Reproducible, high-dimensional imaging in archival human tissue by multiplexed ion beam imaging by time-of-flight (MIBI-TOF)

Abstract

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 serial sections yielded an average correlation of R² = 0.94 ± 0.05. 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.

Figure 1:. MIBI-TOF validation overview

A tissue microarray (TMA) was constructed using human FFPE tissue blocks, and the TMA was serially sectioned for analysis by single-plex chromogenic IHC and MIBI-TOF. The order that each serial section was stained and imaged was randomized. We then analyzed the concordance between IHC and MIBI-TOF, as well as assessed reproducibility of MIBI-TOF between serial sections.

Figure 2:. Representative MIBI-TOF imaging data

High-quality MIBI-TOF imaging data was attained for 16 markers for all serial section TMA slides. Representative images of the various tissue types in the TMA are shown here.

Figure 3:. Concordance of Mean Pixel Intensity by MIBI-TOF

(A) Representative MIBI-TOF images from serial sections of the same TMA core of lymph node tissue. (B) Plot of the average Mean Pixel Intensity (MPI) of all FOVs of the same TMA core vs. the MPI of each FOV. Each color represents a different marker. The gray line is a reference line with slope 1 and intercept 0. We performed least squares linear regression of the average MPI of the core vs the MPI of all six serial sections and found the slope m (C) and coefficient of determination R² (D) for all 16 markers.

Figure 4:. Reproducibility of cell phenotyping by MIBI-TOF

(A) Cells were assigned to a phenotype using an unsupervised clustering approach and manually annotated into eight major cell types. Expression values were z-scored for each marker. High z-scores are an indicator of high marker specificity. (B) The Spearman correlation between all serial sections of each TMA core using the frequency of cell types in each FOV. (C) Representative images of six serial sections of the same TMA core of tonsil tissue. Top row: Cell phenotype map colored according to the eight cell types shown in (A). Middle and bottom rows: MIBI-TOF images showing CD20 and PanCK expression.

Figure 5:. Concordance of MIBI-TOF with single-plex chromogenic IHC

Representative images (CD3: tonsil, CD8: lymph node, Pax5: lymph node) of the comparison of MIBI-TOF images co-registered with single-plex chromogenic IHC stains. Each data point represents the PPP by MIBI-TOF and IHC for a single tissue core. Color of data points indicates tissue type. Shaded area represents 95% confidence interval. Additional markers are shown in Supplementary Figure 9.