The Do’s & Don’ts of Flow Cytometry in the Clinic

Clinical flow cytometry is now a standard tool used by clinical researchers in numerous fields, especially immuno-oncology. Consider these “Do’s & Don’ts” of clinical flow cytometry as you decide to use this tool for clinical research.

Do Perfect Your Panel - A staining panel includes the set of antibodies used to identify different cell subsets within a sample and depends on the use of multiple antibodies conjugated with different fluorochromes. These fluorochromes are excited by specific light wavelengths from the cytometer’s lasers, and the emitted light is analyzed and interpreted as different cell types. Researchers take time to develop staining panels that can reasonably distinguish between cell types and avoid aberrations such as spectral overlap.

Do Vigilant Validation - Clinical flow cytometry assay development must satisfy certain criteria, including accuracy, specificity, limit of detection, reproducibility, precision, linearity, and stability. By satisfying these criteria, researchers are able to have confidence in their clinical data, even if experiments were done at different times or in different locations.

Do Maintenance - Reliable flow cytometry data requires a well-designed and validated protocol as well as a properly maintained cytometer. Routine maintenance is carried out by cytometer users to assure that fluidic systems flow properly, lasers are firing correctly, and the emitted light is detected at the expected wavelength. Infrequent, but more involved, maintenance is carried out by technicians and is essential to the long-term functionality of a cytometer.

Don’t Pick the Wrong Test Tube - The very first step of a clinical flow cytometry protocol is collecting a sample, which is typically peripheral blood, cord blood, or tissue biopsy. These samples can be collected in tubes with preservatives or anti-clotting agents, but some of these additives can alter cell viability. It is worthwhile to evaluate different sample tube types to assure that your cells of interest are not being lost or damaged due to test tube effects.

Don’t Lose your Cells - Clinical flow cytometry protocols rely on the acquisition of thousands to millions of cells per sample in order to have a reasonable number of events to analyze. One essential step to acquiring usable data is the inclusion of a viability dye that discriminates between live and dead cells. Viability dyes come in different formats that work with specific flow cytometer configurations, so it is critical to determine which dye will work with your cytometer.

Don’t Screw Up Your Analysis - After you have completed the laborious process of sample staining and data acquisition on a cytometer, it is essential to execute the appropriate data analysis. Be sure you understand how to use the software needed for analysis, and this may mean including certain controls in your staining and acquisition protocols. Errors in analysis may result in your data being inaccurate or undetected.