Go with the Flow

Laboratory medicine professionals involved in the diagnosis of leukemia and lymphoma will be familiar with the immunohistochemistry (IHC) techniques used to spot these diseases. It’s likely that they are also familiar with flow cytometric immunophenotyping (FCI), an approach that – although less common – carries significant advantages.

In 2013, the FDA held a workshop that brought together laboratorians dealing with the complexities and expense of laboratory-developed flow cytometric tests (LDTs), clinicians relying on correct diagnoses to drive discovery and individual patient treatment, and regulatory agencies concerned with the safety risk posed by non-standardized assays. As a result of that workshop, Beckman Coulter Life Sciences pursued a de novo pathway to bring to market first the original five-color ClearLLab assay and, ultimately, ClearLLab 10C.

Each ClearLLab antibody combination is designed to characterize expected normal cells in a sample, while simultaneously highlighting and characterizing aberrant cells. Every tube contains CD34 and CD45 for the detection of blast populations and their cells of origin, and the tubes’ design also allows maturation sequences to be identified, a key method for detecting abnormal populations. The ClearLLab 10C system’s standardized instrument setup, 10-color control material, and analysis templates reduce the learning curve and make FCI more readily available to laboratories. Mike Keeney, Coordinator for Investigational Hematology and Associate Scientist at the London Health Sciences Center, and Jeannine Holden, Chief Medical Officer and Vice President of Scientific and Medical Affairs at Beckman Coulter Diagnostics, discuss the advantages of this approach.

How does flow cytometry improve on IHC for leukemia and lymphoma testing?

Mike Keeney: Flow cytometry plays a pivotal role in the diagnosis and follow-up of patients with leukemia and non-Hodgkin lymphoma (NHL). Its advantages over IHC include the ability to test a variety of small samples (including fine needle aspirates and body fluids), “gate out” cell fragments and debris from analysis, examine 10 intracellular or surface antigens simultaneously, detect level of antigen expression, and resolve multiple hematopoietic malignancies in the same sample.

IHC still has an important role to play in Hodgkin lymphoma and in situations where bone marrow aspirate results in a suboptimal sample. In addition, IHC preserves the architecture of the sample, which can aid the differential diagnosis of several lymphomas. However, a key advantage of flow cytometry is the speed at which samples can be analyzed. A stat result can usually be provided within hours, whereas the requirement to fix, decalcify, and embed the bone marrow in wax adds a significant delay.

Jeannine Holden: The fundamental advantage of FCI over IHC is FCI’s capacity to simultaneously assess numerous markers. Whereas IHC can generally only assess one or two markers at a time, commercially available clinical flow cytometers can easily assess 10 at once, identifying co-expression of any combination of markers in a single tube. By repeating certain markers across several different tubes (antibody redundancy), the findings can be extrapolated to generate an even more detailed immunophenotype for essentially every single cell in the sample, while simultaneously assessing each cell’s relative volume and cytoplasmic complexity. Finally, FCI can distinguish subtle differences in antigen density, permitting distinction among various normal and pathologic cell types that are difficult or impossible to perform with IHC.

This fundamental difference is the basis for two other advantages: small sample size and small target size. Whereas the serial sections required for detailed IHC quickly consume the small biopsies common in hematolymphoid malignancies, FCI generates a detailed immunophenotype on the first round of testing using a relatively small number of cells. Because the phenotypes of normal cells are reproducibly preserved within and between patients, FCI can distinguish and characterize aberrant cells that represent only 1 percent of the total sample* – and, because of its speed, neither antigen degradation due to tissue processing nor antigen retrieval is a concern (1).

FCI is the standard of care in the US for hematolymphoid malignancies, and in Europe and the rest of the developed world for patients whose disease presents primarily in the bone marrow and/or peripheral blood. Lymph nodes and tissues are increasingly studied with FCI in these geographies as well.

How does the ClearLLab 10C system improve efficiency and reduce error?

JH: By eliminating most of the manual steps involved in staining and/or reagent cocktail preparation, ClearLLab 10C minimizes manual pipetting errors in these steps – as well as reducing the risk of repetitive motion injury from reagent pipetting. From a laboratory efficiency perspective, ClearLLab 10C eliminates the need for panel design and its shelf-stable reagents eliminate the need for cold shipping and cold storage. The waste generated by wet reagent handling is no longer an issue, and low- and high-volume laboratories can use the same system and scale kit orders appropriately as their volumes grow.

