Compensation in Spectral Flow Cytometry: Control Options

01 — Objective: To surface intrinsic limitations of standard blood-based cellular controls, before presenting the client's reagent as an efficacious solution to these known challenges.

02 — Material:

Commercially Available Whole Blood-Based Reagents
At present, there are several commercially available blood controls on the market; these reagents often include preservatives to increase the shelf-life of the product. These blood controls can include erythrocytes, plus specific populations of cells commonly evaluated in the clinic, such as CD34+ leukocytes, CD45+ and CD45- abnormal leukocytes, and mixed white blood cell populations, to name a few. Preserved controls with mixed populations of B- and T-lymphocytes, granulocytes, monocytes, and NK cells are relied on by researchers conducting immunophenotyping studies. 

Although these preserved blood reagents may often be sufficient to act as biologically relevant controls, there are several important caveats of which one should be mindful. First, the availability of these reagents depends directly on the state of the blood supply. In times of national blood shortages, the supply of bio-sourced blood controls quickly falls short of the demand. Similarly, the combined effort of sourcing and processing, plus the demand for this product elevates costs and makes these controls less available for many labs. From an experimental perspective, blood controls sourced from healthy donors do not fully reflect the potentially unique disease states that one may be studying, and positive procedural controls for rare populations and abnormalities will not always be available. Furthermore, these controls cannot be customized or engineered to represent the diversity of internal cytokines and surface markers that are uniquely expressed in diseased cells. Sometimes to avoid completely relying on the human blood supply and reduce the cost of whole blood controls, donor blood is supplemented with blood parts from other organisms, resulting in a blood control that is even farther removed from its experimental counterpart.

Immortalized Cell Lines

Immortalized cell lines can be an attractive alternative to preserved blood controls and can be especially helpful in functional assays. For example, NKL (natural killer line) cells are commonly used in cytotoxicity assays, as they provide an adequate model to evaluate the impact of changes in unique cell surface markers on killing activity.

That said, there are a few limiting factors one must consider when deciding whether to use an immortalized cell line in flow cytometry-based immunology or hematology research. First, cell lines must be well characterized in terms of phenotype and expression of markers and cytokines. Similarly, the expression and phenotype must be known to be stable over time. The time and cost of maintaining these cell lines must also be considered, and it is worth noting that the quality of one’s data is a product of the health of one’s cells. Lastly, although expression in cell lines can be manipulated through common molecular and genetic engineering techniques, it is difficult to address the full array of differentially expressed genes that appears during disease. On the whole, to stably and fully customize cell lines remains a challenging and time-consuming endeavor on top of the effort that must be directed towards designing a comprehensive and well-planned experiment.

Sourcing Biological Controls for Rare Disease Research

Lastly, another consideration is the limited overall commercial availability of options for intracellular markers and specific disease states. Although it is possible to purchase controls that are positive for common cytokines or intracellular markers like TNFα, IL-2, and myeloperoxidate (MPO), for example, what commercially available cytokine-specific controls exist are not customizable, and options for those studying rare diseases or populations are practically non-existent.

03 — Impact: Positioned the client's solution within a genuine research need, while neutralizing potential skepticism through clear, upfront acknowledgement of the drawbacks of existing biological controls, turning balanced examination into credibility.