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Peer-reviewedCC BYCytometry Part A

Measurement and Prediction of Unmixing‐Dependent Spreading due to Collinearity in Spectral Flow Cytometry

  • Peter L. Mage1
  • A Konecny2,3
  • Florian Mair4
4 affiliations
  1. 1Advanced Technology Group Waters Biosciences (formerly BD Biosciences) Milpitas California USA
  2. 2Department of Immunology University of Washington Seattle Washington USA
  3. 3Vaccine and Infectious Disease Division Fred Hutchinson Cancer Center Seattle Washington USA
  4. 4Flow Cytometry Core Facility ETH Zurich Zurich Switzerland
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Abstract

Advances in spectral cytometry instrumentation and fluorescent reagents have led to the possibility of ultra-high-parameter panels exceeding 50 colors. However, panel size is limited in practice by unmixing-dependent spreading (UDS), a phenomenon which leads to a progressive deterioration of unmixed signal-to-noise ratios in panels that contain fluorochrome combinations with significant spectral overlap. So far, choosing spectrally compatible sets of fluorochromes that avoid UDS has been a complex and labor-intensive task involving substantial trial-and-error experimentation. Here, we provide a detailed explanation of UDS and practical strategies for handling UDS in large spectral panels. We describe the empirical hallmarks of UDS, demonstrate how to quantify its impact, and dissect its underlying mathematical cause in terms of spectral collinearity. We present practical tools derived from the regression literature that can be used to select optimal combinations of fluorochromes in a platform-agnostic fashion based on publicly available reference data, providing a general tool for spectral panel design.

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Cite

BibTeXRIS
BibTeX
@article{mage2026measurement,
  title = {{Measurement and Prediction of Unmixing‐Dependent Spreading due to Collinearity in Spectral Flow Cytometry}},
  author = {Peter L. Mage and A Konecny and Florian Mair},
  journal = {Cytometry Part A},
  year = {2026},
  month = jun,
  doi = {10.1002/cyto.a.70044},
  url = {https://doi.org/10.1002/cyto.a.70044},
  note = {CC BY},
}
RIS
TY  - JOUR
TI  - Measurement and Prediction of Unmixing‐Dependent Spreading due to Collinearity in Spectral Flow Cytometry
AU  - Peter L. Mage
AU  - A Konecny
AU  - Florian Mair
PY  - 2026
DA  - 2026/06/01
JO  - Cytometry Part A
DO  - 10.1002/cyto.a.70044
UR  - https://doi.org/10.1002/cyto.a.70044
AB  - Advances in spectral cytometry instrumentation and fluorescent reagents have led to the possibility of ultra-high-parameter panels exceeding 50 colors. However, panel size is limited in practice by unmixing-dependent spreading (UDS), a phenomenon which leads to a progressive deterioration of unmixed signal-to-noise ratios in panels that contain fluorochrome combinations with significant spectral overlap. So far, choosing spectrally compatible sets of fluorochromes that avoid UDS has been a complex and labor-intensive task involving substantial trial-and-error experimentation. Here, we provide a detailed explanation of UDS and practical strategies for handling UDS in large spectral panels. We describe the empirical hallmarks of UDS, demonstrate how to quantify its impact, and dissect its underlying mathematical cause in terms of spectral collinearity. We present practical tools derived from the regression literature that can be used to select optimal combinations of fluorochromes in a platform-agnostic fashion based on publicly available reference data, providing a general tool for spectral panel design.
SN  - 1552-4922
SN  - 1552-4930
ER  -