PRODUCT INFORMATION Dako Omnis ALK, ROS1, RET and MET IQFISH Dako Agilent Pathology Solutions IQFISH on Dako Omnis Panel for Lung Cancer FAST RESULTS
Fast, high-quality FISH Integrated into your IHC workflow on Dako Omnis Dako Omnis, a fully automated, walk-away solution for advanced staining, provides a fully automated, FISH solution with high efficiency and flexibility. Automated slide throughput depends on length of protocol, slide capacity and reagent positions. With the short FISH protocol and high slide and reagent capacity, Dako Omnis processes FISH slides in an IHC-like turnaround time fast and with high quality. IQFISH panel for lung cancer automated on Dako Omnis The IQFISH panel for lung cancer enables the detection of rearrangements involving the ALK, ROS1 and RET genes, and the detection of MET gene amplifications by fluorescence in situ hybridization (FISH). The probes are designed using the groundbreaking, oliogonucleotide-based SureFISH technology and utilize the formamide-free IQFISH fast hybridization buffer. The probes are for use on lung paraffin-embedded (FFPE) tissue sections. It is compelling to move to the Dako Omnis platform from our current because it is automated, and we today do it manually. The result is as good as the one we currently have [from another vendor], but requires less hands-on time. Aleksandra Kolaric, University Örebro, Sweden ALK IQFISH Break-Apart Probe (Dako Omnis) ROS1 IQFISH Break-Apart Probe (Dako Omnis) RET IQFISH Break-Apart Probe (Dako Omnis) MET IQFISH Probe with CEP7 (Dako Omnis) FFPE sample stained with ALK IQFISH dual color, break-apart probe FFPE sample stained with ROS1 IQFISH dual color, break-apart probe FFPE sample stained with RET IQFISH dual color, break-apart probe FFPE sample stained with MET/ CEP7 IQFISH amplification probe Cells show ALK gene rearrangement. Cells show ROS1 gene rearrangement. Cells show RET gene rearrangement. Cells show MET gene amplification.
The short FISH protocol gives you a FISH solution with high efficiency and flexibility. High efficiency IHC-like turnaround time The high-throughput Dako Omnis combined with the IQFISH fast hybridization buffer gives labs an IHC-like turnaround time for FISH. IQFISH fast hybridization buffer reduces hybridization to 75 minutes, making the overall turnaround time for FISH just 4 hours. Workflow alignment across FISH applications The protocols developed for use with the ALK, ROS1, RET, and MET probes on Dako Omnis share ISH Accessories reagents and allow the probes to be run together in the same rack (up to five slides are processed per rack). The ability to batch in groups of five reduces the reagents and time needed to process the different assays. Furthermore, these probes can be run alongside HER2 IQFISH pharmdx (Dako Omnis), maximizing FISH utilization on the instrument. IQ Instant Quality FISH 0 2 4 6 8 10 12 14 16 18 Hours IHC-like TAT for FISH IQFISH hybridization buffer with 75 minutes hybridization 75 minutes IHC protocol Traditional formamidebased buffer Overnight hybridization FISH protocol 0 1 2 3 4 Time (Hours) Deparaffinization Digestion Hybridization Mounting Heat pre-treatment Denaturation Stringent wash Figure 1. The IQFISH fast hybridization buffer reduces hybridization time to 75 minutes compared to overnight hybridization with traditional formamide-based buffer. High flexibility Run FISH simultaneously with IHC Unlike other systems, Dako Omnis is designed for simultaneous FISH and IHC runs. FISH slides can be run with minimal impact to IHC throughput on Dako Omnis, as shown in the chart to the right. The short FISH protocol enables a capacity of up to 45 FISH slides per day with an overnight run. # of slides processed 120 100 80 60 40 20 0 5 FISH 105 IHC 95 IHC 85 IHC Scenario 1 OR OR Scenario 2 Scenario 3 10 FISH Same day* FISH Same day* IHC *8-hour workday Figure 2. Selection of Dako Omnis slide throughput scenario.
