Sudden signal Intensity drop during Helios runs
This is an update to our Signal Drop problem previously mentioned in the "Signal variation upon freezing samples" thread from January. As I mentioned then, we have been experiencing this problem intermittently for some time and it is unrelated to sample preparation. I have attached a PDF showing examples from recent experiments where we ran technical replicates of a PBMC sample on 2 different days.
The cells were stained with Abs, Cisplatin and Iridium (overnight) in 1 batch and then split into 10 tubes. Five were acquired on day 1 on our Helios 1 after a full clean. The other 5 were stored in Ir and acquired on day 2. I have not included all markers - just enough to show that some are affected by a sudden and dramatic signal drop (eg., 89Y, , others are slightly affected while others not all (only a few: 141Pr, 151Eu, 153Eu). Sometimes the drop happens early and sometimes late. Sometimes the signal is clearly split for the entire acquisition (eg. Tube 1 day 2) but that is rarer than the drop partway through.
Finally, sometimes the drop is present for the entire acquisition -ie the signal in the affected channels is abnormally low. We can only know this is a drop (as opposed to poor stain) when we have technical replicates - see the last slide from a run on our 2nd Helios (same cells and Abs). You see that the CD4589Y and CD3-170Er intensities are very low in tubes 1 and 2, but start higher in tube 3 and then drop.
Needless to say this has been very frustrating since it came to light. Not sure when it started but within the last year. We stopped checking Time plots routinely since things had been quite stable. Fluidigm has not identified the problem but thinks it is caused by some sort of (sporadic) cell instability. They asked to test some different acquisition conditions, the details of which I can't divulge. These conditions provided a notable improvement in signal quality (increased tightness of positive peaks) compared to the baseline samples. However, we did not see our particular signal drop problem in the single samples we ran under our conditions.
So the cause remains a mystery. We will now use Fluidigm's new conditions routinely and see if that solves the problem. Will keep you posted!
Cindy
PS - also note the increased background in HLA-DR after 48 h storage in Ir - we have this problem with a few Abs upon storing stained fixed cells - any ideas on how to solve that?