A.

Flow or Mass Cytometry Multiplexed IHC

(Vectra, Codex, IMC)
Basic principle Analysis of dissociated tissue Analysis of intact tissue as
as a single-cell suspension cross-sections on a slide
Field of analysis Entire dissociated tissue Tissue cross-section(s)
Cell identification Single-cell suspension. Cells in close physical proximity.
Unit of analysis is individual cell. Cell identification requires cell
segmentation.
Cell loss Significant, due to tissue No cell loss
processing
Spatial context Destroyed Preserved
Tissue architecture Destroyed Preserved
Rate of event collection Rapid Slow

B.
Data Preprocessing

1. Experimental Design, 2. Region Selection 3. Spectral Unmixing 4. Cell Segmentation

Staining & Imaging
•Experimental question
•Panel design & optimization
•Whole slide scan (4x)
& Visualization
•Region selection
in Phenochart™ •Spectrally unmix (inForm®)
•Region scan (≥20x) •Assign pseudocolors
•Verify staining
•Segment cells (inForm®)
Continue to:
•phenotype in inForm®
•convert .txt file to .fcs
and phenotype using
other methods

5. Cell Phenotyping

Cell phenotyping options:

x,y coordinates
A) Manual inspection B) inForm® C) FlowJo® D) tSNE E) PhenoGraph
Confidence %

Input: inForm® spectrally Input: inForm® defined Input: inForm® defined cell segmentation
unmixed composite images cell segmentation .txt file export, converted to .fcs file

6. mIHC Specific Analysis

Manual Inspection Analysis using FlowJo Analysis in phenoptrReports

•Cell morphology •Structural association •Nearest neighbors

•Cell behavior •Protein expression •Cellular interactions
•Tissue architecture