Stabilization of human whole blood samples for multi-center and retrospective immunophenotyping studies - Mass cytometry, staining Panel B

This .fcs files form the part of the manuscript titled “Stabilization of human whole blood samples for multi-center and retrospective immunophenotyping studies”. The .fcs files presented in this experiment were obtained for mass cytometry (MC) part of the manuscript, using antibody Panel B. To verify long time of storage, 45 aliquots of whole blood from a single donor using PROT protocol were fixed and frozen, and single aliquots were stained regularly using a 34-plex panel that expands both surface and cytokine detection
Conclusion:
The use of a reference sample in MC studies becomes necessary when multiple barcoded batches are run. This reference sample can then be used with confidence to normalize data across multiple runs. Our study provides the way to preserve a reference sample up to 13 months, showing remarkable sample stability in both phenotyping and cytokine studies.
Notes:
The reference blood sample was stimulated in a big volume with 1.25 μg/ml of R848 in the presence of Protein Transport Inhibitor Cocktail. 45 aliquots were fixed and frozen using PROT protocol, and single aliquots were stained regularly using a 34-plex panel that expands both surface and cytokine detection (Table S2, panel B in the manuscript). These files contain normalized, cleaned and live leukocytes events. Detailed gating strategy can be found in figure S9 in the manuscript. Cells were acquired on a mass cytometer (HELIOS, Fluidigm) at an event rate of 300-350 cells/second together with EQ Four Element Calibration Beads (Fluidigm). Raw data were normalized using normCytof function from CATALYST package. After noticing some signal instability in the data, we decided to run two selected functions from the flowAI package (flow_rate_bin, flow_rate_check) to remove regions with unstable flowrate. To make this function applicable to MC data, we adapted the TIMESTEP to 1 bin per 10 seconds. flowCut algorithm was used to remove signal instability with 1000 segments and MaxPercCut to 0.5. FlowJo software v10.0 was used to analyze normalized MC data by manual gating. Normalized, cleaned and live leukocytes events were selected using Pt and exported to this .fcs files.