Leica Oxygen Equipment TA9217 User Manual

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).  
IVD  
Leica Biosystems Newcastle Ltd  
Balliol Business Park West  
Benton Lane  
Newcastle Upon Tyne NE12 8EW  
United Kingdom  
( +44 191 215 4242  
Leica Biosystems Canada  
71 Four Valley Drive  
Concord, Ontario L4K 4V8  
Canada  
( +1 800 248 0123  
Leica Biosystems Inc  
1700 Leider Lane  
Buffalo Grove IL 60089  
USA  
( +1 800 248 0123  
Leica Biosystems Melbourne  
Pty Ltd  
495 Blackburn Road  
Mt Waverly VIC 3149  
Australia  
( +61 2 8870 3500  
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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
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 Herceptinpackage insert). The Leica HER2 FISH System - 30 Test is not intended for use  
to screen for or diagnose breast cancer. All other available clinical information should also be  
taken into consideration, such as tumor size, number of involved lymph nodes, and steroid  
receptor status. No treatment decision for breast cancer patients should be based on HER2  
gene amplification status alone.  
Note: All of the patients in the Herceptin clinical trials were selected using an investigational  
immunocytochemical Clinical Trial Assay (CTA). None of the patients in those trials were  
selected using the Leica HER2 FISH System - 30 Test. The Leica HER2 FISH System - 30 Test  
has been compared to the Abbott Molecular PathVysion®* HER-2 DNA Probe Kit assay on an  
independent set of samples and found to provide acceptably concordant results, as indicated  
in the Clinical Concordance Summary. The actual correlation of the results of the Leica HER2  
FISH System - 30 Test to clinical outcome has not been established.  
* Herceptin® is a trademark of Genentech, Inc. and F. Hoffmann-La Roche Ltd. PathVysion® is a trademark of Abbott Molecular  
Inc. All Rights Reserved. Used under License.  
Required training  
Leica Biosystems will provide training in specimen preparation, assay procedure, and  
interpretation of FISH testing of the HER2 gene for all users.  
Summary and Explanation  
Background  
The HER2 gene, alternatively known as neu or c-erbB2, is located on the long arm of  
chromosome 17 at position 17q11-12 (1). Both the HER2 gene and its 185 kD encoded protein  
have been shown to play a major role in malignant transformation and tumor progression of  
breast cancer (2).  
HER2 functions as a prognostic marker, with gene amplification and protein over expression  
being linked to an increased rate of disease recurrence and higher mortality. HER2  
also functions as a predictive marker for selected systemic chemotherapy and targeted  
treatments (3). Specifically, amplification of the HER2 gene has been shown to be an indicator  
of poor prognosis in node-positive breast cancer (4-8). Furthermore, one study indicates  
the prognostic value of HER2 to be stronger among patients treated with chemotherapy (7).  
However, in predicting disease-free and overall survival in individual patients, other established  
prognostic factors such as tumor size, number of positive lymph nodes and steroid receptor  
status must also be taken into consideration.  
Overexpression of the HER2 oncoprotein, as a result of gene amplification found in breast cancer  
cells, suggests HER2 as a target for an antibody-based therapy (3). Herceptin (trastuzumab),  
a humanized monoclonal antibody (9) that binds with high affinity to the HER2 oncoprotein has  
been shown to inhibit the proliferation of human tumor cells that overexpress HER2 oncoprotein  
both in vitro and in vivo (10–12). Since the development of Herceptin, the detection of both  
the HER2 gene and protein have become essential tools in the assessment of breast tumors,  
directing both therapy selection and subsequent patient management (13,14).  
In both interphase and metaphase cells derived from human breast carcinoma cell lines, FISH  
has been used to show HER2 gene amplification (15-18). For quantification of HER2 gene  
amplification, FISH assesses the level of HER2 gene amplification directly in the tumor cells.  
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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
 
The characteristic morphology of the tissue and the spatial distribution of oncogene copies in  
individual uncultured primary breast carcinomas are retained. Aberrations in chromosome 17  
copy number (aneusomy) are also commonly found in breast tumors. These may present as  
chromosome deletions or gains (polysomy). This chromosomal variation has critical impact on  
the interpretation and reporting of HER2 gene amplification status. Therefore, measurement of  
chromosome 17 copy number in conjunction with HER2 is critically important (4).  
The Leica HER2 FISH System - 30 Test contains the LSI HER2 DNA probe, a 226 Kb  
SpectrumOrangedirectly labeled fluorescent DNA probe specific for the HER2 gene  
locus (17q11.2-q12) and the CEP17 DNA probe, a 5.4 Kb SpectrumGreendirectly labeled  
fluorescent DNA probe specific for the alpha satellite DNA sequence at the centromeric region  
of chromosome 17 (17p11.1-q11.1). The probe solution has been specially formulated and  
validated for use on the Leica BOND-MAX and BOND-III System and should not be modified  
or used in a manual setting.  
Clinical Concordance Summary Leica BOND-MAX System  
The Leica HER2 FISH System - 30 Test was developed to provide a fully automated alternative  
to current methodologies used to determine HER2 gene amplification status. The performance  
of the Leica HER2 FISH System - 30 Test on the Leica BOND-MAX System was evaluated in an  
independent study comparing the results of the Leica HER2 FISH System - 30 Test to the Abbott  
Molecular PathVysion HER-2 DNA Probe Kit Assay on 300 breast tumor specimens. None of  
these tumor specimens were obtained from patients in the Herceptin clinical studies. The results  
indicated a 99.33% concordance in a 2x2 analysis (95% confidence intervals of 97.61–99.92%).  
The concordance data also indicates that a positive result with the Leica HER2 FISH System -  
30 Test is highly likely to correspond with a positive result on the Abbott Molecular PathVysion  
HER-2 DNA Probe Kit assay. The Leica HER2 FISH System - 30 Test is interpreted as negative  
for HER2 gene amplification when the HER2:CEP17 gene ratio is less than 2.0 and positive  
when the HER2:CEP17 gene ratio is greater than or equal to 2.0. Equivocal (borderline) results,  
where the HER2:CEP17 gene ratio is between or equal to 1.8-2.2, should be interpreted with  
caution. Count an additional 20 nuclei and recalculate the ratio.  
Clinical Concordance Summary Leica BOND-III System  
The Leica HER2 FISH System - 30 Test was developed to provide a fully automated alternative  
to current methodologies used to determine HER2 gene amplification status. The performance  
of the Leica HER2 FISH System - 30 Test on the Leica BOND-III System was evaluated in an  
independent study comparing the results of the Leica HER2 FISH System - 30 Test to the Abbott  
Molecular PathVysion HER-2 DNA Probe Kit Assay on 300 breast tumor specimens. None of  
these tumor specimens were obtained from patients in the Herceptin clinical studies. The results  
indicated a 99.67% concordance in a 2x2 analysis (95% confidence intervals of 98.16–99.99%).  
The concordance data also indicates that a positive result with the Leica HER2 FISH System -  
30 Test is highly likely to correspond with a positive result on the Abbott Molecular PathVysion  
HER-2 DNA Probe Kit assay. The Leica HER2 FISH System - 30 Test is interpreted as negative  
for HER2 gene amplification when the HER2:CEP17 gene ratio is less than 2.0 and positive  
when the HER2:CEP17 gene ratio is greater than or equal to 2.0. Equivocal (borderline) results,  
where the HER2:CEP17 gene ratio is between or equal to 1.8-2.2, should be interpreted with  
caution. Count an additional 20 nuclei and recalculate the ratio.  
Principle of Procedure  
TheLeicaHER2FISHSystem-30Testcontainscomponentsrequiredtocompleteauorescence  
in situ hybridization based staining procedure for formalin-fixed, paraffin-embedded tissues.  
Following appropriate pretreatment, incubation with the ready-to-use LSI HER2/CEP17 Dual  
Probe and appropriate stringency washing, tissue sections are then dehydrated and mounted  
with DAPI. Results are interpreted by fluorescence microscopy using the recommended filters  
at the appropriate wavelengths.  
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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
Page 4 of 24  
 
