Post Thu Jul 20, 2023 5:49 pm

Debarcoding purity issues

Hi all,

I ran into a case this week where my debarcoding purity was lower than expected. You can see a summary here (PDF was too big to attach): ... sp=sharing

In short: I was doing a titration on a new CD14 in-house conjugate. I had 2 donors: 399 which is CD33mid/lo on Monocytes, and 1442 which is CD33hi on Monocytes. I've used both these donors several times before, and they're reproducible (and pure) in their respective CD33 staining.

I did a serial dilution of CD14: 1x, 0.5x, 0.25x.
4M viable cells/sample. Surface staining was done on individual samples, then cisplatin livedead, then PFA fix, then methanol, then SBio BC (full 10uL) with no additional perm (estimated 2M/sample left after MeOH).
Then pool 399 and 1442 of each titer point (so, 6 individual samples become 3 pools of 2BC each), then intracellular, then Ir+2% PFA. Acquire on Helios 270-320 events/sec, PSI, NB+MilliQ. SBio EQ4 norm and SBio debarcoding.

While looking at the data, I saw "contamination" of the CD33lo 399 donor in the CD33hi 1442 donor, and vice versa. This happened in all 3 pools. I initially was using the default Minsep=0.20, Mahal=10 (minimum threshold box checked). However, when I went back and tried other combos of Minsep and Mahal on the 1x titer pool, the "contamination" remained in all cases.

The contamination isn't high: of the total Monocyte gate, ~1% are "wrong". And I wouldn't have seen this if my donors weren't different in CD33 expression.

Has anyone else seen this (or other examples of debarcoding purity issues)? If so, how did you address it? 1% isn't much, but if you're looking at rare cells......