Robinson et al. provide a comprehensive description of the development and use of a 24-parameter flow cytometry immunophenotyping panel, which is designed to evaluate treatment-associated receptor expression on immune cells in MS.
While the correlation between immune circulating cells and MS outcomes is well established, the authors’ focus on monitoring treatment-associated receptors in B, T cells, and monocytes offers a valuable approach. However, a few areas of concern should be addressed:

• “Panel Design and Optimization:” The sections on panel design and optimization are overly detailed and more suited for methodological publications. This content could be condensed or referenced from previously established methods.

• Placement of "Optimization Reference Control": The information regarding "Optimization Reference Control" would be better placed in the Materials and Methods section, or in the Supplementary Information, rather than in the Results section, as it does not contribute directly to experimental findings.

• Lack of Results: The paper is primarily technical, focused on the setup of a panel for spectral flow cytometry. However, it lacks experimental results that demonstrate the utility of this panel. Including such data would significantly strengthen the manuscript.

• Small MS Cohort: The current MS cohort is too small to draw meaningful conclusions. I recommend expanding the cohort size and including post-treatment data to assess whether treatment-associated receptors are modulated, which would enhance the relevance and impact of the study.

In conclusion, while the technical details provided are thorough, the paper would benefit from a clearer focus on results and a larger, more diverse MS patient cohort.