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  
Contains ready-to-use LSI HER2/CEP17 Dual Probe.  
Contains <60% (v/v) formamide.  
Post Hybridization Wash 2  
9 mL  
Contains ready-to-use post hybridization wash solution.  
Contains <50% (v/v) formamide.  
Leica BOND Enzyme  
Concentrate 2  
Contains Proteinase K solution at 1.7 mg/mL.  
1 mL  
Leica BOND Enzyme Diluent  
65 mL  
Contains Enzyme Diluent.  
Leica BOND Open Container  
3 x 7 mL  
BOND Open Container used for Enzyme 5.  
Table 1: Leica HER2 FISH System - 30 Test Components  
Refer to individual MSDS for further product safety information, available from  
www.LeicaBiosystems.com/TA9217-IFU  
Directions For Use  
All reagents supplied are formulated specifically for use with this assay and lot numbers are  
specific for each Leica HER2 FISH System - 30 Test. For the assay to be valid, no substitutions  
should be made.  
Storage and Stability  
Store at 2–8 °C. Do not freeze. Return to 2–8 °C immediately after use. Any deviation from these  
conditions will invalidate the assay. Ensure the Leica HER2 FISH System - 30 Test is used within  
its designated expiry date. The signs indicating contamination and/or instability of the Leica  
HER2 FISH System - 30 Test are turbidity of the solutions (except for the probe solution) and  
odor development. The user must verify storage conditions other than those specified above.  
Specimen Preparation  
Standard methods of tissue processing should be used for all specimens (19). It is recommended  
that tissues are prepared in formalin-based fixatives and are routinely processed and paraffin-  
embedded. For example, specimens should be sampled at a thickness of 3–4 mm and fixed  
for 18–24 hours in 10% neutral-buffered formalin. The tissues should then be dehydrated in a  
series of alcohols and cleared through xylene, followed by impregnation with molten paraffin  
wax, held at no more than 60 °C. Tissue specimens should be sectioned between 4–6 µm.  
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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
 
Tissue sections mounted on charged slides (Leica BOND Plus Slides S21.2113) can be held for  
up to 12 months at 2–8 °C before staining. Following sectioning, it is recommended that slides  
are incubated at 60 °C for one hour. Stained sections should be stored at -20 °C to preserve  
fluorescent signal and prevent fading. Allow stored slides to reach room temperature prior to  
reading.  
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.  
Specimens, before and after fixation, and all materials exposed to them, should be handled as  
if capable of transmitting infection and disposed of with proper precautions.  
Never pipette reagents by mouth and avoid contacting the skin and mucous membranes with  
reagents and specimens. If reagents or specimens come into contact with sensitive areas, wash  
with copious amounts of water. Seek medical advice. Consult federal, state or local regulations  
for disposal of any potentially toxic components.  
Minimize microbial contamination of reagents or an increase in nonspecific staining may occur.  
Procedure  
A. Reagents required but not supplied  
Leica BOND Dewax Solution (AR9222)  
Leica BOND Epitope Retrieval Solution 1 (AR9961)  
Leica BOND Wash Solution x10 Concentrate (AR9590)  
Standard solvents used in fluorescence in situ hybridization based assays (eg ethanol,  
absolute and graded)  
Distilled or de-ionized water  
DAPI Counterstain  
Leica HER2 FISH Control Slides (TA9123)  
Leica BOND Aspirating Probe Cleaning System (CS9100)  
B. Equipment required but not supplied  
Pipettes (capable of measuring 1-20 µL and 100 – 1000 µL volumes)  
Charged slides (Leica BOND Plus Slides – S21.2113)  
Leica BOND-MAX (21.0051) or Leica BOND-III (21.2201)  
Leica BOND Universal Covertiles(S21.2001)  
Leica BOND Mixing Stations (S21.1971)  
Leica BOND Slide Label & Print Ribbon (S21.4564)  
Coverslips  
Drying oven (capable of maintaining 60 °C)  
Fluorescence Microscope (60–100x objective) with appropriate light source. Record the  
number of hours that the bulb has been used and replace the bulb before it exceeds the  
rated time. Ensure that the lamp is properly aligned.  
Appropriate Fluorescence Filter Set (SpectrumOrange– Excitation Peak at 559nm,  
Emission Peak at 588nm, SpectrumGreen– Excitation Peak at 497nm, Emission  
Peak at 524nm and DAPI – Excitation Peak at 367nm, Emission Peak at 452nm). Multi-  
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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
Page 6 of 24  
 
