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Optimization of Mitotic Index Quantification Using the Amnis ImageStream Imaging Flow Cytometer.
I V Kholodenko1, K N Yarygin1, Y S Kim2
1Institute of Biomedical Chemistry, Moscow, Russia.
Bulletin of Experimental Biology and Medicine
|November 7, 2025
Summary
A new algorithm offers a more objective way to measure cell proliferation using imaging flow cytometry. This method improves the reproducible quantification of the mitotic index, crucial for cancer and stem cell research.
Area of Science:
- Cell Biology
- Biotechnology
- Oncology
Background:
- The mitotic index quantifies cell proliferation, vital for oncology and stem cell research.
- Imaging flow cytometry on the Amnis ImageStream platform with IDEAS software is a promising method for mitotic index assessment.
- Existing IDEAS algorithms present limitations like operator variability and difficulty in identifying mitotic populations.
Purpose of the Study:
- To critically evaluate the limitations of built-in IDEAS algorithms for mitotic index assessment.
- To propose and validate an alternative, automated algorithm (Mean + xSD) for more objective mitotic index quantification.
- To compare the accuracy and reproducibility of the proposed algorithm against the classical IDEAS approach.
Main Methods:
- Utilized imaging flow cytometry on the Amnis ImageStream platform.
- Critically evaluated the built-in Cell Cycle-Mitosis algorithm within IDEAS software.
- Developed and implemented an alternative Mean + xSD algorithm based on automated quantitative assessment of the Bright Detail Intensity R3 parameter.
- Tested the algorithms on Caco2 and HT-29 cell lines.
Main Results:
- The classical IDEAS algorithm showed operator-dependent variability and challenges in histogram interpretation.
- The proposed Mean + xSD algorithm eliminated the need for manual gating and histogram analysis.
- The Mean + xSD algorithm demonstrated accuracy comparable to the classical IDEAS algorithm.
- In certain cases, the Mean + xSD algorithm provided more reproducible quantification of the mitotic index.
Conclusions:
- The Mean + xSD algorithm offers a more objective and robust tool for analyzing mitotic activity in cultured cells.
- This automated approach overcomes key limitations of existing IDEAS algorithms for mitotic index determination.
- The findings support the adoption of the Mean + xSD algorithm for improved cell proliferation analysis in research settings.
