Flow cytometry has long been the gold standard for cancer diagnostics due to its capability to analyze cell populations at a single-cell level and provide rich data faster compared to conventional methods. 

Clinical research laboratories employ flow cytometry as a key tool to investigate individual cells in liquid samples, such as peripheral blood and bone marrow, to determine cell characteristics and find disease biomarkers. While already helpful for diagnosing leukemia and lymphoma, flow cytometry's value continues to grow as emerging applications expand its capabilities.

Current challenges around increasing sample volume and complexity  

With the revelation of the complex cellular mechanisms involved in disease onset, flow cytometry is increasingly sought after. Tampa General Hospital is one of many research facilities utilizing flow cytometric applications. As a clinical scientist at the hospital, Marci O’Driscoll’s main focus is the improvement and validation of flow cytometry testing. 

Increasing sample volumes and the complexity of testing can be challenging for labs relying on manual operations. The characterization of heterogeneous cell populations requires intricate assay workflows including single-cell suspension, antibody cocktail preparation, cell staining, and analysis. Workflow complexity amplifies with an increased number of antibodies used. These manual steps are often laborious and can be a source of error, imposing challenges on reliable flow cytometric oncology studies. O’Driscoll noted that the shift from a five-color screening for leukemia and lymphoma samples to a 10-color assay was time-consuming, yet necessary, as manual preparation of seven to eight tubes per screening no longer sufficed.

Despite efforts to champion pathology laboratories and clinics through training programs, the need for reliable and rapid flow cytometric assays remains unfulfilled. In fact, there is a nearly 10 percent vacancy rate in the US for expert flow cytometrists. “The gap could get worse because the need for flow cytometry expertise grows more rapidly than the capacity of training programs,” O’Driscoll explains.

Automation to solve time and reproducibility issues in flow cytometry 

Automation of flow cytometry workflows can help maintain the turnaround time and establish reproducibility despite increased sample volumes and reagent scopes. Automated workstations demonstrate the value at various steps of the workflow by allowing the simultaneous processing of multiple tubes, sterilizing pipette tips, and integrating centrifuge automation. Thus, the time spent at the bench is significantly reduced, while human-caused run-to-run variability is mitigated. 

For the Esoteric Lab at Tampa General Hospital—which was running 2,700 leukemia/lymphoma immunotyping analyses per year, as well as screening for paroxysmal nocturnal hemoglobinuria (PNH), fetal hemoglobin testing, and Kleihauer-Betke (KB)—the potential for automation was clear. In 2022, the laboratory decided to enroll in the beta-testing of our automated flow cytometry sample preparation system. 

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One of the highlights of automated sample preparation is the ease of protocol setup, O’Driscoll states: “Only minimal changes were needed for the leukemia and lymphoma screening, such as incubation times, antibody mix volume, and the cell wash protocol.” Nevertheless, the benefits of automation far outweigh the minor challenges. Compared to their initial manual sample preparation protocol, Tampa General Hospital could save up to 51 minutes per one Leukemia & Lymphoma Immunophenotyping assay with automated sample preparation. 

“Flow cytometry automation could help our lab to support the significant increase in both flow case number and case complexity,” says O’Driscoll. “With automated sample preparation, we experienced substantial increase in walkaway time and reduced waste in our liquid antibody inventory. To add to that, introducing custom dried reagents into this workflow significantly improved standardization, reduced errors resulting from manual master mix preparation, and minimized the quality control burden for the team.”

Wet vs. dry: The many advantages of dry antibody panels over liquid cocktails

Reagent reliability is crucial to success in flow cytometry. As O’Driscoll states, the benefits gained from automation were accompanied by the standardization of antibody panels, which further empowers standardization in flow cytometric assays. Laboratories and clinical scientists have been using bespoke liquid antibody cocktails for multicolor flow cytometry due to their precision and volume-based cost savings. 

However, custom-manufactured liquid antibody cocktails come with a few caveats. They generally have a shelf life of approximately one month, preventing their use in longitudinal studies. They need to be stored under refrigeration, often resulting in additional costs for transport and storage. Furthermore, liquid cocktails can lose stability—hence performance and accuracy—over time, even when stored under desirable conditions, which brings their reliability into question. Lyophilization, which involves the removal of water content from the mix after freezing, may be a solution; however, it prolongs sample preparation tremendously and requires expensive equipment.

Now, life sciences companies strive for a more efficient method to transform flow cytometry assays. These companies use proprietary processes to manufacture dry antibodies that form a uniform layer that adheres strongly to the bottom of the tube. This prevents the antibody mix spillage due to electrostatic forces, shaking, or other lab accidents.

Dry antibody panels offer several advantages over liquid or lyophilized reagents, such as ease of storage, long-term stability, and batch-to-batch consistency. Unlike liquid cocktails, dry antibody panels can be stored at room temperature, which eliminates the need for refrigeration. Heat stress experiments also show that they can tolerate temperatures up to 60°C and exhibit consistent staining after seven days, whereas significant degradation is observed in liquid reagents. 

When comparing the performances of new and 200-day-old dry reagents with blood sample tests, consistent staining is observed, meaning that dry reagents can retain their stability long after initial manufacturing. It is clear that dry antibody panels are instrumental to robust flow cytometric assays that can be repeated and reproduced over a long period, e.g., for longitudinal studies evaluating long-term changes in cell populations in response to anticancer compounds.

Other factors to consider are cost and time savings achieved by dry antibody panels. Thanks to their easy integration into automated flow cytometry workflows, they positively contribute to the time reduction enabled by automation. Considering the total time for manual steps, such as specimen preparation, cocktail preparation, and screening, O’Driscoll reports her Tampa General Hospital laboratory has found that using dry antibody panels can save them an average of up to 400 hours of annual bench time for scientists. For an average eight-hour workday, this effectively gives scientists up to 50 additional working days per year to focus on their critical research to enable pioneering new discoveries.

While the initial cost of commercial panels might be of concern to laboratories, the long-term savings compensate for it. Pathology labs must consider a multitude of factors when calculating cost savings, including the number of clinical samples processed, the number of mastermix preparations, annual compensation for the bench technologist, cold shipment, quality control tubes for the validation of liquid cocktails, and the rate of increase in sample size. Because dry antibodies eliminate many of these steps, e.g., QC of antibody panels, since they are pre-validated by the manufacturer, they can significantly reduce annual expenses. 

There can also be significant cost savings. A midsize flow lab running about 200 clinical samples per month could save about $100,000 annually by using dry antibody panels instead of liquid cocktails for a typical 4-tube 10-color leukemia/lymphoma panel.

What is next for flow cytometry laboratories?

The advancements in automation and reagent panels can lead to a wider adoption and confidence in flow cytometry-aided diagnostics. O’Driscoll believes that flow cytometry is at the center of the Esoteric Lab at the Tampa General Hospital Cancer Institute, offering robust diagnostics, treatments, and care to their patients. Her excitement is not limited to the laboratory. 

“Automated flow cytometry enables doing more with less,” she says. “The movement of spectral flow cytometry, advanced visualization, and analysis programs toward the clinical labs will give the potential for additional characterization of cellular populations from smaller sample volumes. Additionally, this will prompt labs to lean more into automation and standardization to increase capacity as many flow labs increase volumes.”