Easy, accurate and fast scoring Spend less time at the microscope thanks to high signal-to-noise ratio and micro-gap probe design High signal-to-noise ratio by repeat and blocker-free design IQFISH are designed in silico and chemically synthesized using Agilent s high-fidelity, oliogonucleotide library synthesis (OLS) technology. FISH probes from other vendors are purified from bacterial library clones (BAC) harboring human genomic DNA fragments. They therefore include repetitive sequences that can bind nonspecifically throughout the nuclei, producing a hazy background. Consequently, BAC-based probes usually come premixed with Cot-1 DNA, which blocks the background signals from repeated sequences, and also suppresses the overall hybridization signal. Agilent IQFISH probes address this by targeting only the unique sequences. During probe design, all repetitive elements are removed. This increases signal specificity and decreases hybridization background. It also eliminates the need for blocking agent and associated signal suppression. The combined effect is a high signal-to-noise ratio and easy visualization. Unique micro-gap design Agilent s oliogonucleotide-based SureFISH technology permits probe placement with base-pair-level precision. This allows a unique micro-gap design for ALK and RET IQFISH probes. Whereas the spacing between child probes with BAC-based probes is typically 100-300 kb, the spacing between Agilent s child probes is only about 0.4 kb. This design methodology is beneficial because the ALK-EML4 fusion is the result of an inversion between genes that are separated by approximately 12 Mb. Similarly, RET fusions with KIF5B are the result of an 11 Mb inversion. Detecting such intra-chromosomal inversions can be challenging due to the limited signal separation compared to a different chromosome. Agilent s micro-gap design provides tighter co-localization of orange-red and green signals in nuclei without the inversion, so cases with the inversion are easier and faster to analyze. Genomic DNA Repetitive & non-unique sequences Oligo FISH probe Standard FISH probe Figure 4. An inversion event. Binds across genome yielding hazy background Figure 3. Oliogonucleotide-based FISH (oligo FISH) probe design strategy. Repetitive elements are identified and removed during probe design process. Figure 5. The IQFISH ALK probe (top left) shows tighter co-localized signals than the ALK BAC probe from vendor (bottom left). Right: Inversion positive nuclei easy to distinguish from negative nuclei with IQFISH ALK probe.
Probe maps A typical signal pattern for negative specimens A typical signal pattern for positive specimens ALK IQFISH probe SHGC-144795 ALK 3 BA 300 kb 0.4 kb ALK 5 BA 599 kb SHGC-85275 Telomere 3' 5' Centromere ALK Chr. 2 ROS1 IQFISH probe SHGC-102962 ROS1 3 BA 1099 kb 137 kb ROS1 5 BA 1153 kb RH93170 Centromere 3' 5' ROS1 Telomere Chr. 6 RET IQFISH probe SHGC-8894 RET 5 BA 668 kb 0.3 kb RET 3 BA 602 kb SHGC-83659 Centromere 5' RET 3' Telomere Chr. 10 MET IQFISH probe STSG597983 7q31.2 MET 400 kb STSG598293 Centromere 5' MET 3' Telomere Chr7 CEP 767kb Chr. 7 Figure 6. Probe map and typical signal pattern for ALK, ROS1, RET and MET IQFISH probes.
Outstanding performance The same high-quality IQFISH on Dako Omnis as manual Performing FISH with the ALK, ROS1, RET, and MET IQFISH (Dako Omnis) probes together with all of the ISH accessory reagents and devices results in equivalent performance compared to the manual procedure. Nine NSCLC samples were processed both manually and on Dako Omnis. Probe signal intensity was evaluated on a 0-3 scale with: 0= no signal, 1= weak signal 2= moderate signal, 3= strong signal. For all samples evaluated, the staining intensity was moderate to strong with Dako Omnis processed samples giving slightly higher scores. Furthermore, processing of the ALK, ROS1, RET, and MET IQFISH (Dako Omnis) probes is both repeatable and reproducible across multiple instruments. The optimized probes and protocols for use on Dako Omnis provide high-intensity staining, work well across multiple tissue types, and provide reproducible results. Together these attributes can lead to reduced analysis time and fewer assay repeats. Manual Dako Omnis Manual Dako Omnis Figure 7. Performing FISH on Dako Omnis gives green and orange-red signals that are equivalent to the manual protocol. Negative specimen Positive specimen Negative specimen Positive specimen ALK RET ROS1 MET Figure 8. Images of IQFISH probes hybridized to negative (left column) and positive (right column) samples using Dako Omnis.
