Table 3.
Advantages and disadvantages for different mfIHC technologies.
| Method Name | Advantage | Disadvantage |
|---|---|---|
| Stain removal technologies | ||
| MELC (Toponome imaging systems) |
Detects hundreds of proteins and high resolution | The multiprobe image is limited to a single microscopic medium-to-high power field and high cost |
| SIMPLE | Easy to perform by whole-slide scanner and can be labeled primary antibodies from same species | Up to 12 biomarkers |
| IBEX | Allows over 65 biomarkers to detect and compatible with over 250 commercial antibodies | Not commercialized and few studies |
| Fluorophore inactivation technologies | ||
| MxIF | Up to 60 biomarkers | Time-consuming and relatively expensive |
| CycIF | Use commonly reagents and instruments | Before the next staining, coverslip should be removed and time-consuming |
| ChipCytometry | Detects unlimited number of biomarkers, long-storage samples, removes autofluorescence and instrument automaticity | Damage the tissue adherence and photobleachable dyes may generate weak signals during imaging processing |
| Multiplexed signal amplification | ||
| Multiplex modified hapten-based | Two-hour fast staining and cocktail antibodies are used in a single slide | Maximal four biomarkers can be labeled per slide and not applied widely |
| TSA | Avoids antibody cross-reactivity and may realize an automated protocol | Nine biomarkers can be labeled per slide |
| QDs | Removes autofluorescence and has much stronger signals | Big size relatively, has toxicity and limited nanocrystals |
| DNA barcoding technologies | ||
| DEI | Short-time staining and applies for most microscopy platforms | Lack of signal amplification, few studies |
| CODEX | Allows 60 biomarkers labeled and can be imaged by conventional fluorescence microscopy, also keeps the morphology of normal and diseased tissues | Longer scanning and lack of signal amplification |
| Immuno-SABER | High multiplexing, sensitivity and 5–180-fold signal amplification | Up to 10-plex and few publications |
| DSP | No-damage staining protocol and performs high multiplexing image on FFPE samples | Chooses ROI manually and is not able to reconstruct images |
| InSituPlex® | Good signal in low-expression antigen, 5.5 h workflow and relatively cheap | Few studies |
| Mass cytometry | ||
| IMC | Removes autofluorescence, reveals the quantity of proteins in subcellular level | Lack of signal amplification, the rate of image acquisition is slow and relatively low resolution in subcellular level |
| MIBI | A large number of metal-antibodies can be labeled spectral overlap and high resolution | Time-consuming, instrument and metal-antibodies are expensive |