.

Innovations in fluorophore technology, in combination with instrumentation, software and workflow advances have driven an increase of cytometric analyses and dramatically expanded the applications of flow cytometry. As the adoption of these tools increases, so do the specific needs of their users, meaning developers need to continue innovating to keep pace with these developments. Flow cytometry is being used in multiple laboratories across the globe, not only to analyze and decipher the cellular and molecular mechanisms underlying COVID-19 pathophysiology and characterize vaccines and drugs in development against this disease, but also to better elucidate pathologic mechanisms for other diseases, such haematological disorders or immuno-deficiencies, finally feeding into the era of precision medicine.

Better Dyes May Help Accelerate Drug Development

Drug development projects, such as those using fluorescent conjugated antibodies to elucidate a drug’s mechanism of action or pharmacodynamics, require the resolution that only bright dyes can provide. However, antibodies can vary from batch to batch, producing dramatically different results.⁴ Similarly, some polymer dye technologies also demonstrate variability between lots, leading to additional workload and potential delays, incurred for example by the frequent need to re-validate each lot, update protocols, change compensation matrices, re-titrate, and adapt to new patterns of data.⁵ This can be mitigated with the use of reagents that are manufactured under controlled and reproducible conditions, i.e., those of current Good Manufacturing Practice (cGMP).

Erroneous data can also occur when dyes stain cell populations other than that of interest.⁶ SuperNova conjugates, for example, are able to curtail this by ensuring the positive staining events are truly positive. This capability is achieved through a proprietary formulation containing unique additives to facilitate minimization of non-specific signals. The improved staining index of this next-generation polymer dye conjugate promises to be especially suitable for studying low abundance and dimly expressed markers.

While clinical laboratories have set quality standards, such as cGMP, and in vitro diagnostics regulations (IVDR), which clearly specify the products they should use, e.g., CE-IVD or analyte specific reagents (ASR), the same is not the case for drug development labs. There are no global quality standards for research use only (RUO) antibody-based products.^7,8 As such, drug development companies should seek products meeting minimum quality, regulatory and manufacturing standards regarding consistent product quality, specifications, and performance, over time and between lots. All products should comply with cGMP and diagnostics regulations, as well as ISO 9001/13485 standards. The application of such compliant products should minimize critical issues, such as a lack of reproducibility, which can arise from the use of reagents with inadequate quality standards regarding batch-to-batch consistency.

For decades, the irreproducibility of preclinical research findings has been an issue for drug development companies, as reported in a Nature paper in 2012, which shockingly found that only 11 studies could be reproduced out of 53 landmark cancer research papers.⁸ Some of this irreproducibility was likely due to technical differences or difficulties.⁸ This could include nonspecific or poorly validated antibodies.

Ultimately, it is worthless to set up state-of-art processes, such as extensive panel design and complex protocols, for drug development assays if the quality of the assay components, namely the conjugated antibodies, are of suboptimal standard. Even with highly skilled staff and robust procedures, insufficient reagent quality will likely lead to failed quality control assessments, challenges in interpreting data, and unnecessary repeats of work – or in a worst case, false results.

References

1. Cossarizza A, De Biasi S, Guaraldi G, et al. SARS-CoV-2, the Virus that Causes COVID-19: Cytometry and the New Challenge for Global Health. Cytometry Part A. 2020; 97A: 340–343.
2. Easwaran A, Tomasulo M, Mukesh Mudgal BHS, et al. SuperNova v428: New Bright Polymer Dye for Flow Cytometry Applications. https://www.beckman.com/gated-media?mediaId=%7B0B9C63D3-1567-404C-883C-CD9665FC83F0%7D (accessed January 2021).
3. Beckman Coulter Life Sciences. SuperNova Fluorescent Polymer Dyes. https://www.beckman.com/reagents/coulter-flow-cytometry/supernova-fluorescent-polymer-dyes (accessed May 2021).
4. Baker M. Reproducibility crisis: Blame it on the antibodies. Nature. 2015; 521: 274–276. doi: 10.1038/521274a.
5. Böttcher S, van der Velden VHJ, Villamor N, et al. Lot-to-lot stability of antibody reagents for flow cytometry.  J Immunol Methods. 2019; 475 :112294. doi: 10.1016/j.jim.2017.03.018.
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7. Uhlen M, Bandrowski A, Carr S, et al. A proposal for validation of antibodies. Nat Methods. 2016; 13: 823–827. doi: 10.1038/nmeth.3995.
8. Couchman JR. Commercial Antibodies: The Good, Bad, and Really Ugly. J Histochem Cytochem. 2009; 57: 7–8. doi: 10.1369/jhc.2008.952820.