When designing panels for flow cytometry, understanding spillover and spread is essential for better resolution of populations and high quality data. Spreading error is due to measurement error and negatively impact the resolution. However while they are related, they are not the same as our friend, Laura Johnston from the University of Chicago explains.
What is Spill?
“Spill refers to when one fluorophore spills into the detector assigned to another fluorophore.” (Johnston, L. 2020) In the figure above the picture on the right shows how emission from the perpetrator, APC-Cy7, spills over to the detector of the victim, PE-Cy7.
What is Spread?
“Spread, known more precisely as spillover-spreading error, refers to an error that is visible after compensation or spectral unmixing has been applied.” If we apply compensation as shown in the right picture above, we “fix” the spillover effect from APC-Cy7 into PE-Cy7 but this causes the “trumpet effect” seen in this picture, which can lead to a loss of resolution in the PE-Cy7 channel
By comparison, in the figure above, the image on the right of two single stain CD4 NovaFluor conjugates showing minimal spillover/spread when detecting the same marker on adjacent channels. This leads to higher biological resolution from each detector, with no trumpet effect.
Laura found that it can be tricky to count photons, particularly from multiple fluorophores in the same detector. Spreading error actually occurs in all flow cytometers including spectral cytometry under these rules:
- More spreading error occurs with more fluorophores in the detector
- More spreading error occurs the more a fluorophore spills into another detector
- More spreading error occurs with brighter fluorophore intensity
So working within these rules Laura’s recommendations are:
- “Assign highly expressed markers to dim fluorophores and low expressed markers to bright fluorophores
- For panels that have many co-expressed markers, avoid using highly similar fluorophores that cannot be distinguished on a conventional flow cytometer.
- For fluorophores that spread significantly into each other (typically pairs that cannot be distinguished by a conventional flow cytometer like APC and AF647), assign them to markers that are on different cell types so that a double positive population is not expected.
- For co-expressed markers, assign fluorophores with minimal spread into each other.
- Manage fluorophores that cause a lot of spreading error” (Johnston, L. 2020)
But what if we didn’t need to manage these fluorophores causing a lot of spread? What if we could replace them with a dye of similar brightness level and a cleaner spectral signature? Not wanting to toot our own horn but this option is now available with Phitonex NovaFluors.
As Theodore Hesburg says “The very essence of leadership is that you have to have vision. You can’t blow an uncertain trumpet.”
Time to bury the trumpet and move to cleaner brighter NovaFluors.
More information about the effect of Spillover and Spread on panel design can be found here:
Johnston, L (March 4, 2020). Understanding the Trumpet Effect: How to Design Aurora Panels Around Spreading Error https://voices.uchicago.edu/ucflow/2020/03/04/understanding-the-trumpet-effect-how-to-design-aurora-panels-around-spreading-error/