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. Methodology  
Prior to undertaking this methodology, users are required to be suitably trained in the  
automated in situ fluorescence technique.  
Each test section stained with the LSI HER2/CEP17 Dual Probe enables same cell  
analysis of both HER2 and centromeric chromosome 17 signals. A subsequent ratio of  
HER2 to chromosome 17 signals will enable a quantitative value to be assigned to the  
sample, indicating a negative (non-amplified) or positive (amplified) result. Equivocal  
(borderline) results (1.8-2.2) should be interpreted with caution. Count an additional 20  
nuclei and recalculate the ratio.  
D. BOND Enzyme Pretreatment  
Prior to staining dilute supplied Leica BOND Enzyme Concentrate 2 at a 1:300 dilution  
using supplied Leica BOND Enzyme Diluent in one of the Leica BOND Open Containers  
provided. For example, to stain 10 slides prepare 3 mL of working enzyme solution by  
diluting 10 µL of Leica BOND Enzyme Concentrate 2 in 2990 µL of Leica BOND Enzyme  
Diluent. It is recommended that the enzyme is freshly prepared before each staining run  
and that a minimum volume of 900 µL be used per run.  
E. Default Staining Protocol  
It is recommended that the Leica HER2 FISH System - 30 Test is used with the  
recommended default staining protocol shown in Table 2 below.  
Protocol Type  
Staining  
Protocol Name  
*FISH Protocol A  
*Dewax  
Preparation  
HIER  
*HIER 25 min with ER1 (97)  
*Enzyme 5 for 25 min  
*D10  
Enzyme  
Denaturation  
Hybridization  
*ISH Hybridization (12Hr)  
Table 2: Default Leica HER2 FISH System - 30 Test Staining Protocol  
F. Procedure Steps  
These instructions should be read in conjunction with the Leica BOND-MAX and BOND-III  
System user manual. A new Leica BOND Universal Covertile should be used with each  
slide.  
The use of Leica BOND Universal Covertiles, which have previously been utilized for either  
immunohistochemical or in situ hybridization staining have not been validated with this test.  
1. On the Leica BOND-MAX and BOND-III System, ensure the bulk and hazardous waste  
containers have enough capacity to perform the required staining runs.  
2. Ensure there is adequate alcohol, distilled or de-ionized water, Leica BOND Dewax  
Solution, Leica BOND Epitope Retrieval Solution 1 and Leica BOND Wash Solution  
in the bulk reagent containers to perform the required staining runs.  
3. Ensure that a clean Leica BOND Mixing Station is installed.  
4. Turn on the Leica BOND-MAX and BOND-III System.  
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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
 
5. Turn on the PC attached to the Leica BOND-MAX and BOND-III System.  
6. Open the Leica BOND software.  
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. Scan in supplied Leica BOND Open Container and register as Bond Enzyme 5.  
10. Go to the Slide setup screen and click Add case.  
11. Enter details for the first case. Ensure the dispense volume is set to 150 µL and the  
preparation protocol is *Dewax. Click OK.  
12. With the case highlighted in the Slide setup screen click Add slide.  
13. First, add patient test slides. Ensure tissue type is set to Test tissue.  
14. Select staining mode Single.  
15. Select process ISH.  
16. Select *LSI HER2/CEP17 Dual Probe – 30 Test from the probe list. The Protocols tab  
defaults to the correct staining protocol (*FISH Protocol A), HIER protocol (*HIER 25  
min with ER1 (97)), EIER protocol (*Enzyme 5 for 25 min), denaturation (*D10) and  
hybridization (*ISH Hybridization (12Hr)).  
17. Repeat steps 10 to 16 until patient test slides and controls (Leica HER2 FISH control  
slides and/or in-house controls) have been created. Print slide labels.  
18. Label slides appropriately.  
19. Open the lids of all Leica HER2 FISH System - 30 Test containers and load the reagent  
tray onto the Leica BOND-MAX and BOND-III System.  
20. Apply new Covertiles to each slide.  
21. Load the slide tray onto the Leica BOND-MAX and BOND-III System and press the  
Load/Unload button.  
22. Confirm that the slides have been scanned and click the Run (Play) button on the System  
status screen to commence the run immediately (for the Leica HER2 FISH System -  
30 Test it is recommended that this assay is run overnight utilizing the delayed start  
functionality).  
23. Ensure that the tray indicator field displays Proc (OK) and batch number and finish time  
are displayed.  
24. When the run is completed press the Load/Unload button and remove the slide tray from  
the Leica BOND-MAX and BOND-III System.  
25. Remove Covertiles and rinse the slides in de-ionized water.  
26. Dehydrate rapidly in two changes of alcohol, air dry.  
27. Dispense 20µL of DAPI directly onto the sample.  
28. Apply coverslip and allow the solution to spread to its full extent, taking care to remove  
any air bubbles.  
29. Seal edge of coverslip with nail varnish, or similar sealant.  
30. Place slides in dark to facilitate signal development before viewing under fluorescence  
microscope.  
31. To preserve signal intensity store stained slides at -20 °C.  
G. Slide Storage  
Store stained slides at -20 °C in the dark. Allow slides to reach room temperature prior to  
viewing following removal from -20 °C.  
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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
Page 8 of 24  
 
