RT Journal Article SR Electronic T1 Reproducible, high-dimensional imaging in archival human tissue by Multiplexed Ion Beam Imaging by Time-of-Flight (MIBI-TOF) JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.10.14.464455 DO 10.1101/2021.10.14.464455 A1 Candace C. Liu A1 Marc Bosse A1 Alex Kong A1 Adam Kagel A1 Robert Kinders A1 Stephen M. Hewitt A1 Sean C. Bendall A1 Michael Angelo YR 2021 UL http://biorxiv.org/content/early/2021/10/16/2021.10.14.464455.abstract AB Multiplexed ion beam imaging by time-of-flight (MIBI-TOF) is a form of mass spectrometry imaging that uses metal labeled antibodies and secondary ion mass spectrometry to image dozens of proteins simultaneously in the same tissue section. Working with the National Cancer Institute’s (NCI) Cancer Immune Monitoring and Analysis Centers (CIMAC), we undertook a validation study, assessing concordance across a dozen serial sections of a tissue microarray of 21 samples that were independently processed and imaged by MIBI-TOF or single-plex immunohistochemistry (IHC) over 12 days. Pixel-level features were highly concordant across all 16 targets assessed in both staining intensity (R2 = 0.94 ± 0.04) and frequency (R2 = 0.95 ± 0.04). Comparison to digitized, single-plex IHC on adjacent serial sections revealed similar concordance (R2 = 0.85 ± 0.08) as well. Lastly, automated segmentation and clustering of eight cell populations found that cell frequencies between replicates yielded an average correlation of R2 = 0.92 ± 0.06. Taken together, we demonstrate that MIBI-TOF, with well-vetted reagents and automated analysis, can generate consistent and quantitative annotations of clinically relevant cell states in archival human tissue, and more broadly, present a scalable framework for benchmarking multiplexed IHC approaches.Competing Interest StatementM.A. and S.C.B. are inventors on patents related to MIBI technology. M.A. and S.C.B. are consultants, board members and shareholders in Ionpath Inc.