The following antibodies were used for immunostaining: 1D5 (Dako, Glostrup, Denmark) for ER, PgR636 (Dako) for progesterone receptor (PgR) and Hercep Test (Dako) for HER2. For evaluation of HER2 gene amplification, dual in situ hybridisation (DISH) was performed with INFORM HER2 Dual ISH DNA Probe Cocktail assay (Ventana Medical Systems, Inc., Tuscon, AZ, USA). ER, PgR and HER2 expression were evaluated in accordance with the American Society of Clinical Oncology and College of American Pathologists (11 (link),12 (link)) criteria. In addition, the degree of ER and PgR staining ≥1% and the specimen was determined as positive. The proportional scores of cells membrane HER2 staining intensity were as follows: scores 0, 1+, 2+ and 3+. HER2 immunostaining with a score of 2+ was subjected to a DISH assay to assess the gene amplification of HER2. A HER2 score of 2+/DISH positive or 3+ was defined as HER2-positive cancer. Patients with ER-positive and/or PgR-positive breast cancer were defined as a hormonal receptor (HR)-positive breast cancer.
Inform her2 dual ish dna probe cocktail assay
Manufactured by Roche
Sourced in United States, Switzerland
The INFORM HER2 Dual ISH DNA Probe Cocktail Assay is a laboratory diagnostic test used to detect the presence and quantify the expression levels of the HER2 gene in tissue samples. The assay utilizes in situ hybridization (ISH) technology to visualize and analyze the HER2 gene within the cellular context.
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Lab products found in correlation
Benchmark xt, by Roche (4 mentions)
Herceptest, by Agilent Technologies (3 mentions)
Benchmark xt machine, by Roche (2 mentions)
Silver ish dinitrophenyl sish, by Roche (2 mentions)
Red ish digoxigenin detection red ish, by Roche (2 mentions)
Pgr 636, by Agilent Technologies (1 mentions)
Envision flex kit, by Agilent Technologies (1 mentions)
Cx21i light microscope, by Olympus (1 mentions)
Ultraview red ish dig detection kit, by Roche (1 mentions)
Mib 1, by Agilent Technologies (1 mentions)
Er 1d5, by Agilent Technologies (1 mentions)
13 protocols using inform her2 dual ish dna probe cocktail assay
1
Immunostaining and HER2 Evaluation in Breast Cancer
2
Dual-Color DISH Assay for HER2 Gene
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This study is performed by using the INFORM HER2 Dual ISH DNA Probe Cocktail Assay from Ventana Medical Systems, which is a dual-color DISH assay. The HER2 gene is detected by a dinitrophenyl (DNP)-labeled probe and visualized using an ultraView silver in situ hybridization (SISH) DNP detection Kit. The CEN17 is targeted using a digoxigenin (DIG)-labeled probe and detected using an ultraView Red ISH DIG detection Kit. Under light microscopy, HER2 shows as discrete black signals, and chromosome 17 appears as red signals. The sections were loaded into the Ventana Benchmark XT machine. A fully automated procedure was carried out with the following basic steps: Deparaffinization, followed by cell conditioning, and protease digestion. Following that, the probe was applied followed by hybridization and application of the SISH Multimer. Following that, the silver chromogen was applied and then followed by the application of Red ISH Multimer and red chromogen. Finally, hematoxylin was used to counterstain the image, which was followed by clearing in xylene and mounting with dibutyl phthalate polystyrene xylene.
3
Her-2 Gene Amplification in Esophageal Neoplasia
4
HER2 Gene Amplification Evaluation
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SISH was performed using the INFORM HER2 Dual ISH DNA Probe Cocktail assay (Ventana Medical Systems) with an automated slide stainer according to the manufacturer’s protocols (BenchMark XT; Ventana Medical Systems). HER2 gene status was determined on the basis of counting its copies (black signals) and detecting the chromosome 17 centromere (red signals) in at least 20 tumour cells. Amplification of the HER2 gene was determined according to the ASCO/CAP guidelines for dual-probe ISH [16 (link)].