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  
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.  
Slide Quality  
- Probe Signal  
Intensity  
Background: The background should be relatively free of fluorescent  
- Background  
particles, and appear dark or black.  
Consult the troubleshooting guide (Table 6) if any of the above features are  
unsatisfactory and repeat the assay if necessary.  
2. Recognition of Target Signals  
Ensure that the correct filters are used for analysis:  
Tissue Overview: Hybridization signals should only be enumerated  
among invasive tumor cells. Tumor cells can generally be distinguished  
from normal cells by size: in general they are larger than normal cells,  
lymphocytes, and epithelial cells. Identify and select target areas by  
H & E stain and mark these areas on the coverslip after the FISH assay  
is performed.  
2. Recognise  
Target Signals  
- Tissue Overview  
- Depth of Focus  
Depth of Focus: Adjust the depth of the focus to determine focal plane  
depth and become familiar with shape and size of the target signals and  
noise (debris).  
3. Nuclei Suitability  
Overview the hybridized area using a 20X objective:  
Nuclei Selection: Locate the target area of interest (tumor cells as  
identified by H & E stain). Avoid areas where nuclear borders are unclear  
or cells are necrotic.  
3. Nuclei  
Suitability  
- Nuclei Selection  
Additionally, signals with weak intensity and non-specific, noisy background,  
or insufficient counterstain to determine the nuclear border should be  
ignored. Only those nuclei with discrete signals should be enumerated.  
4. Signal Enumeration  
Tumor Overview: Scan several areas of tumor cells to account for  
possible heterogeneity, using a 40X objective. Avoid areas of the target  
where signals are weak and select an area of good nuclear distribution.  
Counting: Begin analysis in the upper left quadrant of the selected area  
and, scanning from left to right, count the number of signals within the  
nuclear boundary, using a 100X objective, according to the guidelines  
provided below and in Figure 1.  
4. Signal  
Locate all signals present in the nucleus by focusing up and down (Z  
Enumeration  
axis).  
- Tumor Overview  
Count two signals, that are the same size and separated by a distance  
equal or less than the diameter of the signal, as one signal.  
- Counting  
Nuclei with no signals or with signals of only one color should not be  
scored. Only score those nuclei with one or more FISH signals of each  
color.  
Count the number of HER2 signals and the number of CEP17 signals  
for each nucleus. Alternate between the orange (HER2), green (CEP17),  
green/orange and the DAPI/green/orange filter sets to view both colors, as  
necessary.  
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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
 
Recommended Method for LSI HER2 to CEP17 Ratio Determination  
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. To calculate the final result, use the following calculation:  
Total LSI HER2 signals divided by Total CEP17 signals,  
e.g. 143/48 equals a ratio of 2.98, which is positive for HER2 amplification.  
Important Note: If the LSI HER2 to CEP17 ratio is equivocal (1.80 - 2.20), count an  
additional 20 nuclei and recalculate the ratio.  
Results should be reported as follows:  
1. If the ratio is <2, HER2 gene amplification was not observed  
2. If the ratio is ≥2, HER2 gene amplification was observed  
Important Note: A ratio at or near the cut-off (1.80 - 2.20) should be interpreted with  
caution, as described above.  
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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
Page 10 of 24  
 
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. 1 orange signal is  
diffuse and 1 green signal is diffuse  
Count as 4 orange signals and 2 green signals. 1 orange signal is  
diffuse. Count 2 signals that are the same size and separated by  
a distance equal or less than the diameter of the signal, as one  
signal  
Count as 5 orange signals and 3 green signals. 1 orange signal is  
diffuse  
Countas2orangesignalsand2greensignals. 1orangesignaland  
1 green signal are overlapping  
Do not count. The nuclei are overlapping. It is too difficult to tell  
which nuclei the signals are located in  
Count as approximately 16 orange signals and 2 green signals  
Figure 1: Interpretation Guide  
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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
Leica HER2 FISH System - 30 Test Score Sheet  
20 Nuclei Signal Count  
HER2 Copy  
Number  
CEP17 Copy  
Number  
HER2 Copy  
Number  
CEP17 Copy  
Number  
Nucleus #  
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:CEP17 Amplification ratio  
Total Score 1-20  
Average Per Cell  
Figure 2: Sample Score Sheet  
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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
Page 12 of 24  
 
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.  
Assess control slide quality and perform signal enumeration according to the instructions in the  
Signal Assessment and Enumeration section. The criteria for slide quality must be satisfied  
and the HER2:CEP17 ratio results should be within the established ranges for acceptable test  
performance. See Table 3 for acceptance criteria of the Leica HER2 FISH Control Slides.  
Leica HER2 FISH System - 30 Test  
HER2:CEP17 Acceptance Criteria  
Cell Line  
Bond Oracle  
HER2 IHC  
HER2  
ReceptorLoad  
System Profile per cell*  
3+  
2+  
4.3 x 105  
HER2 amplification is observed  
SKBr-3  
HER2/CEP17 gene ratio should be between  
1.5 – 2.5  
1.4 x 105  
MDA-MB-453  
1+  
0
6.3 x 104  
9.3 x 103  
HER2 amplification is not observed  
HER2 amplification is not observed  
MDA-MB-175  
MDA-MB-231  
*HER2 receptor load analysis as assessed by flow cytometry  
Table 3: Leica HER2 FISH Control Slide Interpretation.  
If assay controls fail, FISH results for that case should not be reported. If control slides fail to  
meet the slide acceptance criteria, the Leica HER2 FISH System - 30 Test may have performed  
inadequately. In this instance, a repeat test with fresh control slides and patient specimen  
slide(s) will be required. If the results are outside of the specified range, but the control slides  
meet the acceptance criteria for quality, repeat screening of the same slide may be appropriate  
since the enumeration may not have been performed correctly. Consult the troubleshooting  
guide (Table 6) in the event of hybridization failure, with either the specimen or control slide(s).  
For clinical specimens, when interpretation of the hybridization signal is difficult and there is  
insufficient specimen sample for re-assay, the test is uninformative. If there are insufficient cells  
for analysis, the test is uninformative.  
Patient specimens should be controlled according to standard laboratory operating procedures.  
Signal quality and enumeration results should be documented on an appropriate report form.  
Limitations  
A. General Limitations  
FISH is a technique that requires specialized training in all aspects of the procedure (including  
the selection of appropriate reagents, tissue, fixation, processing and slide preparation) and  
interpretation. Tissue staining is dependent on the handling, fixation and processing of the  
tissue prior to staining. Improper fixation, freezing, thawing, washing, drying, heating, sectioning  
or contamination with other tissues or fluids may produce morphological artifacts, nucleic acid  
degradation, background fluorescence or false negative results. Inconsistent results may be  
due to variations in fixation, embedding methods, or inherent irregularities within the tissue  
(21). Excessive or incomplete counterstaining may also compromise correct interpretation of  
the results.  
Non-specific staining as a result of unbound probe has a scattered, granular appearance  
and may be visualized at or distant from the expected hybridization site. Use intact cells for  
interpretation of staining results. Necrotic or degenerated cells may stain non-specifically (22).  
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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
 
