Immunotherapy research is a rapidly expanding field with a pipeline of monoclonal antibodies in development to treat a range of cancers and autoimmune diseases. The mechanism of action (MOA) used by an antibody to mediate a therapeutic response must be fully defined to enable a candidate antibody to advance down the preclinical development pipeline. It is also required for all antibodies used in clinical research and regulatory IND filings in order to optimize dosing and assess the risk of detrimental side effects.

Have you ever wondered why certain experiments are done under GLP (good laboratory practices) conditions? GLP is a term that is used frequently in preclinical research, and are a set of guidelines that act as a management control for research laboratories and organizations to ensure the uniformity, consistency, reliability, reproducibility, quality, and integrity of chemicals (including pharmaceuticals) for non-clinical safety tests[1]. Sometimes it’s hard to understand when and why protocols must be done under GLP conditions. In general, GLP conditions must be maintained when an experimental drug or biologic may be used ultimately in humans and will need to be evaluated by regulators like the FDA.

Flow cytometry has been developed and used as a clinical tool since the invention of the first cytometers in the 1970s. At present, flow cytometry is considered essential for many routine clinical diagnostics, including assays for leukemia and lymphoma, stem cell enumeration, solid organ transplantation, HIV infection status, immunodeficiencies, and hematologic abnormalities. Many scientists involved in clinical trials or drug development are faced with developing clinical flow cytometry assays for multiple phases of clinical development. If you find yourself starting to plan a clinical flow cytometry assay, here are the top 3 issues to think about as you plan your experiment.

Toxicology screening is essential to any preclinical study, and flow cytometry-based toxicology assays are a fast and practical approach. Different animal models are used for toxicology studies, including rodents, dogs and non-human primates. One commonly used technique is the micronucleated erythrocyte endpoint assay, which measures DNA damage induced by exposure to experimental drugs or biologics. This method has been adapted into a validated flow cytometry-based assay and can use erythrocytes from different species. Consider these factors when selecting the best toxicology screening method.

Many basic and clinical immunology studies that focus on T cells include proliferation assays in order to determine if T cells are capable of proliferating under different in vitro or in vivo conditions. Flow cytometry is the ideal approach for measuring T cell proliferation and a suite of staining products…

Many scientists performing preclinical and clinical research hit a point when they need to have an assay validated. You may have painstakingly developed and perfected a particular assay, but now you must put it through the rigors of validation for it to be considered a “validated assay.” The basic principles of assay validation were described in an earlier blog post, but how do you know you if you need an assay validated? Use these questions as a guide to help you figure out your validation situation and get a little less vexed about validation.

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.

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.

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].

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.

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.