5
HER2 Gene Amplification Evaluation
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Silver in situ hybridization (SISH) was performed using the INFORM HER2 Dual ISH DNA Probe Cocktail assay (Ventana Medical Systems) with an automated slide stainer according to the manufacturer’s protocols (BenchMark XT; Ventana Medical Systems). Black (HER2) and red (CEP17) signals were quantified in 60 non-overlapping tumor cells. According to the ASCO/CAP guidelines for dual-probe ISH [24 (link)], interpretation of HER2 gene amplification was defined as follows: negative, HER2/CEP17 ratio <2.0 with an average HER2 copy number <4.0 signals/cell; equivocal, HER2/CEP17 ratio <2.0 with an average HER2 copy number ≥4.0 but <6.0 signals/cell; positive, HER2/CEP17 ratio ≥2.0 with an average HER2 copy number ≥4.0 signals per cell or a HER2/CEP17 ratio ≥2.0 with an average HER2 copy number <4.0 signals/cell or a HER2/CEP17 ratio <2.0 with an average HER2 copy number ≥6.0 signals/cell. Additionally, tumors harboring an average copy number of ≥10.0 signals/cell or a HER2/CEP17 ratio ≥5.0 were considered to have high-level amplification.
6
Profiling Germline BRCA and Somatic Oncogene Alterations
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DNA extracted from blood samples from 12 de-identified individuals with germline BRCA1 or BRCA2 deletions and duplications (exonic or whole gene) were obtained from PathWest, Western Australia, and the Australian Ovarian Cancer Study (AOCS) [9 (link)].
Breast cancer samples with >50% tumour purity and known ERBB2 amplifications (6 FFPE tumour samples), and high grade serous ovarian cancer samples with >50% tumour purity and CCNE1 amplifications (5 FFPE tumour and 3 snap-frozen tumour samples from 6 patients), were obtained from the Royal Melbourne Hospital and the Victoria Cancer Biobank (VCB), respectively. ERBB2 amplifications were detected using INFORM HER2 Dual ISH DNA Probe Cocktail Assay (Ventana, Tucson AZ), and CCNE1 amplifications were detected using chr19q12 ISH assay (Ventana).
This project was approved by and conducted under Melbourne University Human Research Ethics Committee project #1238381. Written informed consent for use of samples in future research was previously obtained from individuals enrolled through AOCS and VCB. Consent was waived by the ethics committee for patient samples received from diagnostic laboratories (PathWest, Western Australia and the Royal Melbourne Hospital).
Breast cancer samples with >50% tumour purity and known ERBB2 amplifications (6 FFPE tumour samples), and high grade serous ovarian cancer samples with >50% tumour purity and CCNE1 amplifications (5 FFPE tumour and 3 snap-frozen tumour samples from 6 patients), were obtained from the Royal Melbourne Hospital and the Victoria Cancer Biobank (VCB), respectively. ERBB2 amplifications were detected using INFORM HER2 Dual ISH DNA Probe Cocktail Assay (Ventana, Tucson AZ), and CCNE1 amplifications were detected using chr19q12 ISH assay (Ventana).
This project was approved by and conducted under Melbourne University Human Research Ethics Committee project #1238381. Written informed consent for use of samples in future research was previously obtained from individuals enrolled through AOCS and VCB. Consent was waived by the ethics committee for patient samples received from diagnostic laboratories (PathWest, Western Australia and the Royal Melbourne Hospital).
7
Her-2 Gene Amplification in Esophageal Neoplasia
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Her-2 gene amplification by in situ hybridization was performed using the Ventana INFORM Her-2 Dual ISH DNA Probe Cocktail assay (Ventana Benchmark Ultra Platform). This assay detects the Her-2 gene using a dinitrophenyl-labeled probe and the chromosome 17 centromere using a digoxigenin-labeled probe. The Her-2 gene was visualized as a discrete black signal, using the Ventana ultraview silver ISH dinitrophenyl (SISH). The chromosome 17 centromere was visualized as a red signal using the Ventana ultraview Red ISH digoxigenin detection (Red ISH). Using a ×40 or ×60 objective, analysis of ISH was performed on areas of NM squamous mucosa, BE, LGD, HGD, and ICA. For each nucleus, we conducted a manual count of the number of Her-2 signals and the number of centromere 17 (CEP17) signals using bright-field microscopy. The ratio of the average number of Her-2 gene copies to the average number of Chr17 copies was calculated. Indeterminate results were either due to absence of target cells, no tissue core present, unacceptable nuclear morphology (unable to distinguish NM cells from target cells), unacceptable background (SISH “dust”), or weak/absent ISH staining in target cells. For our analysis, a Her-2-to-Chr17 ratio of <2.0 was considered DISH-negative, whereas a Her-2-to-Chr17 ratio ≥2.0 was considered DISH-positive.
