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Comparative image and flow cytometric TUNEL analysis of fine needle samples of breast carcinoma
Z Maciorowski1, J Klijanienko, E Padoy
1Department of Tumor Biology, Institut Curie, Paris, France. Zofia.Maciorowski@curie.net
Abstract:
The assessment of apoptosis in solid tumors is of interest because of its biological role in tumor evolution and response to therapy. A commonly used method for apoptosis measurement is the TUNEL 3' end-labeling technique, which has shown wide variations in results when applied to solid tumors. Thirty-one fine needle breast carcinoma samples were analyzed by fluorescent TUNEL assay and DNA content using image analysis and flow cytometry. TUNEL positivity, seen both in cells with apoptotic morphology and in a subset of morphologically normal cells, was categorized into five staining patterns and quantitated. Values for patterns of TUNEL-positive cells were compared with TUNEL positivity measured by flow cytometry. Flow cytometric quantitation showed a mean of 24.3% positive cells, which correlated (P < 0.02) with total positive cells (all patterns) measured by image (22.4%). Image analysis quantitation of morphologically apoptotic cells (4.2%) did not correlate with flow cytometric TUNEL positivity and the majority of TUNEL-stained cells were morphologically normal (17%). Image analysis allows discrimination of TUNEL-positive morphologically apoptotic and nonapoptotic cells, which are included in the total number of TUNEL-positive events measured by flow cytometry.
Insights
Assessing apoptosis in breast cancer using TUNEL assays reveals significant variations. Image analysis can differentiate apoptotic from non-apoptotic cells, improving accuracy in solid tumor studies.
Area of Science:
- Oncology
- Cell Biology
- Biomedical Imaging
Background:
- Apoptosis plays a crucial role in tumor evolution and treatment response.
- The Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay is widely used for apoptosis measurement.
- TUNEL assay results in solid tumors often exhibit significant variability.
Purpose of the Study:
- To evaluate the accuracy of TUNEL assay in assessing apoptosis in breast carcinoma.
- To compare TUNEL positivity measured by image analysis versus flow cytometry.
- To investigate the morphological characteristics of TUNEL-positive cells in solid tumors.
Main Methods:
- Analysis of 31 fine needle breast carcinoma samples.
- Fluorescent TUNEL assay combined with DNA content analysis.
- Quantification using both image analysis and flow cytometry.
Main Results:
- Flow cytometry showed a mean of 24.3% TUNEL-positive cells.
- Total positive cells by image analysis (22.4%) correlated with flow cytometry results (P < 0.02).
- Image analysis of morphologically apoptotic cells (4.2%) did not correlate with flow cytometry; most TUNEL-positive cells (17%) were morphologically normal.
Conclusions:
- Image analysis allows discrimination between apoptotic and non-apoptotic TUNEL-positive cells.
- A significant proportion of TUNEL-positive cells in breast carcinoma are morphologically normal.
- Image analysis offers a more detailed assessment of apoptosis compared to standard flow cytometry in solid tumors.