Table 1.

Summary of current multiplex IHC/IF technologies

	Light microscopy		Multiplex IF	Tissue-based mass spectrometry	DSP
	Multiplex chromogenic IHC	MICSSS
Basic description	Simultaneous/sequential application of immunostaining without the removal of previous marker	Iterative cycles of immunostaining, scanning, removal of chromogenic enzyme substrate and blocking previous primary antibody	Iterative cycles of immunostaining using TSA amplification or DNA barcodes	Mass 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 section	3–5	10	5–8 for TSA-based staining; 30–60 for non-TSA-based, cycled staining approaches	40	40–50
Tissue staining time	10–15 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 cycle	Single stain, 12 hours at 4°C	1 hour
Imaging area*	Whole slide	Whole slide	Non-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)
Advantages	Easy 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
Disadvantages	Marker 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.