Among its many clinical uses, fluorescence-based flow cytometry aids laboratories in the diagnosis of blood cancers and other disorders. The Division of Hematopathology within the Department of Laboratory Medicine and Pathology at Mayo Clinic in Rochester, Minnesota, performs both basic and specialized hematology testing via six specialty labs. Its Cell Kinetics Laboratory, in particular, uses flow cytometry as a primary technology to diagnose leukemias and lymphomas from blood, bone marrow, fluid, and tissue specimens. Clinical diagnosis of most hematological diseases, especially malignant forms, requires clinicopathologic correlation, and flow cytometry can play an important role in pathological diagnosis. The processes employed by flow cytometry help distinguish abnormal from normal conditions and provide an expedient method of establishing clonality and aberrant antigen expression on abnormal populations.

The Cell Kinetics Lab employs 28 staff and utilizes 9 flow cytometers to manage its volume demand. The lab analyzes high volumes of mostly malignant samples sent from all over the world in addition to those from patients at Mayo Clinic. All specimens must be preprocessed, and the lab purchases monoclonal antibodies that attach to one type of cell antigen (ie, cluster of differentiation [CD]), multiples of which can be found on each cell surface. These acquired antibodies are pre-conjugated with one or more fluorescent markers, and there are many color options for each CD marker, adding flexibility to panel makeup. Monoclonal antibodies can be expensive but can have a substantial shelf life of 6 to 18 months. The Cell Kinetics Lab stocks anywhere from 70 to 80 different antibodies in refrigeration for use in flow cytometry processes.

The lab’s flow cytometers can detect 6, 8, or 10 colors. A single tube of processed patient cells can be stained with up to 10 antibodies (differentiated by color). The stained cells in each tube are run through the flow cytometer, and by placing multiple antibodies in each patient tube, the lab is able to reduce the number of tubes needed for complex gating and analysis. This efficiency reduces costs to the lab and increases quality, as staining, running, and analyzing 10 individual antibody tubes, and applying a gating structure that makes sense across all 10 would be a difficult and time-consuming task.

Create Efficiencies with Flow Cytometry

Reimbursement from payers is a complex process, as each used antibody is only reimbursed once. If an antibody is used multiple times, say in multiple tubes for a single patient assay, the lab is only reimbursed for the cost of one. For example, if CD45 antibody is needed for cell identification purposes in 10 tubes, only one incidence of CD45 can be reimbursed (the lab must incur the cost of using CD45 in multiple tubes). Conversely, with a 10-color, single-tube assay, the technologist only needs to add CD45 once and will be reimbursed for that antibody. Such advantages make multicolor cytometers highly beneficial to Cell Kinetics Lab operations.

With a 10-color, single-tube panel, as with any multi-antibody panel, the antibodies must either be mixed ahead of time or be individually aliquoted by technologists for each patient test. The latter is a time-consuming, detailed process, which is why the Cell Kinetics Lab prefers to pre-mix or “cocktail” most antibodies prior to use. This mitigates the number of additional antibodies that need to be added to a patient specimen by a busy technologist at the bench. Prior to clinical use, every cocktail has to be validated to ensure all expected antibodies have been added appropriately and the end product is deemed ready for use.

To expedite the cocktailing process, Cell Kinetics has started working with its major supplier, whereby the company produces lyophilized vials (or lyovials, as the laboratory staff refers to them), which have been custom made to contain all desired antibodies. This saves the laboratory hours of labor from cocktailing tubes in-house. The lyovials are more expensive than purchasing liquid antibodies individually, but the time and labor saved (as well as the reduction in repeat testing) overrules the cost, and the lab plans to continue moving more panels in this direction.

Benefits of Layered Analysis

Patients with more complex blood disorder cases tend to require a greater number of antibodies for diagnosis, and multicolor flow cytometers allow technicians to see a larger spectrum of cell types for these complex panels. Layered analysis allows for cell type enumeration and identification of positivity or negativity. It also enables white cell differential and cell type identification according to scatter properties and size. Such layered analysis gives technicians a fingerprint (ie, phenotype) to allow for classification of each cell—known, published phenotypes are commonly used. The cells are then sub-classified into disease states of various leukemias (eg, acute myeloid leukemia, acute lymphoid leukemia, chronic lymphocytic leukemia, T– and B-cell lineage determinations).

In the hematopathology division at Mayo Clinic, the overall diagnostic process is a highly collaborative effort. Technologists run flow cytometry instruments, perform highly complex analysis, and send all material to the pathologists who correlate it with the morphology. Key to this process is pathologist follow-up and discussion with clinicians. Instead of simply passing along a result—such as a positive for CD19 and CD20—to a clinician, the pathologist actively advises the clinician on what the results mean.

