TABLE 4.

Stages, challenge, and possible solutions for the best reproducibility of multiplex immunofluorescence panel.

Stage	Problem	Solution	Advantages of solutions	Disadvantages of solutions
Pre-analytical	Antibody specificity and staining	Use of positive and negative controls. Review of publications and experiences related to the Ab	Comparison with standardized process and other experiences	If the Ab does not have a previous protocol, it could result not reproducible
	Type of antibody and preparation	Preference by monoclonal antibody. Use specification of the vendor to prepare it	Better results	Not always is possible to monoclonal antibodies
	Optimization of panels in mIF	Test and work all the markers previously with IHC.	Comparison between IHC and mIF results	Some markers could not stain as the IHC
Analytical	Interpretation of markers	Standardized the interpretation of the most common markers	Interpretation well established	Some markers do not have protocols
	Consideration of areas of analysis and hotspots	Decide the number of representative areas of analysis and avoid select hotspots	Better representativeness	To have the right representative areas not always is possible. Number of ROIs could change depending on the type of tumor
	Type of image analysis	Do not expect to have the same result in all the different types of analysis technique. Consider the differences between software. Each one has its advantage and disadvantages		Experience-dependent
Post-analytical	Variability of intra- and inter-observer	Create protocols. If still persisting some variability, identify the problem	Standardization	Time-consuming and requires additional effort of the collaborators
	External and internal variability	Publish the results of each project. Take the experience of other laboratories to improve	Share knowledge	The new technologies do not have other experiences in other laboratories because they can be expensive