Table 1

Summary of current multiplex IHC/IF technologies

Light microscopyMultiplex IFTissue-based mass spectrometryDSP
Multiplex chromogenic IHCMICSSS
Basic descriptionSimultaneous/sequential application of immunostaining without the removal of previous markerIterative cycles of immunostaining, scanning, removal of chromogenic enzyme substrate and blocking previous primary antibodyIterative cycles of immunostaining using TSA amplification or DNA barcodesMass spectrometry imaging of primary antibodies tagged with elemental mass reporters.Primary antibodies bound to UV cleavable fluorescent DNA tags. A numerical value is generated that corresponds to the number of antibodies bound
# of markers on a single section3–5105–8 for TSA-based staining;
30–60 for non-TSA-based, cycled staining approaches
4040–50
Tissue staining time10–15 hours for
3–5 markers
1 day (~6 hours) per cycle
10 days for 10 markers
~TSA-based: ~15–20 hours for 6–8 markers; non-TSA-based cycled staining: ~2 hours per cycleSingle stain, 12 hours at 4°C1 hour
Imaging area*Whole slideWhole slideNon-multispectral: Whole slide
Multispectral: ROI=0.66 mm2
(larger areas may be imaged by tiling ROIs)
ROI=1.0 mm2
(larger areas may be imaged by tiling ROIs)
ROI=0.28 mm2
(larger areas may be imaged by tiling ROIs)
AdvantagesEasy use and interpretation
Established guidelines and protocols
Affordable and readily automated
Simple technique very similar to singleplex IHC
Relatively affordable
Whole slide images for each marker
Limited concern for bleed-through, blocking; no autofluorescence
Quantitative marker intensity
Simultaneous measurement of all markers
Autofluorescence correction with multispectral microscope
Quantitative marker intensity
Simultaneous measurement of all markers
No iterative staining cycles
No autofluorescence
Simultaneous measurement of all markers
No iterative staining cycles
No autofluorescence
DisadvantagesMarker intensity assessed semi-quantitatively
Co-expression studies limited and require carefully selected chromogen pairs
Unable to assess marker intensity
Coverslip removal can damage tissue if not careful
Difficulty of coregistration of whole slide images
Slow throughput
Potential fluorophore bleed-through
Potential blocking/umbrella effect with TSA reagents
DNA barcodes require a second round of staining and scanning to increase beyond four markers
Extensive training required
Expensive
Long imaging times
No single cell expression data (i.e., no cell counts or co-expression analysis, less spatial resolution)
Only able to visualize four markers to select ROIs
  • *For the technologies that currently image select ROI, it is possible to tile acquired images and then stitch them to represent whole slide scans.

  • DSP, digital spatial profiling; IF, immunofluorescence; IHC, immunohistochemistry; MICSSS, multiplexed immunohistochemical consecutive staining on single slide; ROI, regions of interest; TSA, tyramide signal amplification.