Challenges in High Complexity Flow Cytometry

Mª Joséfa Marco
Department of Hematology
Hospital Dr. Peset

Please give an overview of high complexity flow cytometry. What contributes to its complexity compared to low complexity flow cytometry?

Many people confuse the term high complexity flow cytometry (HCFC) with the simple increase in the number of parameters that we can measure on a cytometer. For me, this concept is something much more complex. When we refer to HCFC, we’re not only talking about the number of colors, but also about quality, automation, analysis tools, reagents and staff.

Performing HCFC means improving in each of these aspects to obtain more complete, accurate and higher quality results; and, in my case, this directly affects the quality of life of my patients.

Why is high complexity flow cytometry used? What applications are there for this technique?

In the last decade, we’ve experienced a revolution in cancer immunotherapy. Significant advances have been observed not only in the direct targeting of surface tumor antigens via new monoclonal antibodies, like Daratumumab or Bispecific T Cell Engagers (BiTEs) like Blinatumomab, but also in the area of Adoptive Cellular Therapy (ACT) with the development of Chimeric Antigen Receptor T cells (CAR T cells) or the identification of molecules that overcome the inhibitory immune suppression created by tumor cells.

Immunophenotyping by flow cytometry plays a central and very relevant role in many of these therapies. In other words, flow cytometry is not only used for diagnosis and classification of lymphoma and leukemia, but also for detection of minimal residual disease (MRD) after treatment, to study the immune response of patients, and even for progress control of the treatment itself, such as monitoring the number of CAR-T cells that remain active in the patient after infusion.

Due to all these reasons, doctors and pharmaceutical companies demand more precise results from high sensitivity and higher quality testing. And, as I mentioned before, for this, it’s necessary to make improvements in each aspect of HCFC.

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What is minimal residual disease (MRD) detection, and how can flow cytometry be used in its detection?

Minimal (measurable) residual disease (MRD) refers to the small number of cancer cells that remain in the body after treatment. Traditional diagnostic measurements, such as immunohistochemistry and morphology, have detection sensitivities of only 10-2 - 10-3, which do not reliably predict progression-free survival (PFS) or overall survival (OS) after these treatments.

The ability to identify pathological cells can provide valuable information of clinical response and risk of relapse. MRD negativity is consistently associated with improved progression-free and overall survival. If appropriately validated and standardized, it could be used as a surrogate endpoint biomarker in clinical trials evaluating novel therapies. Then, there’s an increasing clinical interest in the measurement and achievement of MRD negativity in several hematological diseases, as for example in multiple myeloma, where MRD procedures are currently more developed.

Multiple myeloma MRD monitoring is currently done with sensitive platforms such as quantitative allele-specific oligonucleotide polymerase chain reaction (ASO-qPCR), next-generation sequencing (NGS), and multiparametric flow cytometry (MFC). Both ASO-qPCR and NGS have excellent detection sensitivities (10-5 - 10-6), but these technologies have lower applicability when compared to MFC. Conventional MFC can easily reach a detection sensitivity of 10-4, but to achieve the high sensitivity required for MRD evaluation, millions of cells have to be acquired and conventional immunophenotyping protocols are unable to achieve these numbers. Current consensus guidelines require a minimum of 2 million and recommend 5 million events be acquired to reach a minimum sensitivity of 10-5. In other words, we have had to increase the specificity and sensitivity of the technique to acquire a greater number of events and to differentiate normal from abnormal plasma cells.

What challenges are there to adopting high complexity flow cytometry?

In my opinion, those challenges can be included at three major stages. We must focus on the improvement of staining, acquisition and analytical processes. Of course, at all levels, elements of accredited quality must be introduced, which in my case must be valid for clinical diagnosis.

If we focus on sample-staining procedures, what we first think about is introducing automation processes to reduce staining times and eliminate human error. Immunophenotyping panels are becoming more complicated in adopting HCFC, and this directly affects the time a technician spends on reagent handling.

