Proliferation/apoptosis determination by tissue cytometry in gastrointestinal fresh frozen sections using triple labeling and automated scanning fluorescence microscopy

摘要

Background: Proliferation/ apoptosis balance is an important information in gastrointestinal ulcerative and malignant diseases. Immunohistochemical staining and visual counting is the routine procedure. Recently we reported a new scanning fluorescence technique for automated motorized microscopes (SFM). Aims: Development of triple fluorescent labeling method for proliferating / apoptotic / resting cells and application of SFM for the automated analysis and counting on gastric biopsy specimen. Materials and methods: Routine antral biopsy specimens by gastroscopy (30) were fresh frozen and 5 micron sections were prepared. Proliferation was detected using a PCNA antibody, anti-mouse-biotin and streptavidin-Texas-Red labeling system. Apoptotic cells was labeled using the TUNEL reaction with FITC bound nucleotids. DAPI nuclear counter staining was applied. The labeled sections were scanned and digitized in the three fluorescent channels. SFM was modified to detect epithelial surface, glands in the biopsy specimen. Automated nuclei detection, PCNA and TUNEL detection was performed, ratio was calculated. In parallel standard biopsy specimen were labeled with PCNA and AEC. TUNEL reaction was also performed. Up to 1000 epithelial cells were manually counted. Results: The mean PCNA labeling in healthy samples were 45,3±12,4%, that significantly increased to 56.4+8.7% in H. Pylori positive cases. Positive TUNEL reaction was found in 2,9±1,1% in H. pylori negative cases, while in the H. Pylori positive cases the apoptotic ratio was significantly increased (14.1±3.2%, p < 0.05). Significant correlation in apoptosis/proliferation ratio between the SFM and routine methods could be observed (p < 0,05). The SFM procedure proved to be more time efficient both in the labeling, both in the detection procedures. Conclusions: Triple fluorescent labeling and automated fluorescence microscopy is an applicable tool for the proliferation, apoptosis determination in fresh frozen samples.