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CompensAID: An Automated Detection Tool for Reference Errors.

Rosan Olsman1, Sarah Bonte2,3, Mattias Hofmans4,5

  • 1Laboratory Medical Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands.

Cytometry. Part a : the Journal of the International Society for Analytical Cytology
|February 23, 2026
PubMed
Summary
This summary is machine-generated.

CompensAID automatically detects reference errors in flow cytometry data, improving quality control. This R-based tool flags marker combinations with potential inaccuracies, reducing manual inspection burdens.

Keywords:
compensationcomputational flow cytometryquality controlreference errorssecondary stain indexunmixing

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Area of Science:

  • Immunology
  • Computational Biology
  • Biotechnology

Background:

  • Flow cytometry data requires mathematical unmixing using reference controls.
  • Inaccurate controls (reference errors) distort fluorochrome abundance estimates and population distributions.
  • Manual inspection of marker combinations for errors is impractical for complex panels and large datasets.

Purpose of the Study:

  • To develop CompensAID, an open-source R-based tool for automatically identifying potential reference errors in flow cytometry.
  • To support and enhance quality control workflows in flow cytometry data analysis.

Main Methods:

  • CompensAID uses density-based cutoff detection to gate negative and positive populations.
  • The Secondary Stain Index (SSI) is computed on segmented positive populations.
  • Marker combinations are flagged if the last segment's SSI is below -1.

Main Results:

  • CompensAID achieved a sensitivity of 0.96 in conventional flow cytometry, identifying 23 out of 24 suspected marker combinations.
  • In spectral flow cytometry, sensitivity was 0.74, flagging 21 out of 28 suspected combinations.
  • False positives were observed, often due to suboptimal gating or low event counts.

Conclusions:

  • CompensAID provides a robust method for detecting potential reference errors in flow cytometry.
  • The tool significantly reduces the need for manual inspection, enhancing data reliability.
  • Integration of CompensAID into quality control pipelines is recommended for improved flow cytometry data analysis.