Flow cytometry: New tools to enhance accuracy and limit non-specific staining

Obtaining accurate and reproducible staining is critical for flow cytometry applications in clinical laboratories. In this exclusive SelectScience interview, we speak with Vishal Gehlot, Senior Development Scientist at Beckman Coulter Life Sciences, about the challenges researchers face in optimizing their flow cytometry staining. Gehlot goes on to provide insight into the newly launched SuperNova polymer dyes*, detailing how non-specific staining can be minimized, without compromising on overall quality.

SS: Tell us more about yourself and the big-picture goals of your work

VG: I contribute to product development at the Beckman Coulter Life Sciences Bangalore Development Center. Our team is one of the three teams across the globe working on designing bright polymer dyes. These single-color reagents are used by our customers – the majority of whom are clinical research laboratories – to build flow cytometry panels optimized to their requirements. The big-picture aim of this work is to enhance the accuracy of flow cytometry results by delivering staining with minimal non-specific results.

SS: What are the challenges your customers are currently facing?

VG: In flow cytometry, accuracy is critical. Events identified as positive should not be due to background staining. A commonly employed parameter called the Staining Index quantifies the separation between negative and positive cell populations as detected using a given conjugated antibody. It takes into consideration the relative brightness as detected on a specific flow cytometer, as well as non-specific staining or background. Dyes with a higher staining index are usually preferred for flow cytometry applications because they can identify dimly expressed cell populations, with greater confidence and with limited non-specific staining.

With the current bright dye reagents available in the market, customers face the issue of significant non-specific staining. This negatively affects the overall staining index. Further, many bright dye reagents cannot be used for clinical research applications. This may be because they are not produced under high-quality standards used in the IVD industry, such as ISO-13485 and current Good Manufacturing Practices (cGMP). Such challenges become tricky to manage as quality, reproducibility, and compliance requirements become increasingly stringent.

SS: Can you tell us more about these new polymer dyes Beckman Coulter Life Sciences has just launched?

VG: SuperNova dyes are a new family of water-soluble polymer dyes for flow cytometry applications. The exceptional brightness of these compounds can be attributed to their structure. The repeating monomer units in the dye have a strong capacity to absorb light (i.e., a high molar extinction coefficient) as well as a high fluorescence efficiency (i.e., a good fluorescence quantum yield). The core polymer dye has excitation and emission maximums at 414 nm and 428 nm respectively, making it detectable using an equivalent 450/50 bandpass filter in a flow cytometer. The dye is also designed such that it can be modified to make suitable tandem dyes, which can be excited at 405 nm and detected with appropriate filters. By controlling the length of the polymers, we ensure that the dye batches are consistent, assuring our customers of reproducibility when they use these conjugated antibodies.

SS: How do these dyes improve flow cytometry staining while overcoming common user challenges?

VG: The SuperNova violet excitable polymer dyes use a proprietary formulation – including a unique additive – which minimizes non-specific staining, considerably improving the staining index. As an example, one of our customers, who happens to be an early adopter of our technology, compared the staining index of a SuperNova v428 conjugated antibody to another Brilliant Violet dye (BV421). For SuperNova v428 conjugated CD19 antibody, the staining index was 134% on average versus the competitor bright dye conjugate¹. Moreover, at Beckman Coulter Life Sciences, we have designed our processes and facilities such that our end product, the bright dye antibody conjugates, can be used in clinical research. We have developed proprietary manufacturing processes to optimize dye stability and prevent issues faced with previous technologies and tandem dyes. The SuperNova** conjugates are manufactured adhering to the highest standards in the industry, such as ISO 13485 and cGMP.

SS: In your opinion, what are the key standout features of this product?

VG: The SuperNova conjugated antibodies are optimal for the assessment of dimly expressed cell populations. These antibodies also generate significantly less non-specific staining, providing greater confidence in the results. Lastly, the SuperNova conjugates are developed to meet the highest quality requirements in the industry, such as ISO-13485 and cGMP.

SS: What do you see as the future developments in this area?

VG: Our SuperNova polymer dyes* are a platform technology and we are working on developing further dyes that can be excited by different lasers of the flow cytometer. We anticipate more dyes being added to the Beckman Coulter Life Sciences catalog in the near future.

Currently, the multicolor panels that our customers build using our dye antibody conjugates are usually in liquid format. I anticipate that we will see dry panels using multiple polymer dye conjugates in the future. Such a development will have the potential to improve the workflow for customers and even enable high-throughput screening.
 
* For research use only - not for use in diagnostic procedures.
** Analyte-specific reagents. Analytical and performance characteristics are not established

References

1. Lassalle, L., Moulard, M., Ezzouaouy, B. Comparison of anti CD19 conjugates and performances of the newly developed SuperNova polymer dyes. [Poster]. CYTO Virtual Interactive, 2021.

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