Cish

Chromogenic In Situ Hybridization

1. Chromogenic in Situ hybridization By- Anubhav Agrawal, 514

2. Contents Introduction Procedure Comparison to similar techniques Services in medical studies Modifications and variations

3. introduction In Situ Hybridization (ISH) is a technique that allows for precise localization of a specific segment of nucleic acid within a histologic section. The underlying basis of ISH is that nucleic acids, if preserved adequately within a histologic specimen, can be detected through the application of a complementary strand of nucleic acid to which a reporter molecule is attached. Visualization of the reporter molecule allows to localize DNA or RNA sequences in a heterogeneous cell populations including tissue samples and environmental samples. Riboprobes also allow to localize and assess degree of gene expression .

4. RNA in situ hybridization - KRT5 and housekeeping gene in human melanoma FFPE tissue section - visualized under brightfield and fluorescence microscope.

5. Chromogenic in situ hybridization (CISH) is a cytogenetic technique that combines the chromogenic signal detection method of immunohistochemistry (IHC) techniques with in situ hybridization. It was developed around the year 2000 as an alternative to fluorescence in situ hybridization (FISH) for detection of HER-2/ neu oncogene amplification. CISH is similar to FISH in that they are both in situ hybridization techniques used to detect the presence or absence of specific regions of DNA.

6. Medical application CISH is frequently applied to assess gene amplification, such as HER-2/ neu status in breast cancer samples. CISH is also used for detection of chromosomal rearrangements and fusions, such as the fusion of ALK tyrosine kinase domain with the promoter and 5’ region of EML4 in lung cancer. Apart from cancers, CISH has also been shown to be useful in detecting human papillomavirus infections.

7. Comparison Compared to FISH- Compared to FISH, CISH has been shown to have a sensitivity of 97.5% and a specificity of 94% for detection of HER-2/ neu gene amplification. CISH is much cheaper and is easier to use because it uses bright-field microscopes instead of fluorescence microscopes. In addition, the CISH reagents are more stable than the FISH reagents so it is possible to store the samples and examine the same sample multiple times. Compared to IHC- A disadvantage of IHC is that it is not possible to identify false-negative and false-positive results. In CISH, if there is no signal for the reference probe, the assay has failed.

8. CISH FISH

9. CISH IHC

10. Variations Silver-enhanced in situ hybridization (SISH) SISH uses a similar method as CISH, but a silver precipitate is the end product rather than a brown product. In this method, a probe tagged with dinitrophenol (DNP) binds to the target sequence. A primary anti-DNP antibody is then added followed by a secondary antibody conjugated to HRP. Silver acetate, hydroquinone, and hydrogen peroxide are subsequently added to the secondary antibody and HRP catalyzes the polymerization of silver in the presence of hydrogen peroxide. Silver metal is consequently deposited into the nuclei of the cell. Amplification of HER-2/ neu is seen as black dots.

11. DuoCISH DuoCISH is a variation of CISH that addresses the need for two different probes on the same slide. It is a well-established technique for HER-2/ neu amplification detection even though it is sometimes reported to be less effective than FISH. In this technique, one probe binds the reference which, in the case of HER-2/ neu amplification detection, is CEN17 (the centromere of chromosome 17) while the other probe binds the target sequence which is HER-2/ neu . DuoCISH combines both FISH and CISH in that it converts signals from FISH probes to chromogenic substrates. It works on the principle that blue cyanine dye is a substrate for HRP and red dye is a substrate for AP.