Signal Amplification of FISH for Automated Detection Using Image Cytometry

Abstract

The purpose of this study was to improve the detection of FISH signals, in order that spot counting by a fully automated image cytometer be comparable to that obtained visually under the microscope. Two systems of spot scoring, visual and automated counting, were investigated in parallel on stimulated human lymphocytes with FISH using a biotinylated centromeric probe for chromosome 3. Signal characteristics were first analyzed on images recorded with a coupled charge device (CCD) camera. Number of spots per nucleus were scored visually on these recorded images versus automatically with a DISCOVERY image analyzer. Several fluochromes, amplification systems and pretreatments were tested. Our results for both visual and automated scoring show that the tyramide amplification system (TSA) gives the best amplification of signal if pepsin treatment is applied prior to FISH. Accuracy of the automated scoring, however, remained low (58% of nuclei containing two spots) compared to the visual scoring because of the high intranuclear variation between FISH spots.

Automated fluorescent in situ hybridization (FISH) analysis of t(9;22)(q34;q11) in interphase nuclei

“…Correct detection of FISH signals is the greatest challenge of automated analysis, since signals demonstrate large variations regarding shape, size, and intensity (21). FISH signals were correctly counted by automated analysis in~69% of nuclei.…”

State‐of‐the‐art FISHing: Automated analysis of cytogenetic aberrations in interphase nuclei

“…By the end of the 90s, the first attempt to analyze FISH-labeled tissue sections in 3D was also made by Adiga et al They quantified CEP7 signals in 10-15 lm thick tissue sections from patients with prostate cancer using confocal microscopy and semi-automated image analysis and reported results of automated counting being almost comparable with visual counting (21,33,80). In parallel with technological development, it has also been recognized that the quality of samples has huge effect on the performance of automated systems thus efforts with more or less success were taken in order to make the FISH spots brighter using various signal amplification methods (64). Apart from early technical comparative studies, results of automated analyses were almost always compared to that of manual scoring considering the latter one as a reference.…”

Fluorescence-based analysis of DNA ploidy and cell proliferation within fine-needle samplings of breast tumors

“…Cells were analyzed as described by Truong et al 16 First, DNA ploidy measurements were performed by exciting the DAPI at 330 nm, then Texas Red was excited with 577 nm red light for BrdU detection. Segmentation of both DAPI and Texas Red emitted light was performed by using an automatic threshold- ing procedure.…”

“…Still, systematic studies using image cytometry on fluorescence-stained samples are relatively few; most of them have analyzed specimen pretreatments and fixation procedures for one parameter only, such as in situ hybridization. 16 BrdU detection of proliferating cells is one of the most widely used methods because of its simple application and detection. Generally, there exists a good correlation between the BrdU labeling index (BLI) and breast carcinoma growth.…”