Fri Mar 09, 2018 6:46 pm by mleipold
Hi Santhosh,
"Is normalization not essential to interpret CyTOF results?"
Short answer: it depends. I say this, based on splitting a sample and running the aliquots 8 hours apart (beginning and end of a long run day; yes, I can share the data if people are interested).
Longer answer: If you are simply comparing Frequencies of Parent or Cell Count, and your panel is reasonably robust (ie, decent separation between all populations), then, formally, no, normalization is not *strictly* required. In other words, if you can resolve your cell populations, those numbers should remain consistent.
However, if you're looking at Median Intensity Values (fold-change in phosphoflow, any kind of algorithm that uses intensity values to define clusters, etc), then it's best that you *do* normalize. This is because the decrease in signal intensity that you get over long runtimes may be enough to affect the numbers: either making extra clusters, or broadening your peaks enough to affect Median values.
Additionally: if you are running long barcoded samples, then you really *do* need to normalize somehow for good analysis. This is part of why the normalization beads were created: you can see in the Finck et al paper that when they run one sample for 2hr, they lose ~30% of their signal start to finish. Assuming the sample is evenly suspended/distributed, the end of the sample should look like the start of the sample, not 30% lower in signal.
Mike