Table 2.

Challenges and current limitations of multiplex IHC/IF approaches.

Challenge	Current limitations
Preanalytical variables	Fixation, cold or hot ischaemic time, sectioning, storage.
The choice of the methodology approach and platform	- Because of the number of methodologies of staining and multiplexed image platforms is challenging to choose which approach would be best for a lab. - Batch controls which are used to normalise quantitative expression signal levels for staining variability. - Pathologists need to consider the advantages and disadvantages of these techniques and the most appropriate use of each technology to be applied for translational research or clinical use.
Choice between chromogenic and immunofluorescence multiplex approach	- The vast majority of pathologists routinely use bright-field microscopes. This approach is certainly more accessible to most pathology laboratories but also has limitations, in particular for the number of possible antibodies to be used per tissue section (on average not more than 5). - Chromogenic approach is not optimal for the analysis of co-expression or precise cellular localisations (nucleus, cytoplasm, membrane). - mIF is better for 3+ plexing, when expression level quantitation and dynamic range is required, and when marker pixel-level colocalization is expected, such as is often the case of cell surface markers. mIHC is better for 1–3 markers that do not colocalize and is primarily for visual assessments, since umbrella affects are significant and hard to mitigate through staining methods.
The choice or identify valuable biomarkers within the tumour	- Given the large amount of recent data, in several solid tumours, the use of several dozen antibodies could be required, but it is impossible to ensure exhaustiveness according to the different molecules of interest.
Real standards for clinical validation	- Optimisation and validation of markers for clinical use can be established only across multiple clinical trials. - Quantification criteria need to be established for accuracy and reproducibility. - Validation of artificial intelligence algorithms is required to have a robust platform.
Turnaround time	- In the context of the treatment of patients, the time required to obtain the results of these complex analyses must be evaluated.
External quality assurance	- Within the framework of external quality assurance controls, it will be necessary to find organisations able to propose external validation tests or to have the possibility of carrying out ring trial studies.
Accreditation/certification	- The accreditation/certification of these different approaches will certainly be a long step to achieve and the issue of the In Vitro Diagnostic Regulation (IVDR) of the European Union will have to be integrated.
Costs	- The fees and reimbursements of these tests will have to be considered promptly in order to allow their diffusion.