Tue Feb 23, 2021 10:17 am
Tuning sensitivity and data quality
We have now for the last two years been concerned about lower tuning results on the Helios machine at our core facility. It was installed in late 2015, and for the first years operated quite steadily at around 1.6-1.4 x10^6 Tb dual counts (DCs). Through some up and down periods, it has now settled at around or just below 1.0 x 10^6 Tb DCs. We consider this to be low sensitivity, as we are used to much higher values. Even in a newly washed machine with brand new cones we cannot get any noticeable (or reproducible) increase in tuning sensitivity.
What is the normal level of 159Tb in the tuning of a Helios machine? Does our machine indeed have a sensitivity lower than what can be expected?
We did after long (!) discussions with Fluidigm get our quadropol changed, and the sensitivity increased to around 1.3 x10^6 Tb DCs immediately. Disappointingly, the machine quickly declined in tuning sensitivity and is now back at a steady 1.0 x 10^6 Tb DCs. However, taking advantage of the planned service, I prepared a four-point titration experiment and froze down two aliquoted sets of samples. One sample set was thawed and run on the machine before changing the quadropol, and the other sample set was run right after, when the machine had a substantially increased sensitivity.
From the titration data (see powerpoint), it is clear that an increased tuning sensitivity of the machine directly translate into increased antigen expression (DCs) on single cell data. Likely, a higher sensitivity also decreases the number of antigens needed on each single cell for it to be detectable. This might result in more antibodies to be usable in our panels, as some antigen could be just in the detection limit.
We showed these data to Fluidigm and argued that lower tuning sensitivity provided poorer data with reduced sensitivity. They were quick to reassure us that we should just use their normalization algorithm which will align (increase) the poor data to their reference bead data, and thereby fixing the lower sensitivity issue. Our immediate concern that increasing the values will potentially drag very low values (potential noise) into an area that might be not be obviously noise, may therefore disturb analysis of the samples who’s lower range extend down into this more uncertain range. Just as a note, we have no problem with the normalization of higher values, here, the algorithm works nicely. But as we and many of my collages are interested in signal transduction antigens, who are often weakly expressed in steady-state/baseline primary material, the lower end of the scale is very important! In a nutshell, higher sensitivity allows us to measure (with increased confidence) lower levels of e.g. pSTAT5 in our patient cells. Indeed, variation between patients will inevitably result in some of the data being on the really low end of the spectrum, and here we believe that it is important to get as much out of the cells as possible (have a look at the 1:800 dilution of CD223 in slide 11 of the attached pdf)
I am just very curious about what the mass cytometry community has to comment on our views on tuning sensitivity and data quality.
What is the normal level of 159Tb in the tuning of a Helios machine? Does our machine indeed have a sensitivity lower than what can be expected?
We did after long (!) discussions with Fluidigm get our quadropol changed, and the sensitivity increased to around 1.3 x10^6 Tb DCs immediately. Disappointingly, the machine quickly declined in tuning sensitivity and is now back at a steady 1.0 x 10^6 Tb DCs. However, taking advantage of the planned service, I prepared a four-point titration experiment and froze down two aliquoted sets of samples. One sample set was thawed and run on the machine before changing the quadropol, and the other sample set was run right after, when the machine had a substantially increased sensitivity.
From the titration data (see powerpoint), it is clear that an increased tuning sensitivity of the machine directly translate into increased antigen expression (DCs) on single cell data. Likely, a higher sensitivity also decreases the number of antigens needed on each single cell for it to be detectable. This might result in more antibodies to be usable in our panels, as some antigen could be just in the detection limit.
We showed these data to Fluidigm and argued that lower tuning sensitivity provided poorer data with reduced sensitivity. They were quick to reassure us that we should just use their normalization algorithm which will align (increase) the poor data to their reference bead data, and thereby fixing the lower sensitivity issue. Our immediate concern that increasing the values will potentially drag very low values (potential noise) into an area that might be not be obviously noise, may therefore disturb analysis of the samples who’s lower range extend down into this more uncertain range. Just as a note, we have no problem with the normalization of higher values, here, the algorithm works nicely. But as we and many of my collages are interested in signal transduction antigens, who are often weakly expressed in steady-state/baseline primary material, the lower end of the scale is very important! In a nutshell, higher sensitivity allows us to measure (with increased confidence) lower levels of e.g. pSTAT5 in our patient cells. Indeed, variation between patients will inevitably result in some of the data being on the really low end of the spectrum, and here we believe that it is important to get as much out of the cells as possible (have a look at the 1:800 dilution of CD223 in slide 11 of the attached pdf)
I am just very curious about what the mass cytometry community has to comment on our views on tuning sensitivity and data quality.
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Titration and Tuning_Cytoforum.pdf- (1.77 MiB) Downloaded 458 times