Signal Amplification of FISH for Automated Detection Using Image Cytometry

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.

[1]  R. Haugland,et al.  Fluorescent rhodol derivatives: versatile, photostable labels and tracers. , 1992, Analytical biochemistry.

[2]  T. Harris,et al.  Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. , 1989, Journal of immunological methods.

[3]  J. Wiegant,et al.  Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides. , 1995, Human molecular genetics.

[4]  A. Raap,et al.  Statistical methods in interphase cytogenetics: an experimental approach. , 1993, Cytometry.

[5]  F. Ramaekers,et al.  Interphase cytogenetics of tumours , 1992, The Journal of pathology.

[6]  J W Gray,et al.  Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[7]  M N Bobrow,et al.  The use of catalyzed reporter deposition as a means of signal amplification in a variety of formats. , 1992, Journal of immunological methods.

[8]  J. Wiegant,et al.  Improved localization of fluorescent tyramides for fluorescence in situ hybridization using dextran sulfate and polyvinyl alcohol. , 1996, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[9]  D. Pinkel,et al.  Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Sample preparation and in situ hybridization techniques for automated molecular cytogenetic analysis of white blood cells. , 1996, Cytometry.