Leukocyte-Derived Extracellular Vesicles in Blood with and without EpCAM Enrichment
Abstract
:1. Introduction
2. Materials and Methods
2.1. Immunofluorescence Image Data Sets of EpCAM-Enriched Cells and Extracellular Vesicles of 25 Healthy Individuals and 75 Metastatic Cancer Patients
2.2. Blood Samples of 10 Healthy Individuals
2.3. Immunofluorescence Imaging of Cells and Extracellular Vesicles in Whole Blood Samples
2.4. Automated Enumeration of Objects in Immunofluorescence Images Using the Open-Source ACCEPT Software
3. Results
3.1. Detection of ldEVs in EpCAM-Enriched Blood Samples of Healthy Individuals and Metastatic Cancer Patients
3.2. Detection of Cell and Extracellular Vesicle Classes in the Blood Of Healthy Individuals without EpCAM Enrichment
3.3. ACCEPT Gates for the Automated Enumeration of Different Classes in the Blood with and without EpCAM Enrichment.
3.4. Absolute and Relative Frequencies of Leukocytes and ldEVs in 7.5 mL of EpCA- Enriched Blood of Healthy Individuals and Metastatic Cancer Patients.
3.5. The Reproducibility of Measurements by Fluorescence Imaging and the Correlation with the Frequencies of Blood Cells by Hematology Analyzer
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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A. EpCAM Enrichment | B. No Enrichment | ||||
---|---|---|---|---|---|
Leukocytes | DAPI/Hoechst a | Mean Intensity | >30 | Mean Intensity | >30 |
Max Intensity | >50 | Max Intensity | >50 | ||
Size | >16 μm2 | Size | >16 μm2 | ||
CD45 | Mean Intensity | >30 | Mean Intensity | >30 | |
Max Intensity | >50 | Max Intensity | >50 | ||
Size | ≤400 μm2 | ||||
CK | Standard Deviation | ≤5 | n/a b | ||
CD61 | n/a b | Standard Deviation | ≤5 | ||
CD235a | n/a b | Standard Deviation | ≤5 | ||
Extra channel | Standard Deviation | ≤5 | Standard Deviation | ≤5 | |
ldEVs | DAPI/Hoechst a | Standard Deviation | ≤5 | Standard Deviation | ≤5 |
CD45 | Mean Intensity | >30 | Mean Intensity | >30 | |
Max Intensity | >50 | Max Intensity | >50 | ||
Perimeter | >5 pixels | Perimeter | >5 pixels | ||
Size | ≤150 | Size | ≤150 μm2 | ||
Eccentricity | ≤0.85 | Eccentricity | ≤0.85 | ||
CK | Standard Deviation | ≤5 | n/a b | ||
CD61 | n/a b | Standard Deviation | ≤5 | ||
CD235a | n/a b | Standard Deviation | ≤5 | ||
Extra channel | Standard Deviation | ≤5 | Standard Deviation | ≤5 | |
Platelets | CD45 | n/a b | Standard Deviation | ≤5 | |
CD61 | Mean Intensity | >30 | |||
Max Intensity | >50 | ||||
Perimeter | >5 pixels | ||||
Size | ≤150 μm2 | ||||
Eccentricity | ≤0.85 | ||||
CD235a | Standard Deviation | ≤5 | |||
Standard Deviation | ≤5 | ||||
Extra Channel | Standard Deviation | ≤5 | |||
Red blood cells | Hoechst | n/a b | Standard Deviation | ≤5 | |
CD45 | Standard Deviation | ≤5 | |||
CD61 | Standard Deviation | ≤5 | |||
CD235a | Mean Intensity | >30 | |||
Max Intensity | >50 | ||||
Perimeter | >5 pixels | ||||
Extra Channel | Standard Deviation | ≤5 |
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Nanou, A.; Zeune, L.L.; Terstappen, L.W.M.M. Leukocyte-Derived Extracellular Vesicles in Blood with and without EpCAM Enrichment. Cells 2019, 8, 937. https://doi.org/10.3390/cells8080937
Nanou A, Zeune LL, Terstappen LWMM. Leukocyte-Derived Extracellular Vesicles in Blood with and without EpCAM Enrichment. Cells. 2019; 8(8):937. https://doi.org/10.3390/cells8080937
Chicago/Turabian StyleNanou, Afroditi, Leonie L. Zeune, and Leon W.M.M. Terstappen. 2019. "Leukocyte-Derived Extracellular Vesicles in Blood with and without EpCAM Enrichment" Cells 8, no. 8: 937. https://doi.org/10.3390/cells8080937