Leica HER2 User Manual
English
Bond™ Oracle™ HER2 IHC System
Instructions For Use
For use on Leica Biosystems’ BOND™ fully automated, advanced staining system.
Product Code TA9145 is designed to stain 60 tests (150 slides):
60 test slides with HER2 Primary Antibody
60 test slides with HER2 Negative Control
15 HER2 Control Slides with HER2 Primary Antibody
15 positive in-house tissue controls with HER2 Primary Antibody
IVD
Leica Biosystems Newcastle Ltd
Balliol Business Park
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
Page 1 of 23
Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN-CE-Rev_E 18/06/2013
Contents
Intended Use .............................................................................................................................3
Summary and Explanation ......................................................................................................3
Background ..........................................................................................................................................3
Expression of HER2 .............................................................................................................................3
Clinical Concordance Summary ...........................................................................................................3
Principle of Procedure .............................................................................................................4
Components Provided ..........................................................................................................................4
Directions on Use .................................................................................................................................5
Storage and Stability ............................................................................................................................5
Specimen Preparation ..........................................................................................................................5
Warnings and Precautions ...................................................................................................................5
Procedure ..................................................................................................................................6
A. Reagents required but not supplied .................................................................................................6
B. Equipment required but not supplied ...............................................................................................6
C. Methodology ....................................................................................................................................6
D. Slide Layout .....................................................................................................................................6
E. Procedure Steps ..............................................................................................................................7
Quality Control ..........................................................................................................................9
HER2 Control Slide – HER2 Primary Antibody ..................................................................................10
In-house Positive Control Tissue – HER2 Primary Antibody ..............................................................10
In-house Negative Control Tissue Component – HER2 Primary Antibody .........................................10
Patient Tissue – HER2 Negative Control ..........................................................................................10
Patient Tissue – HER2 Primary Antibody ..........................................................................................10
Assay Verication ..............................................................................................................................10
Interpretation of Staining .................................................................................................................... 11
Slide Screening Order Rationale ..........................................................................................12
1. HER2 Control Slide – HER2 Primary Antibody ..............................................................................11
2. In-house Positive Control Tissue – HER2 Primary Antibody ..........................................................12
3. In-house Negative Control Tissue Component – HER2 Positive Control .......................................12
4. Patient Tissue – stained using the HER2 Negative Control ...........................................................12
5. Patient Tissue – stained using the HER2 Primary Antibody ...........................................................12
Limitations ..............................................................................................................................12
A. General Limitations ........................................................................................................................12
B. Product Specic Limitations ...........................................................................................................13
Cell Line Data ..........................................................................................................................14
Clinical Concordance of Bond Oracle HER2 IHC System to Dako HercepTest ................14
2x2 Concordance Results ..................................................................................................................15
3x3 Concordance Results ..................................................................................................................15
Clinical Concordance of Bond Oracle HER2 IHC System to PathVysion DNA HER-2 Probe
Kit.......................................................................................................................................................16
2x2 Concordance Results ..................................................................................................................16
Immunoreactivity – Normal Panel .........................................................................................17
Reproducibility Study ............................................................................................................18
Within and Between Precision Testing ...............................................................................................18
A. Within Run Precision Testing .........................................................................................................18
B. Between Run Precision Testing .....................................................................................................18
C. Lot-to-Lot Reproducibility ...............................................................................................................18
D. Between Laboratory Reproducibility ..............................................................................................19
E. Inter-Observer Reproducibility .......................................................................................................19
F. Between Instrument Precision (BOND-MAX vs BOND-III)..............................................................20
Troubleshooting .....................................................................................................................21
References ..............................................................................................................................22
Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN-CE-Rev_E 18/06/2013
English
Page 2 of 23
Intended Use
For in vitro diagnostic use
Bond Oracle HER2 IHC System is a semi-quantitative immunohistochemical (IHC) assay to
determine HER2 (Human Epidermal Growth Factor Receptor 2) oncoprotein status in breast
English
cancer tissue processed for histological evaluation. The Bond Oracle HER2 IHC System is
indicated as an aid in the assessment of patients for whom Herceptin® (trastuzumab) treatment
is being considered (see Herceptin® package insert).
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 Bond Oracle HER2 IHC System. The Bond Oracle HER2 IHC System has
been compared to the Dako HercepTest™ 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 Bond Oracle HER2 IHC System to clinical outcome has not been
established.
