English Leica HER2 FISH System - 30 Test Instructions For Use For use on Leica Biosystems’ BOND-MAX and BOND-III System. TA9217 is a fluorescence in situ hybridization product designed to stain 30 tests (30 slides stained with LSI HER2/CEP17 Dual Probe).
Intended Use................................................................................................................................................................................3 For in vitro diagnostic use............................................................................................................................................................................... 3 Required training....................................................................................................
English Intended Use For in vitro diagnostic use The Leica HER2 FISH System - 30 Test is designed to detect amplification of the HER2/neu gene via fluorescence in situ hybridization (FISH) in formalin-fixed, paraffin-embedded human breast cancer tissue specimens. The Leica HER2 FISH System - 30 Test is indicated as an aid in the assessment of patients for whom Herceptin®* (trastuzumab) treatment is being considered (see Herceptin package insert).
The Leica HER2 FISH System - 30 Test contains the LSI HER2 DNA probe, a 226 Kb SpectrumOrange™ directly labeled fluorescent DNA probe specific for the HER2 gene locus (17q11.2-q12) and the CEP17 DNA probe, a 5.4 Kb SpectrumGreen™ directly labeled fluorescent DNA probe specific for the alpha satellite DNA sequence at the centromeric region of chromosome 17 (17p11.1-q11.1).
English The Leica HER2 FISH System - 30 Test is for use only on the Leica BOND-MAX and BOND-III System. Components Provided The materials listed below (Table 1) are sufficient to stain 30 tests (30 slides stained with LSI HER2/CEP17 Dual Probe). LSI HER2/CEP17 Probe 6.6 mL Post Hybridization Wash 2 9 mL Leica BOND Enzyme Concentrate 2 Contains ready-to-use LSI HER2/CEP17 Dual Probe. Contains <60% (v/v) formamide. Contains ready-to-use post hybridization wash solution. Contains <50% (v/v) formamide.
Warnings and Precautions For professional users only. One or more components in the product are hazardous and may cause harm to the unborn child. As a rule, persons under 18 years of age are not allowed to work with this product. Users must be carefully instructed in the proper work procedure, the hazardous properties of the product and the necessary safety instructions.
English bandpass fluorescence microscope filter sets optimized for use with the Leica HER2 FISH System - 30 Test are available for most microscope models. The recommended filter sets for the Leica HER2 FISH System - 30 Test are the DAPI/9-Orange dual bandpass, DAPI/ Green dual bandpass, Green/Orange(V.2) dual bandpass and the DAPI/Green/Orange (V.2) triple bandpass. C.
5. Turn on the PC attached to the Leica BOND-MAX and BOND-III System. 7. For a new Leica HER2 FISH System - 30 Test kit, scan the reagent tray barcode with the handheld scanner to enter the system into the Leica BOND reagent inventory (Single barcode only). 8. Prepare Leica BOND Enzyme 5 in the supplied Leica BOND Open Container at a dilution of 1:300. For example, for 10 slides add 10µL of Leica BOND Enzyme Concentrate 2 to 2990 µL of Leica BOND Enzyme Diluent. 9.
English Signal Assessment and Enumeration To assess the signal quality and enumerate the HER2 and CEP17 signals follow the process below: 1. Assessment of Slide Quality Evaluate slide quality using the following criteria: 1. Assess Slide Quality - Probe Signal Intensity - Background • Probe Signal Intensity: The signals should be bright and easy to evaluate. Signals should be in either bright, compact oval shapes or stringy, diffuse oval shapes.
To determine the LSI HER2 to CEP17 ratio, use the following method: 1. Record and determine the number of LSI HER2 and CEP17 signals in 20 nuclei (see Figure 2 Leica HER2 FISH System - 30 Test score sheet below). 2. Total all of the LSI HER2 signals. This represents the total LSI HER2 signals for the count, e.g. 143. 3. Total all of the CEP17 signals. This represents the total CEP17 signals for the count, e.g. 48. 4.
English Leica HER2 FISH System - 30 Test Interpretation Guide Count as 2 orange signals and 1 green signal Do not count. Nuclei with no signals or with signals of only 1 color should not be scored. Only score those nuclei with one or more FISH signals of each color Count as 1 orange signal and 2 green signals Count as 3 orange signals and 2 green signals Count as 5 orange signals and 4 green signals.
