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.

Introduction of CAR-T Therapy T lymphocytes are engineered with synthetic receptors known as chimeric antigen receptors (CAR) in CAR-T Cell therapy. The CAR-T cell is an effector T cell that recognizes and eliminates specific cancer cells, independent of major histocompatibility complex molecules. (Zhai et al. 2018). Chimeric antigen receptors (CARs) cells have recombinant receptor constructs expressed in T cells to target cells expressing specific antigens.

Flow cytometry is a powerful tool for surveying the cellular landscape during preclinical development of drugs and biologics. But flow cytometry can go beyond immunophenotyping to actual functional measurements that can contribute to understanding the true potential of a therapeutic candidate. To make the most of your flow cytometry studies, consider these other assays as you plan the next phase of preclinical development.

Flow cytometry is a powerful tool because it allows users to analyze the characteristics of millions of cells with relative speed and precision. A single cell suspension of fluorescently labeled sample travels through the cytometer for excitation by lasers, and the emitted light photons are measured by different detectors. Having a single cell suspension is essential to measuring cell fluorescence accurately, and many types of cell or tissue samples must be specially processed to make this suspension. Two different methods can be used for single cell suspensions: mechanical dissociation of tissue or enzymatic dissociation of tissue. This processing step is typically carried out before cells are stained and both methods have benefits and caveats.

Memory is a characteristic of the immune system that provides humans and other vertebrates with long term protection against infectious diseases and other “non-self” antigens such as those associated with tumor cells. In the context of T cells, memory responses occur when a naïve T cell encounters an antigen bound to a major histocompatibility complex molecule and is activated to undergo differentiation into an effector cell or a memory cell. Memory T cell populations can persist in the body for months to years and can be stimulated to respond specifically and rapidly to a foreign antigen upon re-exposure.

Flow Cytometry utilizes fluorescently labeled antibodies to detect specific biomarkers on the surface and within cells, and over the past few years, there has been a surge in reagents available for flow cytometry applications. Most of these have been developed using monoclonal antibodies raised in mice and conjugated to a range of fluorophores. However, there are still instances where suitable monoclonal antibody reagents/conjugates are not commercially available, and small-scale conjugations are not practical. In these instances, so-called indirect staining may be employed, where the binding of an unconjugated primary antibody is detected using a secondary anti-IgG antibody conjugate.

Flow cytometry is a powerful technique for characterizing immune responses to vaccines, immunotherapeutic drugs, and other clinical interventions. But many preclinical and clinical studies may take place at sites that are not in the same location as the flow cytometry lab. That’s why it’s critical to determine how clinical specimens should be collected, processed, stored, and shipped to assure that cells will be viable and abundant enough for flow cytometry analysis.

With the rapid progress of immune-monitoring drug development, flow cytometry has found itself increasingly at the forefront of clinical trial assessment of safety and efficacy. This is not without challenges since flow cytometry analysis can be complicated and expensive, too often employs idiosyncratic experimental and analytical methods. So how can a platform without standardized methods and processes, be successfully applied to evaluate clinical endpoints?

The flow cytometry market is filled with an abundance of products for mouse and human samples. But what if your studies use different species? Fortunately, many antibodies for standard cell markers can work on multiple species, and more species-specific reagents are becoming available.

Flow cytometry assays are important for preclinical and clinical research, however, it is vital to understand the level of compliance required for the stage of research you are completing.  Flow Cytometry assays completed for toxicology and safety assessments are required to be in compliance of Good Laboratory Practices (GLP), on the other hand, basic research or discovery/exploratory studies can be non-GLP.  GLP refers to a set of standards for laboratory studies to be planned, performed, monitored, reported, and archived. Preclinical and clinical studies must be GLP-compliant in order to be submitted for review by regulatory agencies like the FDA. Consider these three points if you find yourself in need of a GLP-compliant flow cytometry assay. 

Data by FlowMetric All flow cytometry experiments begin with similar basic set up protocols to assure that the equipment is functioning properly and that samples can be measured accurately. Using a flow cytometry gating strategy is an essential step during this set up phase as it assures that the correct cell populations are being measured. Here are factors to consider as you determine how you want to establish your flow cytometry gating strategy for your next experiment.