Hi Greg,
New prep of a Historical Conjugate:
The main thing that we do to validate new preps of historical conjugates is running them against a "known" sample. For example, we use cryoPBMC controls on most of our plates; we get those controls by processing an LRS chamber, so we get high tens to low hundreds of vials from the single draw/LRS. Therefore, we typically have "historical" data from that marker-conjugate's previous batch(es) from previous plates. If there are stims involved then obviously you need to stim, etc.
But in general this has worked for us. I'll admit to being a bit lazy and not often running full titration curves on new preps: 9/10 times, the titer for the new prep is the same as the old prep. 9/10 of the remaining 1/10 time, it's within a factor of 2, and I test that in a subsequent experiment if the staining seems low, high, or streaky. But I don't run customer samples until I'm happy with the titer (including occasionally tossing and starting over with a conjugate with really off-titer signal or background). And, from Daniel's comments here (
viewtopic.php?f=4&t=1414 ) on how very tight titering (along with stronger fixation) has resolved Sinai's intermittent signal intensity issues, I might change my plans.
Development of a New Conjugate:
This is a trickier question. Hopefully you have some idea of where to start: previous flow cytometry datasets, previous papers, etc. So, you'll know whether you have to Stim (and how) to find the marker; whether sample processing has depleted (ie, few/no Grans in PBMCs), etc.
Hopefully you also know which cell types the marker *should* be on, and just as importantly, which ones the marker should *not* be on (negative control beyond MMO). This is particularly important in the case of dimmer and/or rarer markers. For example, CD85j (ILT2/LILRB1) staining on CD8+ and NK cells is often dim and highly donor-dependent. However, it's expressed on many/most Monocytes and B cells, and virtually no CD4+ cells (especially in Healthy people, but even most disease states I've examined). Therefore, I can feel confident in a shoulder or dim peak on CD8+ and NK cells, because of having inherent Positive (B cells, Monocytes) and Negative (CD4+) controls inherently in the sample and exposed to the same staining conditions.
There's also the ugly/wonderful matter of biological variability between donors. This can be both in terms of Frequency (my current Healthy Donor PBMC control has virtually no CD57+ CD8+ cells, but does have CD57+ NK cells) as well as Expression/Marker Intensity (1-2% of of the donors I've seen have CD33dim Monocytes that are nicely CD14+). This is one reason why I try to stain at least 3 different donors when doing development work: you could still get misled, but it's increasingly unlikely that you'll get 3 weird donors for a given marker.
As an example: when I first joing the HIMC back in 2010, my first job was to adapt the HIPC Lyo panel to the CyTOF platform. A few clones had to be changed along the way. In particular, I had problems with CD28 at that time. I finally found a clone that gave good staining on T cells, and little or no staining on biologically negative cells. The T cell staining matched the fluorescent flow staining on the same Healthy Control donor that a labmate performed. So, I thought I was done.
Unbeknownst to me, that Healthy Control donor was weird in having a very high percentage of CD28+ CD8+ cells. And my limited immunology knowledge at the time didn't raise a flag when most of the donors in the plates I started running *also* showed a high Freq of CD28+ CD8+. Someone from a different lab analyzing the data noticed the issue, and I had to go back and change the CD28 clone again. I then talked with the manufacturer about that CD28 clone (how it would only stain T cells, but would give a false positive on most CD8+); they then admitted that other people had reported that problem.
Had I tried 3 different donors and compared them to the flow experiments my labmates were doing, I would have caught this earlier.
In summary:
1. Check multiple donors.
2. Check all major cell populations for false positives/false negatives.
Mike
Wed Feb 10, 2021 3:42 pm