I am looking to detect a rare population of 150 cells via flow cytometry and analyze a subset of this population with one cell-surface marker. Sample size will be about 150,000 to 500,000 cells in total. Is this possible? What is the limit of detection (in terms of cell number) for flow cytometry?
Technically, there is no limit of Flow Cytometry detection in terms of population percentage. There is detection limit in terms of how many molecules of an antigen per cell can be detected (but I will leave that for another topic).
When looking at rare populations (in your case, the frequency seems to be 0.03% - 0.1%), it is highly recommended to record as many cells as possible. Would you be able to start with more cells than just 150K-500K?
If you really have paucity of cells, and cannot run more than 150k-500K total cells, then I strongly suggest to be very careful with sample preparation and staining process. You do not want to lose some/all of your 150 cells of interest as dead cells and debris.
Often times when people can manage to have millions of cells to start with, an enrichment step (AutoMACS or simple magnetic pull down of non-desired cells) is highly recommended. This will significantly reduce the sample acquisition (recording) time while getting enough counts of rare population(s). For example, when people sort LSK/SLAM rare populations, they perform a Lineage depletion to reduce sorting time and collect more cells.
Hope this answered your question! Please feel free to reply with any further questions. All the best!!
Technically, there is no limit of Flow Cytometry detection in terms of population percentage. There is detection limit in terms of how many molecules of an antigen per cell can be detected (but I will leave that for another topic).
When looking at rare populations (in your case, the frequency seems to be 0.03% - 0.1%), it is highly recommended to record as many cells as possible. Would you be able to start with more cells than just 150K-500K?
If you really have paucity of cells, and cannot run more than 150k-500K total cells, then I strongly suggest to be very careful with sample preparation and staining process. You do not want to lose some/all of your 150 cells of interest as dead cells and debris.
Often times when people can manage to have millions of cells to start with, an enrichment step (AutoMACS or simple magnetic pull down of non-desired cells) is highly recommended. This will significantly reduce the sample acquisition (recording) time while getting enough counts of rare population(s). For example, when people sort LSK/SLAM rare populations, they perform a Lineage depletion to reduce sorting time and collect more cells.
Hope this answered your question! Please feel free to reply with any further questions. All the best!!
The first thing that came to my mind was that you need enrichment.
You should detect minumum 10 cells as a cluster of target cell. How much total cell is required? This depends on 10cells and its ratio in suspension. If population is below %1, artefacts and resulting false positivity are usually most important problems. In this levels as in minimal resudual disease, you should use artefact cleaning methods such as doublets, apoptotic/dead cells, discreminating other cells with other CDs...
At least 1000 cells could be recognized as precise data to consider in flow cytometry. yes, you can detect the cells that you want to. But it only helps you to detect, not to quantify them precisely. you can not get better data until you have a more sensitive machine.
One additional recommendation for rare events is to run cytometer on low speed to improve data resolution. Explanation here: https://expert.cheekyscientist.com/optimize-flow-cytometry-hardware-for-rare-event-analysis/
- Badges
- Science topic
- Similar topics
- Mathematics
- Statistics
- Sampling Design
- Sample Size