Brandon Walling, PhD, from Champions Oncology, answers key questions from his recent webinar.
1) I am hearing more about spectral flow cytometry, what is this, and what benefits does it provide?
Spectral flow cytometry differs from conventional flow cytometry through utilizing all detectors to capture the entire spectra for each fluorophore. This provides a “fingerprint” for each fluorophore which can allow for extremely similar emission spectra to be separated (ex. Alexa647 and APC). This can increase the resolution of the sensitivity of your study. Spectral flow cytometry also has cell autofluorescence detection and subtraction capability, which can improve dim marker’s separation from high auto-FL background.
2) Does Champions Oncology use Spectral or conventional Flow Cytometry instruments?
Champions has the option to run either spectral flow cytometry using the Cytek Northern Lights (3 lasers 30 colors) or conventional flow cytometry using the BD FACSymphony A3 (5 lasers 24 colors).
3) Are there different types of validations?
Yes, depending on the use of the assay different validation strategies may be applied. The level of rigor required for a research or exploratory endpoint will differ greatly from that of an assay used for diagnostic purposes. The type of assay will also dictate which test parameters are required as something such as a lower limit of detection may only be required if testing low levels of material. Many of these conditions and recommendations have now been outlined in the H62 Guidelines: Validation of Assay Performed by Flow Cytometry.
4) On the aspect of compensation caused by spillover, how could you validate compensation for each AML sample?
Compensation and the associated spillover values should be calculated during panel development with panel design playing a key role in minimizing spillover. The establishment of the spillover values should be done using the same lot and identical fluorophore to what will be used. Cells and beads can both be used for compensation controls, but the positive and negative populations must have the same background. You should ensure that your positive population is at least as bright, if not brighter than your samples. Finally, you should ensure you capture enough events. The use of cells is always preferable but the limitation on sample amount can require the use of other controls such as beads or surrogate cells. If these practices are followed, compensation should not need to be adjusted on a sample-to-sample basis.
5) Why is it important to use Viability dye in your experiments?
Viability dyes allow for the distinguishing of live and dead cells. This is critical to flow cytometry experiments as dead cells tend to not only have higher autofluorescence but also exhibit non-specific binding. Generally, without first excluding non-viable cells, you may be interpreting a false positive signal.
6) What is the sensitivity of the PhenoSeek AML Flow Cytometry Panel?
The Phenoseek AML panel developed at Champions has sensitivities for all major cell populations with as few as 400 cells detected.
7) How can I ensure quality between sites?
The quality between sites starts with ensuring consistent training procedures, methods, and documentation between labs, this can include cross-site training for an assay. Cytometers should have the same configuration and be harmonized using controls such as hard-dyed beads. Following normalization, routine QC should be performed to ensure the cytometers remain within specifications. For the assay itself, one of the three common strategies is typically used: Comparative Testing, Co-Validation, and Validation. These can be selected based on the stability of the assay substrate, the timeline to perform the assay, and whether the assay will be developed in a single lab before transfer. These methods will typically include testing the same or similar samples, control samples, and standard QC tools (preserved whole blood) at all sites.
8) What controls do you typically use in your assays?
Controls may be broken out into those for the equipment and those for the experiment. QC controls can include hard-dyed beads and tools such as preserved whole blood or lyophilized cells to routinely evaluate the equipment, assay, and staining process. Other experimental controls such as fluorescence minus one (FMO) controls are critical for the gating of samples and compensation controls to establish a spillover matrix. Universal negative controls such as unstained beads, unstained cells, isotype antibodies, secondary antibody controls, positive or negative control cells, or the treatment control group will be assay specific.
9) How do you determine the number of replicates or donors needed for an assay?
For most assays, three to four replicates will generally be sufficient for a %CV of less than 10%. This may need to be extended for rare populations or depending on the assay use. The H62 Guidelines provide excellent recommendations for replicate and donor numbers depending on the assay.
