Palladium barcode artifact when staining PBCs (not PBMCs)
We were recently testing the difference in staining patterns on peripheral blood cells (PBCs) in comparison with peripheral blood mononuclear cells (PBMCs). PBCs were created simply by spinning whole blood and separating the buffy coat from serum/plasma. PBMCs were created through standard gradient density separation using Ficoll. Both were stained fresh with our standard panel in separate tubes. We included another tube with our cryopreserved PBMC reference (Ref1). All tubes started out with 3 x 10^6 cells. During overnight intercalation they were barcoded with the Pd-based barcoding kit from FDG as we normally do. They were run separately and the only reason I wanted to barcode them was to see if there was a differential uptake of barcodes between mononuclear cells, residual red cells, granulocytes, and debris.
When I inspect the files using [Pd channels] vs time it becomes clear that the PBC tube has very low barcode signal on the expected channels (104/106/108). This is not seen on the other tubes:
Although possible, it is very unlikely we failed to add adequate amount of barcode to the PBC tube. Besides, when I inspect the signal on the iridium channels and on CD45 I see that the Ir signal is very low and there is a sizable populations of CD45low cells which could represent RBCs (PBC sample did not undergo red cell lysis step):
I hypothesize that the red cells in the PBC tube are causing interference and quenching both the Ir intercalator and barcode. Not sure how that could be possible as far as the iridium intercalator goes since mature RBCs are not supposed to have much nucleic acid left. I am puzzled and would like to know if anyone else has seen this phenomenon. Any thoughts or comments are welcomed.
Best
JC
When I inspect the files using [Pd channels] vs time it becomes clear that the PBC tube has very low barcode signal on the expected channels (104/106/108). This is not seen on the other tubes:
Although possible, it is very unlikely we failed to add adequate amount of barcode to the PBC tube. Besides, when I inspect the signal on the iridium channels and on CD45 I see that the Ir signal is very low and there is a sizable populations of CD45low cells which could represent RBCs (PBC sample did not undergo red cell lysis step):
I hypothesize that the red cells in the PBC tube are causing interference and quenching both the Ir intercalator and barcode. Not sure how that could be possible as far as the iridium intercalator goes since mature RBCs are not supposed to have much nucleic acid left. I am puzzled and would like to know if anyone else has seen this phenomenon. Any thoughts or comments are welcomed.
Best
JC