Summary and Explanation
Background
The Bond Oracle HER2 IHC System contains the mouse monoclonal anti-HER2 antibody, clone
CB11. Clone CB11, originally developed by Corbett et al (1) and manufactured by Novocastra
Laboratories Ltd (now Leica Biosystems Newcastle Ltd), is directed against the internal domain
of the HER2 oncoprotein.
In a proportion of breast cancer patients, the HER2 oncoprotein is overexpressed as part of the
process of malignant transformation and tumor progression (2). Overexpression of the HER2
oncoprotein found in breast cancer cells suggests HER2 as a target for an antibody-based
therapy. Herceptin® is a humanized monoclonal antibody (3) that binds with high afnity to the
HER2 oncoprotein and has been shown to inhibit the proliferation of human tumor cells that
overexpress HER2 oncoprotein both in vitro and in vivo (4–6).
Since the rst immunoperoxidase technique, reported by Nakane and Pierce (7), many
developments have occurred within the eld of immunohistochemistry, resulting in increased
sensitivity. A recent development has been the use of polymeric labeling. This technology has
been applied to both primary antibodies and immunohistochemical detection systems (8). The
Compact PolymerTM detection system utilized by the Bond Oracle HER2 IHC System is part of
a family of novel, controlled polymerization technologies that have been specically developed
to prepare polymeric HRP-linked antibody conjugates. As this polymer technology is utilized in
the Oracle product range, the problem of nonspecic endogenous biotin staining, which may be
seen with streptavidin/biotin detection systems, does not occur.
Expression of HER2
The HER2 oncoprotein is expressed at levels detectable by immunohistochemistry in up to 20%
of adenocarcinomas from various sites. Between 10% and 20% of invasive ductal carcinomas
of the breast are positive for HER2 oncoprotein (9). 90% of cases of ductal carcinoma in situ
(DCIS) of comedo type are positive (10), together with almost all cases of Paget’s disease of
the breast (11).
Clinical Concordance Summary
The Bond Oracle HER2 IHC System was developed to provide an alternative to the investigational
Clinical Trial Assay (CTA) used in the Herceptin® clinical studies. The performance of the
Bond Oracle HER2 IHC System for determination of HER2 oncoprotein overexpression was
evaluated in an independent study comparing the results of the Bond Oracle HER2 IHC System
to the Dako HercepTest on 431 breast tumor specimens, of US origin. None of these tumor
specimens were obtained from patients in the Herceptin® clinical trials. The results indicated
a 92.34% concordance in a 2x2 analysis (95% condence intervals of 89.42% to 94.67%) and
86.54% in a 3x3 analysis (95% condence intervals of 82.95% to 89.62%) between the results
from the two assays.
Page 3 of 23
Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN-CE-Rev_E 18/06/2013
Principle of Procedure
The Bond Oracle HER2 IHC System contains components required to complete an
immunohistochemical staining procedure for formalin-xed, parafn-embedded tissues.
Following incubation with the ready-to-use HER2 Primary Antibody (clone CB11), this
system employs ready-to-use Compact Polymer technology. The enzymatic conversion of
the subsequently added chromogen results in the formation of a visible reaction product at
the antigenic site. The tissue sections may then be counterstained, dehydrated, cleared and
mounted. Results are interpreted using light microscopy. Control slides with four formalin-xed,
parafn-embedded human breast cancer cell lines are provided to validate staining runs. The
four cell lines demonstrate HER2 oncoprotein expression at 0, 1+, 2+ and 3+ intensities. The
staining intensity of these cell lines correlates to both HER2 oncoprotein receptor load per cell
and HER2 gene amplication status.
The Bond Oracle HER2 IHC System (product code TA9145) is for use on the Leica Biosystems’
BOND fully automated, advanced staining system.
Components Provided
The materials listed below (Table 1) are sufcient to stain 150 slides (60 test slides incubated with
HER2 Primary Antibody, 60 corresponding test slides incubated with HER2 Negative Control,
15 HER2 Control Slides incubated with HER2 Primary Antibody and 15 in-house positive tissue
controls incubated with HER2 Primary Antibody). The number of tests is based on the use of
a 150 µL automated dispense per slide. The kit provides materials sufcient for a maximum of
15 individual BOND staining runs.