8
Immunohistochemical Profiling of Breast Cancer
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Immunohistochemical staining for the ER, PR and HER2 status was performed on tissue slices with standard methods30 (link),31 (link). A cut-off value of ≥ 1% positively stained nuclei was used to define ER and PR positivity using the Envision FLEX Kit (DAKO, Glostrup, Denmark). HER2 staining using the Hercep Test TM (DAKO, Glostrup, Denmark) was scored as 0, 1+, 2+, or 3+ according to the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines31 (link). HER2 immunostaining was considered positive when strong (3+) membranous staining was observed whereas cases with 0–1+ were regarded as negative. In cases with a HER2 2+ result, silver in situ hybridization (SISH) was performed using the INFORM HER2 Dual ISH DNA Probe Cocktail Assay (Ventana Medical Systems, Tucson, AZ) with an automated slide stainer according to the manufacturer’s protocols. HER2 gene amplification was defined with a HER2 gene/chromosome 17 copy number ratio ≥ 2.0 or a HER2 gene/chromosome 17 copy number ratio < 2.0 with an average HER2 copy number ≥ 6.0 signals/cell according to the ASCO/CAP guidelines.
9
Dual-ISH Analysis of HER2 Status
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D-DISH assay was carried out using the INFORM HER2 Dual ISH DNA Probe Cocktail Assay from Ventana Medical Systems. With this, HER2 gene gets detected by a dinitrophenyl (DNP)-labeled probe and visualized using ultraView silver in situ hybridization (SISH) DNP detection Kit. The chromosome 17 centromere is targeted with a digoxigenin (DIG)-labeled probe and detected using ultraView Red ISH DIG detection Kit. On light microscopy, HER2 appears as discrete black signals and chromosome 17 as red signals. The 2-μm-thick sections were loaded into the Ventana Benchmark XT machine. On board, a fully automated procedure was carried out as per the company recommended protocol with the following basic steps of deparaffinization followed by cell conditioning and protease digestion. Subsequently, the probe was applied followed by hybridization and application of the SISH Multimer. This was followed by application of silver chromogen, and then Red ISH Multimer and red chromogen. Finally, counterstain with hematoxylin was followed by clearing in xylene and mounting with dibutylphthalate polystyrene xylene. The slides were evaluated on Olympus CX21i light microscope using the 60× objective lens for better visualization and counting of the signals.
10
HER2 Status Determination in Invasive Breast Carcinomas
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One hundred and twenty specimens of invasive breast carcinoma (no special subtype; NST) were retrospectively investigated using routinely HER2 stained biopsies diagnosed within one year at the Institute of Pathology Nordhessen, Kassel, Germany (Example photomicrographs: Fig. 1 , Additional file 1 : Figure S1). HER2 status was determined according to the 2013 updated ASCO/CAP recommendations [4 (link)]. Accordingly, carcinomas classified as IHC 2+ were subsequently tested with dual-color chromogenic in situ hybridization (ISH) for amplification of the HER2/Neu Gene (INFORM HER2 Dual ISH DNA Probe Cocktail Assay, Ventana Medical Systems Inc., Tucson, USA). Anonymized cases were scored by three pathologists and the consensus score for each taken as the final IHC HER2 status.![]()
Her2-IHC scoring categories reflect DAB-precipitate widths. Table: Microscope objectives have a fixed resolution that depends on the numerical aperture (Range: Values of common objectives). DAB-precipitates in HER2-IHC differ in width according to the intensity score.
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