Expression of Concern: Development of a 43 color panel for the characterization of conventional and unconventional T-cell subsets, B cells, NK cells, monocytes, dendritic cells, and innate lymphoid cells using spectral flow cytometry

Fairooz Sahir, Jericha Miles Mateo, Martin Steinhoff, Kodappully Sivaraman Siveen (2020), Development of a 43 color panel for the characterization of conventional and unconventional T-cell subsets, B cells, NK cells, monocytes, dendritic cells, and innate lymphoid cells using spectral flow cytometry. Cytometry Part A, https://doi.org/10.1002/cyto.a.24288.

This Expression of Concern is for the above article, published online on 18 December 2020 in Wiley Online Library (https://onlinelibrary.wiley.com/doi/10.1002/cyto.a.24288), and has been published by agreement between the journal's Editor-in-Chief, Attila Tárnok, the International Society for Advancement of Cytometry, and Wiley Periodicals, LLC.

The Expression of Concern has been agreed due to concerns raised by the cytometry community about the validity and reproducibility of the results, and overinterpretation of the data by the authors. However, the premise of the manuscript is that a 43-color panel is possible, with the conclusion that although not optimized, it nonetheless establishes an attempt at demonstrating the possibility of analyses at this depth of dimension.

With respect to panel design and assay optimization, the authors do not provide sufficient information regarding criteria for fluorochrome selection, spillover spread characterization, unmixing control optimization, unmixing accuracy, and differences in resolution between the single stained controls and the corresponding marker in the multicolor tube. The authors mention specific metrics used for panel design without explaining their meaning or relevance. In some instances, the gating strategy was found non-standard and the positioning of the gates arbitrary. Several populations display unusual patterns compared to what is expected in normal donors, and this may result from incorrect labeling of populations, poor data quality or limited familiarity with the tools used for analysis (e.g., high percentage of cells labeled as “MAIT”, and too close proximity of monocyte and dendritic cell populations in Suppl. Fig. 1). As the settings for the algorithms used have not been provided, reproducibility and verification of the accuracy of the data proves challenging. Lastly, the discussion of the figures and supplementary data is inaccurate or overinterpreted, in particular concerning the claims of satisfactory resolution using highly overlapping dyes when comparing the fully stained sample to the single-color stained sample (Suppl. Fig. 2).

The authors were informed about the concerns raised and asked to provide an explanation. Unfortunately, the concerns have not been satisfactorily addressed by the authors in the course of the investigation. Therefore, the journal has decided to issue an Expression of Concern to inform and alert the readers.