Table 4.

Analytic challenges of multiplex in situ methods.

Multiplexing Level
High	Challenging panel validation, increase in image analysis labor/difficult cell phenotyping
Panel Validation
Section thickness	Affects staining intensity and tissue autofluorescence
Staining sequence of primary antibodies	Can deal with epitope instability and cross reaction of primary antibodies
Fluorophores, Co-localization in the same cellular compartment, Low abundance epitopes	Selection of spectrally separated fluorophores; selection of more intense fluorophores
Staining pattern	For each antibody, staining pattern in multiplex image should be identical to single-plex immunohistochemistry
Regions of interest
Number	Whole tissue section resemblance: as many as possible evaluation
Prior selection	Potential selection bias
Statistical analysis
Optimal statistical method	Hierarchical linear modeling: statistical power improvement over t-test.
Cut-off	Biomarkers expressed by various cell types: establishment of single positivity threshold is difficult
Image Analysis
Storage	Extreme data sizes
Cell phenotyping	Difficult cellular segmentation: 1. Mixed cells with different shapes and sizes 2. High multiplexing level
Distance analysis	Densities can be confounding factor: Possible solution proximity analysis between areas with similar levels of cellular infiltration
Bias	Inter-center and inter-vendor variability, as well as intra- and inter-observer variability either during tissue sample preparation or during image acquisition.