Unexpected FISH staining, or variations in the staining, may be a result of alterations in the  
expression levels of the encoding genes. Any change in expected staining patterns should be  
interpreted in association with all other diagnostic investigations. Staining interpretation should  
be complemented by morphological studies and the use of suitable control material, and should  
be evaluated within the context of the patient’s clinical history and any other diagnostic tests, by  
a qualified pathologist.  
The performance of the assay (i.e. assessment of adequacy of control materials) and the  
interpretation of any staining or its absence must be carried out in an appropriately accredited/  
licensed laboratory under the supervision of a suitably qualified and experienced pathologist,  
who is responsible for the overall assessment of the in situ hybridization assay and its  
interpretation. False positive results in FISH may be due to cross-reactivity of the probe to other  
nucleic acid sequences and/or nonspecific binding. Appropriate controls must be employed and  
documented, and tests should take into account all relevant expiration dates.  
Technical and interpretational variation may also be seen when FISH is utilized on cell line  
derived materials (23).  
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. The  
use of any other type of fixative may invalidate the assay.  
Tissue sections cut outside of the recommended thickness range have not been validated. The  
use of any other section thickness may invalidate the assay.  
Clinical Concordance of HER2 FISH System - 30 Test to Abbott  
Molecular PathVysion HER-2 DNA Probe Kit  
This study examined the suitability of the Leica HER2 FISH System - 30 Test for use as an aid  
in determination of treatment for Herceptin (trastuzumab) therapy. The study was designed to  
examine the concordance between the Leica HER2 FISH System - 30 Test and a previously  
approved diagnostic device, theAbbott Molecular PathVysion HER-2 DNAProbe Kit, considered  
as the ‘gold standard’ for this assay. The acceptance criterion for testing was that the lower limit  
of the 95% one-sided confidence interval is above 90% between the Leica HER2 FISH System  
- 30 Test and the manual Abbott Molecular PathVysion HER-2 DNA Probe Kit, between positive  
(amplified) and negative (non-amplified) formalin-fixed, paraffin-embedded (FFPE) invasive  
breast cancer cases.  
The study was conducted as a three-site, masked evaluation of clinical invasive breast  
carcinoma samples. Each of the investigational sites were supplied with archived formalin-fixed,  
paraffin-embedded invasive breast carcinoma tissue blocks with known HER2 oncoprotein  
expression levels. A cohort of 300 specimens consisting of 75, 0/1+ previously characterized  
IHC cases; 150, 2+ previously characterised IHC cases; and 75, 3+ previously characterized  
IHC cases were selected, and split equally across each of the three investigational trial sites.  
All cases were stained with the manual Abbott Molecular PathVysion HER-2 DNA Probe Kit  
Assay according to the manufacturer’s instructions for use, as specified in the package insert.  
Sequential sections from each case were then stained with the Leica HER2 FISH System - 30  
Test on the Leica BOND-MAX and BOND-III System.  
All stained slides were masked and scored in a randomized fashion by a single trained  
observer at each of the three investigational trial sites. Scores were interpreted as negative  
with a calculated HER2/CEP17 gene ratio of <2.0 and positive with a calculated HER2/CEP17  
gene ratio of ≥2.0. Data was then analyzed for concordance, positive staining agreement and  
negative staining agreement.  
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Page 14 of 24  
 
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 97.61–99.92% for the Leica BOND-MAX System  
The percentage Positive Agreement (sensitivity) or the ability of the Leica HER2 FISH System  
- 30 Test to correctly identify Abbott Molecular PathVysion HER-2 DNA Probe assay positive  
cases (the percentage of specimens scored positive by both the Leica HER2 FISH System -  
30 Test and manual Abbott Molecular PathVysion HER-2 DNA Probe Kit out of all the Abbott  
Molecular PathVysion HER-2 DNA Probe Kit positive cases) was 99.03% (102/103).  
The percentage Negative Agreement (specificity) or the ability of the test to correctly identify  
Abbott Molecular PathVysion HER-2 DNA Probe Kit negative cases (the percentage of  
specimens scored negative by the Leica HER2 FISH System - 30 Test and Abbott Molecular  
PathVysionHER-2 DNA Probe Kit out of all the Abbott Molecular PathVysion HER-2 DNA Probe  
Kit negative cases) was 99.49% (196/197). See Table 4.  
Abbott Molecular PathVysion HER-2 DNA Probe Kit  
Negative (<2.0)  
Positive (≥2.0)  
Totals  
197  
Leica HER2 FISH  
System - 30 Test  
Leica BOND-MAX  
196  
1
1
Negative (<2.0)  
Positive (≥2.0)  
Totals  
102  
103  
103  
197  
300  
Overall Concordance (95% CI) = 99.33% (97.61 – 99.92%)  
Table 4. 2x2 concordance of Leica HER2 FISH System - 30 Test on the Leica BOND-MAX System with Abbott Molecular  
PathVysion HER-2 DNA Probe Kit.  
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Page 15 of 24  
Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
 