Improvement of acquisition processes is, perhaps, more directly linked to the development of high-performance flow cytometers with many parameters for the acquisition of complex panels. They must also have a high capacity for data processing, and, to facilitate quality control, they must make it easy to monitor performance, calibration, and compensation and standardization processes between cytometers.

Finally, we need qualified staff, as well as analysis software that allows us to carry out complex analysis strategies, and that can process a large number of events and parameters.

How can avalanche photo diodes (APDs) be used in place of photomultiplier tubes (PMTs) to help simplify the technology used?

I have had extensive experience using CytoFLEX cytometers in the lab for intra-laboratory research projects in oncology, pneumology and nephrology. This cytometer includes APDs (Avalanche Photo diodes) instead of PMTs (Photomultipliers tubes) as a photon detector. My feedback about APDs is really positive.

First, it has shown a great linearity between measured intensities (MFI) and detector gain settings. In a practical point of view, the software can automatically recalculate spillover values in real time as the gains are adjusted. This has allowed me to use the same compensation experiment in panels with different gain settings. So, no extra time is needed to do a compensation calibration as opposed to traditional flow cytometers using PMT detectors.

Another remarkable feature is their fluorescence sensitivity and low electronic noise. Sensitivity is the capacity to measure low-intensity signals—in other words, to see dim and bright populations in the same sample. When acquiring high numbers of events for MRD studies, sensitivity is crucial because antigen expression patterns of abundant normal populations may overlap with expression patterns of rare abnormal cell populations.

So, in order to get the clearest discrimination between normal and abnormal cells, I recommend high complexity flow cytometry (HCFC), where innovative staining techniques and APD technology flow cytometer are adopted.

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How can software and compensation to particular test parameters help researchers with high complexity flow cytometry?

I´m convinced that, for most flow cytometry users, any tool that facilitates automatic compensation through intuitive and easy-to-use software is essential today. This is especially true when carrying out high complexity flow cytometry studies, which require a greater number of multi-colored panels, where the combination of all fluorescence generates a large compensation matrix.

In our DxFLEX evaluation, we realized compensating 13 colors is very easy (we usually worked with between 8 to 10 colors). It´s only necessary to stain individual tubes for each fluorescence to be studied, acquire them using the automatic compensation module, and set the software to calculate the compensation matrix. This compensation matrix can be saved in a compensation library that can be used in combination with the catalog gain settings to create any combination of settings in a panel.

These features save a lot of time in the creation of experiments, and in monitoring performance of the cytometers, in addition to facilitating workflows.

How are analyte-specific reagents (ASRs) used in high complexity flow cytometry? Why is it important to use these, and what extra challenges do they bring?

Today, we´re under increasing pressure from government administration and health institutions to comply with a series of laboratory quality control regulations and accreditations to ensure the quality and reliability of results. Within this context, and under the premises even of public tenders, IVD marking is increasingly necessary for equipment, but also for reagents. One of our biggest challenges, therefore, is to increase the number of reagents with this qualification, since, in addition to facilitating accreditation, they minimize internal validation and give reliable results to avoid repetition of tests.

For example, IVD-labelled antibodies ensure quality products, the high performance of tandem fluorochromes, and lot-to-lot consistency, all important features in HCFC experiments. Finding the manufacturer with the largest catalog of these products will make a difference.

What can be done with sample preparation to simplify high complexity flow cytometry?

To simplify preparation of high complexity samples, you can choose to automate the process or use predesigned tubes with a cocktail of unitized antibodies.

In our laboratory we have extensive experience with Beckman Coulter DURAClone tubes in research projects. These tubes contain a combination of monoclonal antibodies predispensed and dried at the bottom of the tube. Using them requires less hands-on time and minimizes pipetting errors. These features have enabled us to significantly improve our laboratory workflows and lower costs due to a reduction in errors.

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What does Beckman Coulter Flow Cytometry offer to help overcome challenges in high complexity flow cytometry?

One of my biggest concerns in the daily routine of our laboratory was the need to acquire a sufficiently large number of events to achieve a sensitivity of 10-6 in the MRD study for multiple myeloma. With DxFLEX we have achieved this, as we have been able to acquire up to 25 million events per acquisition.

