Advantages and drawbacks of cytometry techniques. This table lists the most representative advantages and disadvantages of the different immunophenotyping procedures, as well as some references that collect applications of these techniques in DILI research

Immunophenotyping techniquesDescription and applicationsAdvantagesDrawbacksApplications in DILI research
IFCIFC platform combines features of flow cytometry and fluorescent microscopy with data-processing algorithms, allowing for the evaluation of morphological and fluorescent data and analysing protein expression in single cells in heterogeneous cell populations

  • Spatial information beyond a single cell

  • Static or kinetic analysis of the sample

  • Reduced overlapping of fluorescence

  • Different cellular structures can be simultaneously visualized

  • Challenging analysis

  • A high percentage of acquired images are eliminated after processing

[29]
Multicolor flow cytometryTechnology that uses multiple fluorescent markers to identify and characterize cellular subpopulations of interest with the possibility of isolating pure, viable populations by cell sorting

  • Analysis of live and/or fixed cells

  • Cells can be retrieved for further analysis

  • Automatic data acquisition

  • Viability analysis can be performed

  • Possible overlapping of fluorophores’ emissions

  • The number of cell parameters that can be quantified is very limited

[30–33]
Mass cytometryMass cytometry utilizes elemental mass spectrometry to detect metal-conjugated antibodies bound intracellularly or extracellularly to antigens of interest on single cells, allowing the simultaneous analysis of a great number of cellular features

  • The use of rare metal isotopes instead of fluorochromes highly reduces the overlap between different signals

  • More than forty proteins can be detected in a single experiment

  • After the analysis, the cells used cannot be recovered for further studies

  • Cell function cannot be detected

  • Slow analysis

[33–35]
Hyperspectral flow cytometrySingle-cell analysis technique that combines ultrafast optical spectroscopy and flow cytometry. This technology uses diffraction gratings or prism-based monochromators to disperse fluorescence signals from multiple labels onto linear detector arrays

  • The capture signal covers the entire spectrum

  • Combination of chemometric methods of data processing and spectroscopy hardware with the single cell flow cytometry system

  • Intracellular spatial and quantitative information is obtained

  • Expensive technique

  • A wide hypercube data which requires precise and complex data processing is generated

  • Slow analysis

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