CONCLAVE: CONsensus CLustering with Annotation-Validation Extrapolation for cyclic multiplexed immunofluorescence data

Nazari, P.; Arnould, A.; Andhari, M. D.; Fontecha, M.; Hernandez, J. M.; De Moor, B.; Pey, J.; De Smet, F.; Bosisio, F. M.; Antoranz, A.

doi:10.1101/2025.11.13.688190

bioRxiv · 10.1101/2025.11.13.688190

CONCLAVE: CONsensus CLustering with Annotation-Validation Extrapolation for cyclic multiplexed immunofluorescence data

Nazari, P.·Arnould, A.·Andhari, M. D.·Fontecha, M.·Hernandez, J. M.·De Moor, B.·Pey, J.·De Smet, F.·Bosisio, F. M.·Antoranz, A.

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Abstract

High-dimensional cyclic multiplexed immunofluorescence (cMIF) enables single-cell phenotyping within intact tissues. Cell annotations rely on a multi-step pipeline involving normalization, sampling, dimensionality reduction, and clustering, but the absence of standardized benchmarks for method selection--especially at the clustering stage--leads to inconsistent and less reproducible phenotyping. To address this, we developed CONCLAVE, a consensus-clustering-based workflow that optimizes upstream steps and integrates results from multiple clustering algorithms retaining only those cell labels supported by at least two independent methods. Through in-silico simulations and real-world cMIF datasets, CONCLAVE consistently outperformed single-clustering-method approaches in accuracy, reproducibility, and robustness, with improvements becoming more evident when mapped within spatial tissue contexts. Additionally, CONCLAVE includes a scoring module that flags regions likely to contain unreliable or inconsistent data, facilitating targeted quality control. In summary, CONCLAVE offers a robust framework for cell annotation in cMIF datasets, enhancing the reliability of downstream spatial proteomics analyses. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/688190v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@14ddfecorg.highwire.dtl.DTLVardef@1a83a17org.highwire.dtl.DTLVardef@17dfc74org.highwire.dtl.DTLVardef@493c07_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Nazari, P., Arnould, A., Andhari, M. D., Fontecha, M., Hernandez, J. M., De Moor, B., Pey, J., De Smet, F., Bosisio, F. M., Antoranz, A.. 2025-11-14. CONCLAVE: CONsensus CLustering with Annotation-Validation Extrapolation for cyclic multiplexed immunofluorescence data. https://doi.org/10.1101/2025.11.13.688190

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