In addition, we have been able to test its maximum configuration with 13-color panels that provide greater specificity and complexity to the analysis, while reducing the number of tubes per study panel.

To all this, we must add that we have improved workflows in sample processing with DURAClone technology, and in the acquisition process with the automatic compensation, standardization, daily QC and monitoring performance modules included in DxFLEX software.

When I first became a flow cytometry user I worked with another commercial company. When I started working with Beckman Coulter Life Sciences, I was pleasantly surprised by their technological knowledge, and also the quality of the human and scientific support behind their products, including staff training and a willingness to help us implement new techniques.

What is the future of high complexity flow cytometry?

I believe that the natural evolution of HCFC will continue to require new tools to improve staining, acquisition and analytical processes. In this way, I´m happy to see that Beckman Coulter Life Sciences is developing products that provide solutions for each of these points, such as, for example, DURAClone technology to improve staining processes, and advanced equipment such as DxFLEX to improve acquisition processes.

However, I think that one of the biggest challenges will be when processing data, as soon we will be more capable of increasing the number of samples processed and the number of parameters analyzed. This will gradually move us toward high-dimensional single-cell analysis, which presents its own challenges in terms of data management, data visualizations, result reproducibility, computational power and information sharing.

In this sense, I have high expectations of Beckman Coulter as a result of their acquisition of Cytobank software, which enables cloud computing for flow cytometry analysis. Ongoing development by Cytobank and Kaluza software teams is promising, and I hope to include these new tools in future research.

Where can readers find more information?

• International Myeloma Working Group consensus criteria for response and minimal residual disease assessment in multiple myeloma. Kumar S, Paiva B, Anderson KC, Durie B, Landgren O, Moreau P, et al. Lancet Oncol. (2016) 17: e328–46. doi: 10.1016/S1470-2045(16)30206-6
• Consensus guidelines on plasma cell myeloma minimal residual disease analysis and reporting. Arroz M, Came N, Lin P, Chen W, Yuan C, Lagoo A, et al. Cytometry B Clin Cytom. (2016) 90:31–9. doi: 10.1002/cyto.b.21228
• Next Generation flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma. Flores-Montero J, Sanoja-Flores L, Paiva B, Puig N, Garcia-Sanchez O, Böttcher S et al. 2017 Leukemia Oct;31(10):2094-2103. doi: 10.1038/leu.2017.29
• Measurable residual disease by Next-Generation flow cytometry in Multiple Myeloma. Paiva B, Puig N, Cedena MT, Rosiñol L, Cordón L, Vidriales MB, et al. 2020 J Clin Oncol. Mar 10;38(8):784-792. doi: 10.1200/JCO.19.01231
• Practical Guidelines for Optimization and Characterization of the Beckman Coulter CytoFlexTM Platform. Bhowmick D, Sheridan RTC, Bushnell TP, Spalding KL. 2020 Cytometry part A. doi: 10.1002/cyto.a.23998
• A novel Semiconductor-Based flow cytometer with enhanced Light- Scatter Sensitivity for the Analysis of Biological nanoparticles. Brittain GC, Chen YQ, Nartinez E, Tang VA, Tylre M R, Langlois MA, Gulnik. Sci Rep 2019 Nov 5;9(1):16039. doi: 10.1038/s41598-019-52366-4

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About Mª Joséfa Marco 

Dra. Mª Joséfa Marco is a hematologist at the Department of Hematology, Hospital Dr. Peset, in Valencia, Spain. After completing her medical degree at the University of Valencia, Spain, she gained extensive experience in flow cytometry diagnosis involving the immunophenotyping of leukemia and lymphoma. In the specialized hematology laboratory, she has always had healthcare responsibility in flow cytometry, and she has been a proponent of introducing technological adaptations that are possible and necessary to improve patient care. Her research has focused on clinical-biological, diagnostic and minimal residual disease aspects with high complexity flow cytometry — mainly on monoclonal gammopathies — and she assists with design and development of clinical trials of immune status in patients with cancer and organ transplants.