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[The detection of apoptosis cells by TUNEL stain in peripheral blood smears]
1School of Allied Health Sciences, Yamaguchi University, Ube.
Abstract:
We have determined the fixative conditions for TUNEL stain in peripheral blood smears. Murine IL-3 dependent B cell line (BAF-3) was used as positive control. The intensity of fixing solutions effected on the positivity/negativity for TUNEL stain. Alive cells showed positivity for TUNEL stain (false positive), when 70% methyl alcohol, acetone or carnoy fluid was used. In contrast to the strong fixation by high concentrated glutaraldehyde and the phosphate buffer, denatured cells showed to be negative (false negative). The fixation by 0.4% glutaraldehyde and 70% methyl alcohol for 15 min at room temperature, followed by the treatment by 20 micrograms/ml proteinase K for 15 min at 37 degrees C was most suitable for TUNEL stain to detect the apoptotic cells in peripheral blood smears. The frequency of TUNEL positive cells increased significantly in peripheral blood cells treated by apoptosis induced reagents, or infected by CMV. This is a new approach for the techniques to detect the apoptotic cells in peripheral blood smears.
Insights
Optimizing fixation is crucial for accurate TUNEL staining in peripheral blood smears. A novel method using 0.4% glutaraldehyde and proteinase K effectively detects apoptotic cells, avoiding false positives and negatives.
Area of Science:
- Cell Biology
- Immunology
- Histology
Background:
- The Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stain is vital for detecting apoptosis.
- Standard fixation protocols can lead to inaccurate TUNEL staining results in peripheral blood smears, causing false positives or negatives.
Purpose of the Study:
- To determine optimal fixative conditions for TUNEL staining in peripheral blood smears.
- To establish a reliable method for identifying apoptotic cells in blood samples.
Main Methods:
- Tested various fixatives (methyl alcohol, acetone, carnoy fluid, glutaraldehyde, phosphate buffer) and conditions.
- Utilized a murine IL-3 dependent B cell line (BAF-3) as a positive control.
- Optimized fixation with 0.4% glutaraldehyde and 70% methyl alcohol, followed by proteinase K treatment.
Main Results:
- Fixative intensity significantly impacted TUNEL staining results.
- 70% methyl alcohol, acetone, or carnoy fluid caused false-positive TUNEL staining in live cells.
- Strong fixation with high glutaraldehyde or phosphate buffer resulted in false-negative staining in denatured cells.
- The optimized protocol (0.4% glutaraldehyde, 70% methyl alcohol, proteinase K) accurately detected apoptotic cells.
Conclusions:
- The developed fixation protocol is highly suitable for TUNEL staining of apoptotic cells in peripheral blood smears.
- This method improves the accuracy of apoptosis detection in blood samples.
- The optimized TUNEL staining technique shows increased sensitivity for detecting apoptosis induced by reagents or viral infections (e.g., CMV).