Two IQFISH protocols Two IQFISH protocols are available for processing on Dako Omnis: The IQFISH and IQFISH (Extra Wash). The ALK IQFISH protocol provides a slightly faster run-time and allows for the processing of HER2 IQFISH pharmdx (Dako Omnis) slides in the same Dako Omnis Slide Rack. Using the IQFISH (Extra Wash) protocol may result in lower background on challenging samples. (Note that the two protocols cannot be run in the same Slide Rack.) Microscope objective recommendation For identifying the relevant tissue/regions for scoring, an oilimmersion 20x objective with a minimum numerical aperture (N.A.) of 0.75 is recommended. For scoring signal patterns, an oil-immersion 60x or 100x objective with a numerical aperture (N.A.) of 1.4 is recommended. Step Reagent Time and Temperature Dewaxing Clearify (clearing agent) 10 minutes, 38 C Target retrieval ISH Pre-Treatment Solution (Dako Omnis) 15 minutes, 97 C Wash ISH Ethanol Solution, 96% (Dako Omnis) 2 3 minutes, 32 C Digestion ISH Pepsin (Dako Omnis) 30 minutes Drying Denaturation 15 minutes, 45 C 10 minutes, 66 C Hybridization ALK IQFISH Break-Apart Probe (Dako Omnis) 75 minutes, 45 C Stringent wash ISH Stringent Wash Buffer (Dako Omnis) 10 minutes, 61 C Cool down wash ALK IQFISH protocol: ISH Stringent Wash Buffer (Dako Omnis) ALK IQFISH (Extra Wash) protocol: Wash Buffer (Dako Omnis) Brief rinse, 1 cycle Brief rinse, 2 cycles Table 1. Simplified overview of the automated staining protocols for ALK IQFISH performed onboard Dako Omnis. Filter recommendations Filters are individually designed for specific fluorochromes and for each microscope. For the interpretation of IQFISH staining assays, the following combination of filters should be used: Fluorochrome Excitation Wavelength Emission Wavelength FITC 495 nm 517 nm Cy3 547 nm 565 nm Specific DAPI filter High-quality Cy3/FITC double filter (alternatively specific Cy3 and FITC single filters)
Ordering information Product Code Vol. & tests per unit ALK IQFISH BA Probe (Dako Omnis) G111800-2 2 ml, 20 tests ROS1 IQFISH BA Probe (Dako Omnis) G111801-2 2 ml, 20 tests RET IQFISH BA Probe (Dako Omnis) G111802-2 2 ml, 20 tests MET IQFISH Probe with CEP7 (Dako Omnis) G111803-2 2 ml, 20 tests ISH Pepsin (Dako Omnis) GM302 7 ml, RTU, 20 tests ISH Ethanol Solution, 96 (Dako Omnis) GM300 14 ml, RTU, 20 tests ISH Pre-Treatment Solution (20x) (Dako Omnis) GM301 175 ml for 3.5 L bulk, 5-25 tests FISH Stringent Wash Buffer (20x) (Dako Omnis) GM303 175 ml for 3.5 L bulk, 5-25 tests Fluorescence Mounting Medium (Dako Omnis) GM304 0.8 ml, 20 tests ISH Cleaning Solution (Dako Omnis) GC207 10 ml, 100 tests Dako Omnis FISH Lid GC102 Box of 5, 25 tests Dako Omnis Mixing Device GC116 1 pcs Companion devices Dako Omnis Mixing Device Dako Omnis FISH Lids Cy3/FITC double filter Dako Agilent Pathology Solutions www.dako.com Represented in more than 100 countries Australia +61 2 9922 0700 Austria +43 1 408 43 34 0 Belgium +32 (0) 16 38 72 20 Brazil +55 11 50708300 Canada +1 905 335 3256 China +86 21 3612 7091 Denmark +45 44 85 97 56 Finland +358 9 348 73 950 France +33 1 64 53 61 44 Germany +49 40 69 69 470 Ireland +353 1 479 0568 Italy +39 02 58 078 1 Japan +81 3 5232 9970 Korea +82 2 2194 9577 The Netherlands +31 20 42 11 100 Norway +47 23 14 05 40 Poland +48 58 661 1879 Spain +34 93 344 57 77 Sweden +46 8 556 20 600 Switzerland +41 41 760 11 66 United Kingdom +44 (0)1 353 66 99 11 United States of America +1 805 566 6655 PR7000-0192 Agilent Technologies, Inc. 2016 Printed in USA, June 1, 2016 5991-7018EN 29150 2016JUN01