2x2 Concordance Results Leica BOND-III 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  
99.67% (299/300) with a 95% CI of 98.16–99.99% for the Leica BOND-III System.  
The percentage Positive Agreement (sensitivity) or the ability of the Leica HER2 FISH System  
- 30 Test to correctly identify Abbott Molecular PathVysion HER-2 DNA Probe Kit assay positive  
cases (the percentage of specimens scored positive by both the Leica HER2 FISH System -  
30 Test and manual Abbott Molecular PathVysion HER-2 DNA Probe Kit out of all the Abbott  
Molecular PathVysion HER-2 DNA Probe Kit positive cases) was 99.03% (102/103).  
The percentage Negative Agreement (specificity) or the ability of the test to correctly identify  
Abbott Molecular PathVysion HER-2 DNA Probe Kit negative cases (the percentage of  
specimens scored negative by the Leica HER2 FISH System - 30 Test and Abbott Molecular  
PathVysion HER-2 DNA Probe Kit out of all the Abbott Molecular PathVysion HER-2 DNA Probe  
Kit negative cases) was 100% (197/197). See Table 5.  
Abbott Molecular PathVysion HER-2 DNA Probe Kit  
Negative (<2.0)  
Positive (≥2.0)  
Totals  
Leica HER2 FISH  
System - 30 Test  
Leica BOND-III  
197  
1
198  
Negative (<2.0)  
Positive (≥2.0)  
Totals  
0
102  
103  
102  
300  
197  
Overall Concordance (95% CI) = 99.67% (98.16 – 99.99%).  
Table 5. 2x2 concordance of Leica HER2 FISH System - 30 Test on the Leica BOND-III System with Abbott Molecular  
PathVysion HER-2 DNA Probe Kit.  
In conclusion, the data generated in this study demonstrates that the Leica HER2 FISH System  
- 30 Test can be used as an aid in the assessment of patients for whom Herceptin (trastuzumab)  
treatment is being considered, based upon its high concordance with Abbott Molecular  
PathVysion HER-2 DNA Probe Kit, a previously approved diagnostic test for this indication.  
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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
Page 16 of 24  
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 formalin-  
fixed paraffin-embedded breast cancer cases. The use of TMAs for the determination of  
within run precision enabled a larger volume of cases covering a wider range of HER2  
expression within a single run on a single instrument.  
On enumeration of the slides stained in the Within Run Precision Study, 532/540 cases  
evaluated demonstrated a concordant result giving an overall concordance of 98.52% with  
a lower 95% CI of 97.10%.  
B. Within Instrument Precision Study  
The within instrument precision study was performed in a randomized and blinded fashion.  
Within instrument precision testing of the Leica HER2 FISH System - 30 Test was evaluated  
at a single investigational site on 1620 previously HER2 characterized TMA samples  
containing formalin-fixed paraffin-embedded breast cancer cases. The use of TMAs for the  
determination of within instrument precision enabled a larger volume of cases covering a  
wider range of HER2 expression within multiple runs on a single instrument.  
On enumeration of the slides stained in the Within Instrument Precision Study, 1620/1620  
cases evaluated demonstrated a concordant result giving an overall concordance of 100%  
with a lower 95% CI of 99.82%.  
C. Between Run Precision Study  
The between run precision study was performed in a randomized and blinded fashion.  
Between run precision testing of the Leica HER2 FISH System - 30 Test was evaluated  
at a single investigational site on 900 previously HER2 characterized TMA samples  
containing formalin -fixed paraffin-embedded breast cancer cases. The use of TMAs for the  
determination of between run, day-to-day precision testing enabled a larger volume of cases  
covering a wider range of HER2 expression to be tested between runs on different days.  
On enumeration of the slides stained in the Between Run Precision Study, 894/900 cases  
evaluated demonstrated a concordant result giving an overall concordance of 99.33% with  
a lower 95% CI of 98.55%.  
D. Between Laboratory Precision Study  
The between laboratory precision study was performed in a randomized and blinded  
fashion. Between laboratory precision testing of the Leica HER2 FISH System - 30 Test  
was evaluated between three investigational sites on 513 previously HER2 characterized  
TMA samples containing formalin-fixed paraffin-embedded breast cancer cases. The use of  
TMAs for the determination of between laboratory precision testing enabled a larger volume  
of cases covering a wider range of HER2 expression to be tested between runs on multiple  
instruments.  
On enumeration of the slides stained in the Between Laboratory Precision Study, 510/513  
cases evaluated demonstrated a concordant result giving an overall concordance of 99.42%  
with a lower 95% CI of 98.30%.  
E. 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.  
On enumeration of the slides stained in the Between Observer Precision Study, 53/54 cases  
evaluated demonstrated a concordant result giving an overall concordance of 98.15% with  
a lower 95% CI of 90.11%  
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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
 
