High-Throughput Copy Number Analysis of 17q23 in 3520 Tissue Specimens by Fluorescence in Situ Hybridization to Tissue Microarrays

Context.— Fluorescence in situ hybridization (FISH) and brightfield in situ hybridization (ISH) are 2 clinically approved laboratory methods for detecting ERBB2 (HER2) amplification in breast cancer. Objective.— To compare the performance of FISH and brightfield ISH on proficiency testing administered by the College of American Pathologists Laboratory Accreditation Program. Design.— Retrospective review was performed on 70 tissue core samples in 7 separate proficiency testing surveys conducted between 2009 and 2013. Results.— The samples included 13 consensus-amplified tissue cores, 53 consensus-nonamplified cores, and 4 cores that did not reach consensus for FISH and/or brightfield ISH. There were 2552 individual responses for FISH and 1871 individual responses for brightfield ISH. Consensus response rates were comparable for FISH (2474 of 2524; 98.0%) and brightfield ISH (2135 of 2189; 97.5%). The FISH analysis yielded an average HER2 copy number per cell that was significantly higher (by 2.86; P = .02) compared with brightfield ISH for amplified cores. For nonamplified cores, FISH yielded slightly, but not significantly, higher (by 0.17; P = .10) HER2 copy numbers per cell. There was no significant difference in the average HER2 to control ratio for either consensus-amplified or consensus-nonamplified cores. Participants reported “unable to analyze” more frequently for brightfield ISH (244 of 2453; 9.9%) than they did for FISH (160 of 2684; 6.0%). Conclusions.— Our study indicates a high concordance rate in proficiency testing surveys, with some significant differences noted in the technical performance of these assays. In borderline cases, updated American Society of Clinical Oncology/College of American Pathologists cutoff thresholds that place greater emphasis on HER2 copy number per cell could accentuate those differences between FISH and brightfield ISH.

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Interphase cytogenetics using fluorescence in situ hybridization: an overview of its application to diffuse and solid tissue

Brazilian Journal of Genetics ◽

10.1590/s0100-84551997000100018 ◽

1997 ◽

Vol 20 (1) ◽

pp. 97-106 ◽

Cited By ~ 4

Author(s):

Christine Hackel ◽

Marileila Varella-Garcia

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Bone Marrow Cells ◽

Isolated Cells ◽

Interphase Cytogenetics ◽

Tissue Samples ◽

Solid Tissue ◽

Tissue Sections ◽

Tissue Specimens

Interphase cytogenetics, utilizing fluorescence in situ hybridization (FISH) techniques, has been successfully applied to diffuse and solid tissue specimens. Most studies have been performed on isolated cells, such as blood or bone marrow cells; a few have been performed on cells from body fluids, such as amniotic fluid, urine, sperm, and sputum. Mechanically or chemically disaggregated cells from solid tissues have also been used as single cell suspensions for FISH. Additionally, intact organized tissue samples represented by touch preparations or thin tissue sections have been used, especially in cancer studies. Advantages and pitfalls of application of FISH methodology to each type of specimen and some significant biological findings achieved are illustrated in this overview.

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High-density pile-up array is a reliable tool for the evaluation of high throughput screening in fluorescence in situ hybridization

10.26226/morressier.596dfd59d462b80292387d2d ◽

2017 ◽

Author(s):

Souda Masakazu

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

High Throughput ◽

High Throughput Screening ◽

High Density ◽

Reliable Tool ◽

Pile Up

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TERT and AURKA Gene Copy Number Gains Enhance the Detection of Acral Lentiginous Melanomas by Fluorescence in Situ Hybridization

Journal of Molecular Diagnostics ◽

10.1016/j.jmoldx.2013.10.009 ◽

2014 ◽

Vol 16 (2) ◽

pp. 198-206 ◽

Cited By ~ 19

Author(s):

Alba Diaz ◽

Joan Anton Puig-Butillé ◽

Alexandra Valera ◽

Concha Muñoz ◽

Dolors Costa ◽

...

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Copy Number ◽

Gene Copy Number ◽

Gene Copy

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Telomere Length Analysis of Human Mesenchymal Stromal Cells by High-Throughput Quantitative-Fluorescence in situ Hybridization (HT Q-FISH)

Cytotherapy ◽

10.1016/j.jcyt.2016.03.253 ◽

2016 ◽

Vol 18 (6) ◽

pp. S131

Author(s):

P. Teng-umnuay ◽

P. Yanudom ◽

T. Mitupatum ◽

J. Rongdet ◽

T. Sripradisvarakul

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Telomere Length ◽

Mesenchymal Stromal Cells ◽

High Throughput ◽

Stromal Cells ◽

Human Mesenchymal Stromal Cells ◽

Length Analysis ◽

Quantitative Fluorescence

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An Automated High-Throughput Fluorescence In Situ Hybridization (FISH) Assay Platform for Use in the Identification and Optimization of siRNA-Based Therapeutics

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555220960045 ◽

2020 ◽

pp. 247255522096004

Author(s):

Hui H. Dou ◽

Rommel Mallari ◽

Andrew Pipathsouk ◽

Amrita Das ◽

Mei-Chu Lo

Keyword(s):

In Situ Hybridization ◽

Gene Silencing ◽

Fluorescence In Situ Hybridization ◽

High Throughput ◽

Messenger Rna ◽

Great Promise ◽

Small Interfering Rnas ◽

Response Curves ◽

Vitro Assay

Since the revolutionary discovery of RNA interference (RNAi) more than 20 years ago, synthetic small interfering RNAs (siRNAs) have held great promise as therapeutic agents for treating human diseases by the specific knockdown of disease-causing gene products. To facilitate the development of siRNA therapeutics, a robust, high-throughput in vitro assay for measuring gene silencing is imperative during the initial siRNA lead sequence identification and, later, during the lead optimization with chemically modified siRNAs. There are several potential assays for measuring gene expression. Quantitative reverse transcription PCR (qRT-PCR) has been widely used to quantitate messenger RNA (mRNA). This method has a few disadvantages, however, such as the requirement for RNA isolation, complementary DNA (cDNA) generation, and PCR reaction, which are labor-intensive, limit the assay throughput, and introduce variability. We chose a high-content imaging assay, bDNA FISH, that combines the branched DNA (bDNA) technology with fluorescence in situ hybridization (FISH) to measure gene silencing by siRNAs because it is sensitive and robust with a short reagent procurement and assay development time. We also built a fully automated liquid-handling platform for executing bDNA FISH assays to increase throughput, and the system has a capacity of generating 192 concentration–response curves in a single run. We have successfully developed and executed the bDNA FISH assays for multiple targets using this automated platform to identify and optimize siRNA candidate molecules. Examples of the bDNA FISH assay for selected targets are presented.

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