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A method for simultaneous quantification of monoclonal antibody Ki-67 and DNA content by flow cytometry. Application
M C Gorisse1, L Venteo, M Pluot
1Department of Biology, Institut Jean Godinot, Reims, France.
Insights
A new flow cytometry method simultaneously assesses Ki-67 immunostaining and DNA content. This technique accurately evaluates tumor proliferative activity, offering a fast and sensitive approach for pathology departments.
Area of Science:
- Oncology
- Immunohistochemistry
- Flow Cytometry
Background:
- Ki-67 identifies a nuclear protein in nonquiescent tumor cells.
- The Ki-67 antigen is labile with standard fixation methods.
- Accurate assessment of tumor proliferation is crucial for diagnosis and treatment.
Purpose of the Study:
- To develop a novel method for simultaneous Ki-67 immunostaining and DNA content analysis.
- To enable rapid and sensitive quantification of tumor cell proliferation.
- To validate the technique for assessing breast carcinoma and other solid tumors.
Main Methods:
- Developed a flow cytometry technique preserving the labile Ki-67 antigen using saponin permeabilization.
- Quantified nuclear Ki immunofluorescence and DNA content simultaneously in unfixed tumor cells.
- Calculated Ki-positive subpopulations by subtracting non-specific fixation histograms.
Main Results:
- Observed significant correlations between Ki-67 staining and S-phase fraction.
- Aneuploid tumors showed significantly higher mean Ki-67 labeling rates than diploid tumors.
- Found significant differences in Ki-67 labeling rates among histologic types.
Conclusions:
- The developed flow cytometry technique is fast, sensitive, and optimal for evaluating proliferative activity.
- This method is suitable for routine use in pathology departments for breast carcinomas and other solid tumors.
- Simultaneous assessment of Ki-67 and DNA content provides valuable prognostic information.
Objective:
To seek a method assessing Ki-67 immunostaining and DNA content by flow cytometry simultaneously.
Study Design:
The murine monoclonal antibody Ki-67 (Ki) identifies a nuclear protein complex expressed by all nonquiescent tumor cells. Since the antigen detected by Ki is quite labile in most fixation and embedding protocols, a new method for simultaneous quantification of nuclear Ki immunofluorescence and DNA content by flow cytometry was developed. Unfixed, solid tumor cells are permeabilized only with saponin to preserve Ki antigen. The percentage of Ki-positive cell subpopulations calculated by subtraction of the related aspecific fixation histogram gives optimal results more rapidly than by cytogram analysis. Application to breast carcinoma shows the feasibility of the method.
Results:
Significant correlations between Ki staining and the S-phase fraction were observed. Mean Ki labelling rates of aneuploid tumors were significantly higher than those of the diploid tumors, and significant differences between histologic types were found.
Conclusion:
This technique can be considered a fast, sensitive and optimal method to evaluate the proliferative activity of breast carcinomas and possibly of other solid tumors in a department of pathology.
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