F. Lot-to-Lot Precision Study  
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. The use of TMAs for the  
determination of lot-to-lot reproducibility enables a larger volume of cases covering a wider  
range of HER2 expression to be tested between lots.  
On enumeration of the slides stained in the Lot-to-Lot Precision Study, 534/540 cases  
evaluated demonstrated a concordant result giving an overall concordance of 98.89% with  
a lower 95% CI of 97.60%.  
Precision Testing – Leica BOND-III System  
G. 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 formalin-  
fixed paraffin-embedded breast cancer cases. The use of TMAs for the determination of  
within run precision enabled a larger volume of cases covering a wider range of HER2  
expression within a single run on a single instrument.  
On enumeration of the slides stained in the Within Run Precision Study, 540/540 cases  
evaluated demonstrated a concordant result giving an overall concordance of 100% with a  
lower 95% CI of 99.45%.  
H. Within Instrument Precision Study  
The within instrument precision study was performed in a randomized and blinded fashion.  
Within instrument precision testing of the Leica HER2 FISH System - 30 Test was evaluated  
at a single investigational site on 1620 previously HER2 characterized TMA samples  
containing formalin-fixed paraffin-embedded breast cancer cases. The use of TMAs for the  
determination of within instrument precision enabled a larger volume of cases covering a  
wider range of HER2 expression within multiple runs on a single instrument.  
On enumeration of the slides stained in the Within Run Precision Study, 1620/1620 cases  
evaluated demonstrated a concordant result giving an overall concordance of 100% with a  
lower 95% CI of 99.82%.  
I. Between Run Precision Study  
The between run precision study was performed in a randomized and blinded fashion.  
Between run precision testing of the Leica HER2 FISH System - 30 Test was evaluated  
at a single investigational site on 900 previously HER2 characterized TMA samples  
containing formalin-fixed paraffin-embedded breast cancer cases. The use of TMAs for the  
determination of between run, day-to day precision testing enabled a larger volume of cases  
covering a wider range of HER2 expression to be tested between runs on different days.  
On enumeration of the slides stained in the Between Run Precision Study, 891/900 cases  
evaluated demonstrated a concordant result giving an overall concordance of 99.00% with  
a lower 95% CI of 98.11%.  
J. Between Laboratory Precision Study  
The between laboratory precision study was performed in a randomized and blinded  
fashion. Between laboratory precision testing of the Leica HER2 FISH System - 30 Test  
was evaluated between three investigational sites on 513 previously HER2 characterized  
TMA samples containing formalin-fixed paraffin-embedded breast cancer cases. The use of  
TMAs for the determination of between laboratory precision testing enabled a larger volume  
of cases covering a wider range of HER2 expression to be tested between runs on multiple  
instruments.  
On enumeration of the slides stained in the Between Laboratory Precision Study, 511/513  
cases evaluated demonstrated a concordant result giving an overall concordance of 99.61%  
with a lower 95% CI of 98.60%.  
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Page 18 of 24  
 
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.  
On enumeration of the slides stained in the Between Observer Precision Study, 53/54 cases  
evaluated demonstrated a concordant result giving an overall concordance of 98.15% with  
a lower 95% CI of 90.11%.  
L. Lot-to-Lot Precision Study  
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 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. The use of TMAs for the  
determination of lot-to-lot reproducibility enables a larger volume of cases covering a wider  
range of HER2 expression to be tested between lots.  
On enumeration of the slides stained in the Lot-to-Lot Precision Study, 540/540 cases  
evaluated demonstrated a concordant result giving an overall concordance of 100% with a  
lower 95% CI of 99.45%.  
Assay Robustness  
Robustness studies were performed on the Leica BOND-MAX and BOND-III System to determine  
the assay tolerance range for heat retrieval time and temperature; enzyme retrieval  
time, temperature and concentration; denaturation time and temperature; hybridization time and  
temperature; and stringency wash time and temperature. Robustness studies using the default  
Leica BOND-MAX and BOND-III System protocol were also performed outside the  
recommended limits as defined in the FDA/ORA guidance document ORA LAB5.3 Rev1.7 for  
temperature and humidity.  
• No difference in amplification status was observed when the default temperature for each  
heat dependent step was raised by 4 °C or decreased by 4 °C, when compared to the default  
LeicaHER2FISHSystem-30Testprotocol.Highestqualityratingswereobservedatthedefault  
temperatures and these temperatures are recommended.  
• No difference in amplification status was observed when the heat induced epitope retrieval  
(HIER) time was performed for 20 minutes and 30 minutes at 97 °C with Leica BOND ER1  
solution, when compared to the default Leica HER2 FISH System - 30 Test protocol. Highest  
quality ratings were observed at the default time of 25 minutes and this incubation time is  
recommended.  
• No difference in amplification status was observed when the enzyme induced epitope retrieval  
(EIER) time was performed for 15 minutes and 35 minutes at 37 °C, when compared to the  
default Leica HER2 FISH System - 30 Test protocol. Highest quality ratings were observed at  
the default time of 25 minutes and this incubation time is recommended.  
• No difference in amplification status was observed when the enzyme induced epitope retrieval  
(EIER) enzyme concentration was performed with enzyme concentrate/enzyme diluent  
ratios of 1:200 and 1:500 using the default Leica HER2 FISH System - 30 Test protocol.  
Highest quality ratings were observed at the default concentration of 1:300 and this dilution  
is recommended.  
• No difference in amplification status was observed when the denaturation time was performed  
for 5 minutes and 15 minutes, when compared to the default Leica HER2 FISH System - 30  
Test protocol. Highest quality ratings were observed at the default time of 10 minutes and this  
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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
 
denaturation time is recommended.  
• No difference in amplification status was observed when the hybridization time was performed  
for 9 hours and 15 hours when compared to the default Leica HER2 FISH System - 30  
Test protocol. Highest quality ratings were observed at the default time of 12 hours and this  
hybridization time is recommended.  
• 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.  
• No difference in amplification status was observed when the Leica HER2 FISH System - 30  
Test was performed at 28 °C and 30% relative humidity and 16 °C and 80% relative humidity,  
when compared to the default Leica HER2 FISH System - 30 Test protocol performed at  
ambient conditions.  
Operations performed outside of the tested assay robustness recommended parameters have  
not been validated. The use of any other testing parameter may invalidate the assay.  
The above text describes the conditions tested and the results from the study. Note that Leica  
did not test all possible combinations of conditions and does not recommend using non-default  
ranges for all conditions. The default Leica HER2 FISH Staining Protocol is outlined in Table 2.  
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Page 20 of 24  
 