English Leica HER2 FISH System - 30 Test Score Sheet 20 Nuclei Signal Count Nucleus # HER2 Copy Number CEP17 Copy Number Nucleus # 1 11 2 12 3 13 4 14 5 15 6 16 7 17 8 18 9 19 10 20 Total 1-10 Total 11-20 HER2 CEP17 HER2 Copy Number CEP17 Copy Number HER2:CEP17 Amplification ratio Total Score 1-20 Average Per Cell Figure 2: Sample Score Sheet Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013 Page 12 of 24
English Quality Control Use of Control Slides It is recommended that a Leica HER2 FISH Control Slide is included in each test run to monitor assay performance and to assess the accuracy of signal enumeration. Control slides should be run for each staining batch on the Leica BOND-MAX and BOND-III System and with each new reagent lot. In addition, individual users may choose to use their own control material.
B. Product Specific Limitations This product is not designed for use in any other DNA-based diagnostic assay. Do not replace Leica HER2 FISH System - 30 Test reagents with any other components either supplied by Leica Biosystems or by other manufacturers. To do so will invalidate the assay. The user must validate any deviation from the recommended procedures. It is recommended that tissues fixed only in formalin-based fixatives be used in the assay.
English 2x2 Concordance Results Leica BOND-MAX System Data was grouped as negative (<2.00) or positive (≥2.00) for a 2x2 analysis The observed agreement for 300 samples between the two tests in a 2x2 analysis show a concordance of 99.33% (298/300) with a 95% CI of 97.61–99.
Data was grouped as negative (<2.00) or positive (≥2.00) for a 2x2 analysis The observed agreement for 300 samples between the two tests in a 2x2 analysis show a concordance of 99.67% (299/300) with a 95% CI of 98.16–99.99% for the Leica BOND-III System.
English Precision Testing – Leica BOND-MAX System A. Within Run Precision Study The within run precision study was performed in a randomized and blinded fashion. Within run precision testing of the Leica HER2 FISH System - 30 Test was evaluated at a single investigational site on 540 previously HER2 characterized TMA samples containing formalinfixed paraffin-embedded breast cancer cases.
The lot-to-lot precision study was performed in a randomized and blinded fashion. Lot-to-Lot precision was determined on three independently manufactured lots of the Leica HER2 FISH System - 30 Test, manufactured under Good Manufacturing Practice (GMP). Each lot was tested at a single investigational site on 540 previously HER2 characterized TMA samples containing formalin-fixed paraffin-embedded breast cancer cases.
English K. Between Observer Precision Study The between observer precision study was performed in a randomized and blinded fashion. Between observer reproducibility testing of the Leica HER2 FISH System - 30 Test was evaluated between three investigational sites. A single experienced observer at each investigational site was used. Eighteen whole section breast cancer cases were used for between observer precision, reflecting samples types used in the clinical setting.
• No difference in amplification status was observed when the post hybridization wash time was performed for 2 minutes, 5 minutes, and 7 minutes, when compared to the default Leica HER2 FISH System - 30 Test protocol. Highest quality ratings were observed at the default time of 4 minutes and this post hybridization wash time is recommended.
Troubleshooting English Problem No or weak fluorescent signal/staining Probable Cause Inappropriate fixation or processing of test specimen Ensure a formalin-based fixative is used and that processing schedules are suitable for the specimen undergoing testing. Leica HER2 FISH System - 30 Test is being used outside its expiry date Ensure the Leica HER2 FISH System - 30 Test used is within its specified expiry date.
Nonspecific background fluorescent signal/staining Poor preservation of tissue morphology Probable Cause Remedial Action Inadequate post hybridization washing Increase post hybridization wash incubation Inappropriate bulk reagents dispensed Ensure all Leica BOND reagents have been allocated into appropriate bulk containers and placed into appropriate positions on the instrument. Inadequate deparaffinization of slides Ensure Dewax is selected in the Preparation field of the Add slide dialog.
English References 1. Bargmann CI, Hung MC, Weinberg RA. The neu oncogene encodes an epidermal growth factor related protein. Nature 1986;319:226–30. 2. Lonardo F, Di Marco E, King CR, Pierce JH, Segatto O, Aaronson SA, et al. The normal erbB2 product is an atypical receptor-like tyrosine kinase with constitutive activity in the absence of ligand. New Biologist 1990; 2: 992–1003. 3. Wolff A.C., Hammond E.H., Schwartz J.N., et al.
This product contains PathVysion FISH probes supplied by Abbott Molecular Inc. PathVysion, LSI and CEP are a trademark of Abbott Molecular Inc. All Rights Reserved. Used under License. Amendments to Previous Issue Product name, Sponsor information, Summary and Explanation, Procedure.