Fortunately, flow cytometry allows the Cell Kinetics Lab to meet both internal and external demands for a short turnaround time (often within the same day) on sub-classifications for leukemia diagnosis, thereby allowing clinicians to commence appropriate patient therapy as soon as possible.

Minimal Residual Disease Testing & Sorting

Recently, the Cell Kinetics Lab has increased its focus on minimal residual disease testing using flow cytometry, which entails collecting data for many more events (cells) than a diagnostic leukemia lymphoma. Moving forward, order volumes for minimal residual disease testing are expected to increase. The lab also has recently added cell sorting to its flow cytometry services. Last year, the lab started sorting cells prior to performing molecular assays that focus on specific cell types, such as plasma cells. Similarly, cell sorting is expected to expand and become a large part of the laboratory’s function in the future.

Justifying the Acquisition of Flow Technology

Given the expense of purchasing flow cytometry technology (as well as the ongoing cost of antibodies), acquisition proposals must be well justified. This usually begins with the hospital or health system gaining the infrastructure and volume to support flow cytometry, in-house. Assuming these two conditions are met, the technology itself requires specialty training. Flow cytometry technology has critical diagnostic utility in hematological diseases (as well as important prognostic utility in a subset of hematological diseases), and the equipment requires dedicated, well-trained technologists to properly perform analysis and interpretation.

In the Cell Kinetics Lab, technologists are trained to identify which antibody colors work best for certain applications. Panels are created and tested, and adjustments may be necessary for a variety of reasons. For example, a different color antibody may need to be ordered or utilized in a different instrument channel in order to optimize each panel. Instrument settings and compensation are necessary to show that all stained cells in a tube have proper separation between positive and negative and do not interfere with other cell types or fluorochromes. These adjustments for compensation speak to the detailed knowledge that staff need to ensure a successful outcome.

Both high internal volumes and a variety of outreach samples can help create a patient population volume that represents a broad base of case types. A large sample variety will help technologists gain proficiency in ensuring the antibodies are working properly. Labs with limited flow cytometry volumes may struggle to qualify that their antibodies are functioning properly due to low exposure to lesser-known abnormalities. Accordingly, patient sample volume and variety are important factors in flow technology acquisition proposals.

Remaining Challenges

Whereas many biochemistry and hematology laboratories have a variety of manufacturing controls in place for assays, including low-, medium-, and high-range controls, many clinical labs newly introduced to flow cytometry do not have this luxury (other than for certain assays, such as CD34, CD4, and CD8). This lack of standardized controls is largely due to the fact that each leukemia lymphoma lab uses a different type of panel or group of antibodies. Given the Cell Kinetics Lab’s stock of approximately 95 antibodies at any given time, it remains a challenge to maintain proper controls for all of them. This illustrates that the more comprehensive the flow cytometry lab, the more details must be attended to.

Another ongoing challenge to flow cytometry labs (as well as clinical labs in general) is recruiting qualified and apt technologists. Thus, it can be quite valuable to review current staff for potential candidates displaying the desire to manage flow cytometry operations at the level of a developmental technologist. This requires working closely with lab directors and pathologists in the flow assay development process.

Regardless of these challenges, flow cytometry produces reliable, high-quality results, and with a wide range of client samples, the hematopathology group and the Cell Kinetics Lab will continue to develop new assays and panels to meet a growing demand.

Christophe Bahn is a freelance writer specializing in clinical research and discovery for Mayo Medical Laboratories in Rochester, Minnesota.

Connie Penz, MBA, is the supervisor of the Cell Kinetics Laboratory at Mayo Clinic in Rochester, where flow cytometry is the primary platform. She earned a BS in chemistry from the University of Wisconsin–Eau Claire and an MBA from Cardinal Stritch University.

Cecelia Meyers is an operations manager in the hematopathology division of the Department of Laboratory Medicine and Pathology at Mayo Clinic in Rochester. She is a former technologist and supervisor of Mayo’s Flow Cytometry Laboratory.

Min Shi, MD, PhD, is an assistant professor in the Department of Laboratory Medicine and Pathology at Mayo Clinic, and is co-director of the Cell Kinetics Laboratory. She received her MD from Beijing Medical University and her PhD from the University of Massachusetts Medical School.

Dragan Jevremovic, MD, PhD, is a hematopathologist and co-director of the Cell Kinetics Laboratory. He graduated from Belgrade University in Serbia and received his PhD and performed his residency and fellowship training at Mayo Clinic in Rochester.