Troubleshooting  
Problem  
Probable Cause  
Remedial Action  
No or weak  
fluorescent  
signal/staining  
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.  
Incorrect protocol selection  
Ensureappropriatedefaultto*FISHProtocol  
AinthestainingprotocolfieldoftheAddslide  
dialog.  
Inappropriate bulk reagents  
dispensed  
Ensure all Leica BOND reagents have been  
allocated to appropriate bulk containers  
andplacedintoappropriatepositionsonthe  
instrument.  
Inadequate deparaffinization  
of slides  
Ensure *Dewax mode is selected in the  
Preparation field of the Add slide dialog.  
Inappropriate pretreatment  
Ensure default pretreatment (HIER and  
EnzymaticDigestion)protocolsareselected.  
Adjust pretreatment protocol (HIER or  
Enzymatic Digestion) if required.  
Inadequate denaturation  
Inadequate hybridization  
Ensureappropriatedefaultdenaturation*D10is  
selected.  
Ensureappropriatedefaulthybridization*H12is  
selected.Extendhybridizationtimeifrequired.  
Excess post hybridization  
washing  
Decreaseposthybridizationwashincubation  
time.  
Run aborted prior to  
completion  
Using Leica BOND software, confirm the  
presence of any reportable errors during the  
staining run and address as instructed by  
the Leica BOND software.  
Inappropriate fluorescence  
microscopy equipment  
Ensure that all fluorescence microscopy  
equipment used is appropriate for the assay  
being performed, confirm:  
Inappropriate filter set  
Incorrect lamp  
Appropriate filter set  
Appropriate lamp  
Expired lamp  
Good lamp strength  
Incorrect oil type  
Appropriate oil for use in oil immersion  
microscopy  
Over exposure to UV light  
(photobleaching)  
Store slides before and after assessment in  
the dark to preserve fluorescent signals. To  
maintain signal for long term storage store  
slides at –20 ºC.  
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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
Problem  
Probable Cause  
Remedial Action  
Nonspecific  
background  
fluorescent  
Inadequateposthybridization  
washing  
Increaseposthybridizationwashincubation  
time.  
signal/staining  
Inappropriate bulk reagents  
dispensed  
Ensure all Leica BOND reagents have been  
allocated into appropriate bulk containers  
andplacedintoappropriatepositionsonthe  
instrument.  
Inadequate deparaffinization  
of slides  
Ensure Dewax is selected in the Preparation  
field of the Add slide dialog.  
Nonspecific cross-reaction  
with areas of tissue necrosis  
Ensure a formalin-based fixative is used  
and that processing schedules are suitable  
for the specimen undergoing testing. If  
possible, retest case using another block.  
If this is not possible, assess in conjunction  
with a corresponding H&E stained section,  
and select areas which show best fixation  
patterns.  
Sections adhered to slides  
using alternative adhesives  
Use Leica BOND Plus Slides (S21.2113).  
Poorpreservation  
of tissue  
morphology  
Inadequate tissue fixation and  
processing  
Ensure a formalin-based fixative is used  
and that processing schedules are suitable  
for the specimen undergoing testing. If  
possible, retest case using another block.  
If this is not possible, assess in conjunction  
with a corresponding H&E stained section,  
and select areas which show best fixation  
patterns.  
Inappropriate pretreatment  
Adjust pretreatment protocol (HIER or  
Enzymatic Digestion).  
Tissue detached  
from patient/  
control slide(s)  
Use of incorrect type of slides  
or inadequate draining of  
section  
Ensureappropriateslidesareusedforpatient/  
control sections (e.g. Leica BOND Plus Slides  
S21.2113). Ensure slides receive adequate  
drainingandareincubatedfor1hourat60°C.  
Table 6. Leica HER2 FISH System - 30 Test Troubleshooting Guide.  
If any problems associated with the Leica HER2 FISH System - 30 Test fall outside the scope  
of the troubleshooting guide please contact your local Leica Biosystems Technical Services  
Department or Distributor for assistance.  
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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
Page 22 of 24  
References  
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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 erbB-  
2 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. American Society of Clinical Oncology/College of  
American Pathologists Guideline recommendations for human epidermal growth factor receptor 2  
testing in breast cancer. Journal of Clinical Oncology 25, 1-28, 2007.Slamon DJ, Clark GM,  
4. Wong SG, et al. Human breast cancer: correlation of relapse and survival with amplification of the  
HER-2/neu oncogene. Science 1987;235:177-182.  
5. Gusterson BA, Gelber RD, Goldhirsch A, et al. Prognostic importance of c-erbB-2 expression in  
breast cancer. Journal of Clinical Oncology 1992;10:7:1049-1056.  
6. Gullick WJ, Love SB, Wright C, et al. c-erbB-2 protein overexpression in breast cancer is a risk  
factor in patients with involved and uninvolved lymph nodes. British Journal of Cancer 1991;63:434-  
438.  
7. Borg A, Tandon AK, Sigurdsson H, et al. HER-2/neu amplification predicts poor survival in node-  
positive breast cancer. Cancer Research 1990;50:4332-4337.  
8. Tandon AK, Clark GM, Chamness AU, et al. HER-2/neu oncogene protein and prognosis in breast  
cancer. Journal of Clinical Oncology 1989; 7:1120-1128.  
9. Carter P, Presta L, Gorman CM, Ridgway JBB, Henner D, Wong WLT, et al. Humanization of an  
anti-p185HER2 antibody for human cancer therapy. Proceedings of the National Academy of Science  
USA 1992; 89: 4285–9.  
10.Hudziak RM, Lewis GD, Winget M, Fendly BM, Shepard HM, Ullrich A. p185HER2 monoclonal  
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necrosis factor. Molecular & Cell Biology 1989; 9: 1165–72.  
11.Lewis GD, Figari I, Fendly B, Wong WL, Carter P, Gorman C, et al. Differential responses of human  
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12.Baselga J, Norton L, Albanell J, Kim Y-M, Mendelsohn J. Recombinant humanized anti-HER2  
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13.Ellis I.O., Bartlett J., Dowsett M., Humphreys S., Jasani B., Miller K., Pinder S.E., Rhodes A. and  
Walker R. Best practise No. 176: Updated recommendations for Her-2 testing in the UK. Journal of  
Clinical Pathology 57; 233-237, 2004.  
14.Walker R.A, Bartlett, J., Dowsett, M., Ellis, I., Hanby, A., Jasani, Miller, K., Pinder, S. HER2 Testing  
in the UK – further update to recommendations. Journal of Clinical Pathology 2007.054866  
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940 West Valley Road, Suite 1400, Wayne, Pennsylvania 1999; 19087–1898: USA  
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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013  
 
License Agreement  
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.  
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08 April 2013  
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Herceptin is a trademark of Genentech, Inc. and F. Hoffmann-La Roche Ltd.  
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