An Improved Barcoding Method Based On Ratiometry For Mass Cytometry Detection

For immunological flow cytometry analysis on large clinical cohort,researchers are commonly required to barcode their samples and perform the analysis simultaneously to minimize batch-to-batch variance and enhance data repeatability.Conventional flow cytometry adopts fluorescent tags to label antibodies.Since the number of available fluorescent tags are limited,it is extremely challenging to barcode a big cohort and,at the same time,leave enough channels to label target signaling markers.Mass cytometry mass has permitted high-throughput and multiplex approaches to acquire single-cell information of immunological status for clinical diagnostics.This project aims to develop a novel barcoding strategy for large scale clinical cohort diagnostics.Mass tags with different ratios will be applied to barcode common expressed antigens on cell surface（such as CD45）.Compared to conventional flow cytometry,mass cytometry（also named as CyTOF）can simultaneously label more than 100 channels.This capability mitigates the bottleneck that conventional technique can hardly barcode large clinical cohort and spare enough channels for target protein detection.To demonstrate the application of this novel strategy,three metal tags namely ¹⁶⁶Er,¹⁶⁹Tm and ¹⁷³Yb with only three ratios were able to barcode at least 19 clinical samples and allow simultaneous detection within the same batch.The gold standard method was utilized for double-label verification,and the debarcoding accuracy was above 85%.After barcoding,the median intensity signals of non-barcoded surface markers（such as CD3,CD4,CD8）among samples were getting close,which meant barcoding method facilitated the reduction of staining and background differences.Also,the sample identity was maintained with high accuracy for the correlation coefficients between barcoded and individual samples were all above 0.9.Moreover,the presented method minimizes the overall labeling operation process down to 40 minutes and avoids the expensive commercial barcoding buffer reagents.