Anyone who starts an investigation of acute myeloid leukemia (AML) soon finds out the complexity of this disease. Although daunting initially, it soon becomes apparent the need for complex classifications for AML subtypes and different mechanisms for formation. AML forms from a wide variety of DNA mutations leading to numerous phenotypic changes in the blood makeup. Early on there were the French-American-British classifications in the 1970s (FAB) but in present day, AML type is being broken down to genetic markers. For the most part, this is due to the advancement of scientific-technical capability. Conversely, being able to clearly define AML by mechanistic function, allows for clinicians to state, with some certainty, treatment and survival options for their patients.

Tumor-infiltrating lymphocytes (TILs) are now understood to be key players in anti-tumor responses. These cells are found in solid tumors such as those observed in breast cancer, ovarian cancer, melanoma, and lung cancer. TILs have now been harnessed to treat cancer through adoptive cell therapy protocols. As TILs are a major area of focus for both basic and clinical research, flow cytometry applications for identifying and characterizing TILs are increasingly important. Consider these key points if you are pursuing TIL research and plan to use flow cytometry.

T cells are well known for their roles in combating cancer and infection, but chronic exposure to antigens and inflammation can cause T cells to enter a state of “exhaustion[1].” Exhausted T cells lose critical effector functions including cytokine production, the ability to proliferate and memory T cell differentiation is also compromised. Exhausted T cells also express inhibitory receptors and become unresponsive to IL-7 and/or IL-15-driven self-renewal. This progression toward T cell exhaustion results in diminished control of chronic infection or cancer. Exhaustion can occur in both CD4+ and CD8+ T cell populations and the phenotypes of these subsets is somewhat heterogeneous. Nonetheless, T cell exhaustion is reversible and various immuno-oncology interventions have been examined or are currently being evaluated in order to improve outcomes in cancer and chronic infection[2].

Flow cytometry is an elegant and powerful tool that has been critical to understanding the immune system and advancing the development of immune-based therapies. Critical to many studies, and essential for FDA filings, is the development and documentation of a validated assay. While most flow cytometric assays fall into the “quasi-quantitative” category according to FDA guidelines, there are some assays that can be quantitative and even qualitative.

Fluorescent-Activated Cell Sorting (FACS) is a flow cytometry-based technique in which cells are stained with fluorescently labelled antibodies and sorted based on pre-defined staining parameters that are specific to different cell types. FACS users must consider multiple factors when designing and running a FACS experiment. Consider these three factors as you plan and carry out your next FACS experiment.

Fluorescence-activated cell sorting (FACS) is a powerful technique for obtaining a relatively pure cell population for downstream applications. This technique uses fluorescently labelled antibodies to stain cells that express specific markers, and these stained cells can be sorted into separate subsets using a cell sorter and can even be separated into individual cells. FACS is especially useful for gene expression analysis of individual cells or pure cell populations.

The immune system is comprised of a multitude of unique cell subsets. Each cell type, from B and T cells, to macrophages, monocytes and dendritic cells, have been phenotypically subdivided into unique subsets as we learn more about the phenotypic signatures that define these cells. Flow cytometry has been the central tool in evaluating and defining cell subsets, and major advances in immunophenotyping have occurred recently as more parameters can be measured during a single run on newer flow cytometers.

In a healthy person, immune cells like B cells, T cells, and macrophages are typically surveilling the body for abnormal cells or infectious agents. These cells don’t fire up their inflammatory toolbox unless they recognize one of these foreign entities. The potent inflammatory mediators activated during these responses include cytokines, free radicals, prostaglandins, and clotting factors, which must be tightly regulated to avoid wreaking havoc on healthy tissue. This exquisitely controlled activation of inflammatory molecules means that when you look for them in cells by flow cytometry, they may be very difficult to detect.

The idiom, garbage in, garbage out applies to many areas of scientific research, including flow cytometry. Good sample preparation is critical to accurate and sensitive cytometry analysis of cells, wherever their origin. 

Immunotherapeutic molecules currently being used in the clinic are powerful immune modulators, but their effectiveness can be inconsistent between patients. Clinicians and scientists use different assays to evaluate why immunotherapies fail in the clinic. The flow cytometry-based receptor occupancy (RO) assay is a critical tool for evaluating the effectiveness of immunotherapies in the clinic. Here are three features of flow cytometry-based RO assays that give them clinical value.

For most applications flow cytometry is used to identify cell populations and define bivariant terms of positive and negative sub-populations according to specific biomarkers, through the binding of fluorescently tagged monoclonal antibodies (mAbs). Typically, the cutoff between these populations is set relative to a control unstained population. Since the fluorescent intensity of a signal is proportional to the amount of monoclonal antibody bound to that cell target, this signal is directly related to the expression level of that target. However, for flow cytometry endpoints to be considered truly quantitative and fulfill the rigor of clinical utility, several obstacles needed to be overcome. In this blog, we explore the rationale behind quantitative flow cytometry, and the tools that are now being implemented to help achieve standardization.

What are receptor occupancy assays?