MK: Because most flow cytometry laboratories use their own LDTs, it’s difficult to compare results across centers. Additionally, the level of expertise in choosing the right antibodies and fluorochromes for leukemia and lymphoma immunophenotyping is challenging and requires highly trained personnel – especially as the number of colors increases to as many as 10. The time spent validating different antibody clones, preparing and validating cocktails, and ensuring that cocktailed reagents do not degrade is significant. The ClearLLab 10C system standardizes the flow cytometry approach from instrument setup and compensation to 10-color control material and analysis templates – areas where many labs without flow cytometry experience struggle.

How does it improve standardization and validation of results?

JH: Unlike previous attempts at LDT standardization, intra- and inter-laboratory standardization among ClearLLab 10C users is straightforward and does not require direct coordination. The pathologist can consequently concentrate on analyzing and interpreting the data, rather than worrying about whether the assay design was robust or if deviations from manual laboratory protocols may have introduced new artifacts.

MK: With one fixed set of reagents, validation is reduced to comparison with current reagents or lot and verifying quality control results.

What is significant about the antibody panel at the heart of the ClearLLab 10C system?

JH: The ClearLLab 10C system employs 10-color reagent combinations of antibodies designed to characterize both normal and aberrant cells. The normal cells, or “normal internal control,” are the key to FCI for hematolymphoid malignancies. The more detailed the normal immunophenotype, and the more familiar the pathologist with both the normal immunophenotype and characteristic deviations typical of various malignancies, the easier it becomes to recognize even subtle aberrations. To that end, the ClearLLab 10C system includes a casebook of normal samples as well as some common hematolymphoid malignancies to help users gain familiarity with expected staining patterns.

MK: The antibody combinations in the ClearLLab 10 C system have been designed to identify the majority of leukemia and NHL cases seen in standard practice. The reagents are formulated in a dry-down format that can be stored at 18–30°C for 12 months without degradation. This allows a full year’s supply of reagent to be ordered as a single lot and stored on the shelf. This single lot of reagent can be validated once received; then, no further validation is required other than monitoring quality control samples and internal populations in samples under investigation.

The ClearLLab 10C system has four targeted tubes: one to characterize B cells, one for T cells, and two for myeloid. Each tube covers one lineage and shows the maturation sequence in each cell line, making it easier to detect “different from normal” changes. I’ve found that, because the system comes with fixed antibody combinations and standard analysis templates, pattern recognition is easily acquired. This kind of consistency can lead to quick and accurate detection of an abnormal population. The analysis templates cover all main cell populations, making it unlikely that an unexpected secondary abnormal population would be missed.

How does the system conform to the 2006 Bethesda guidelines?

JH: The 2006 Bethesda Guidelines (2) described a common set of antibody specificities that had been used for the characterization of hemolymphoid neoplasms for approximately 10 years at that point and are still in use today. Due to the variability among vendors as regards antibody clones, fluorescent tags, and cytometer design, the Bethesda participants could not agree on consensus standardized panels. The lack of clinical flow cytometers that could assess more than five or six markers simultaneously at the time was a significant barrier, as most of the participants had preferred combinations that didn’t agree perfectly with those of other participants. With the more recent availability of 10-color flow cytometers, this barrier has largely disappeared. ClearLLab 10C incorporates a core set of antibodies and some more recently described markers, using strategic antibody redundancy to permit cross-tube comparison.

MK: The guidelines were an attempt to find consensus on hematological disease types and the antibody panels most effective in defining those conditions. All of the antibodies that met consensus in the Bethesda guidelines are included in the ClearLLab 10C system. Additionally, the B cell tube contains CD200, which – although not in the guidelines – is an excellent discriminator between B-CLL and mantle cell lymphoma. The addition of TCR γδ to the T cell tube is useful for detecting mature T cell malignancies that may express this receptor (3).

How do systems like this change the work of laboratory medicine professionals?

JH: On a day-to-day basis, ClearLLab 10C users should expect to spend less time on error-prone manual tasks and more on value-added work, such as data analysis and interpretation. From the research perspective, cases studied with ClearLLab 10C, even if done in different laboratories in different places, should generate comparable results.

MK: Consistent antibody combinations enable retrospective analysis of patterns that may not have been obvious on initial diagnosis. If new data appears linking such information to different disease states, it will be much easier to review previous cases due to the consistent antibody combinations in the ClearLLab 10C system.

* This number depends on how different the aberrant immunophenotype is from the normal cells, the number of events collected, and the number of informative markers in the tube.