English
HER2 Control Slides,
(x15)
Sections of formalin-xed, parafn-embedded, human breast cancer
cell lines that demonstrate HER2 oncoprotein expression at 0, 1+, 2+ and
3+ staining intensities when stained in accordance with the protocol
provided. These sections are fully adhered and do not require further
baking.
HER2 Primary
Antibody, 13.5 mL
HER2 Negative
Control, 9 mL
Peroxide Block,
22.5 mL
Post Primary, 22.5 mL
Polymer, 22.5 mL
DAB Part 1, 2.25 mL
DAB Part B (x2),
22.5 mL
Hematoxylin, 22.5 mL
Table 1. Bond Oracle HER2 IHC System components
Contains ready-to-use, afnity-puried, mouse monoclonal IgG antibody,
clone CB11 and 0.35% ProClin™ 950 .
Contains ready-to-use mouse IgG at an equivalent concentration to the
HER2 Primary Antibody and 0.35% ProClin™ 950.
Contains 3-4% hydrogen peroxide.
Rabbit anti-mouse IgG (<10 μg/mL) in Tris-buffered saline containing 10%
(v/v) animal serum and 0.09% ProClinTM 950.
Poly-HRP goat anti-rabbit IgG (<25 μg/mL) in Tris-buffered saline
containing 10% (v/v) animal serum and 0.09% ProClinTM 950.
Contains 66 mM 3,3’-diaminobenzidine tetrahydrochloride, in a stabilizer
solution.
Contains ≤0.1% (v/v) hydrogen peroxide.
Contains <0.1% hematoxylin.
Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN-CE-Rev_E 18/06/2013
Page 4 of 23
Directions on Use
All reagents supplied are formulated specically for use with this assay and lot numbers are
specic for each Bond Oracle HER2 IHC System. For the assay to be valid, no substitutions
should be made.
English
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 Bond Oracle HER2 IHC System used is within its
designated expiry date. The signs indicating contamination and/or instability of the Bond Oracle
HER2 IHC System are: turbidity of the solutions, odor development, and presence of precipitate.
Storage conditions other than those specied above must be veried by the user.
Specimen Preparation
All specimens must be prepared to preserve the tissue for immunohistochemical staining.
Standard methods of tissue processing should be used for all specimens (12).
It is recommended that tissues are prepared in formalin-based xatives and are routinely
processed and parafn-embedded. For example, resection specimens should be blocked into
a thickness of 3–4 mm and xed 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 parafn wax, held at no more than 60 °C. Tissue specimens should
be sectioned between 3–5 µm.
The slides required for HER2 oncoprotein evaluation and tumor verication should be prepared
at the same time. To preserve antigenicity, tissue sections mounted on slides (Leica BOND
Plus Slides – product code S21.2113) should be stained within 4–6 weeks of sectioning when
held at room temperature (20–25 °C). Following sectioning, it is recommended that slides are
incubated for 12–18 hours (overnight) at 37 °C. Sections which require additional adherence
may be incubated at 60 °C for a further hour.
In the USA, the Clinical Laboratory Improvement Act of 1988 requires in 42 CFR 493.1259(b)
that “The laboratory must retain stained slides for at least ten years from the date of examination
and retain specimen blocks at least two years from the date of examination”.
Warnings and Precautions
For professional users only.
One or more components in the product are hazardous.
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.
Symptoms of overexposure to ProClin™ 950, the preservative used in the Oracle reagents, may
include skin and eye irritation and irritation to mucous membranes and upper respiratory tract.
The concentration of ProClin™ 950 in this product is up to a maximum of 0.35%. These solutions
do not meet the OSHA criteria for a hazardous substance. A Material Safety Data Sheet is
available upon request or from www.LeicaBiosystems.com.
Specimens, before and after xation, 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 nonspecic staining may occur.
Page 5 of 23
Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN-CE-Rev_E 18/06/2013
Procedure
A. Reagents required but not supplied
• BOND Dewax Solution (product code AR9222)
• BOND Epitope Retrieval Solution 1 (product code AR9961)
• BOND Wash Solution x10 Concentrate (product code AR9590)
• Standard solvents used in immunohistochemistry ( e.g. ethanol, absolute and graded)
• Xylene (or xylene substitutes)
• Mounting medium
• Distilled or de-ionized water
B. Equipment required but not supplied
• Leica Biosystems’ BOND-MAX and BOND-III fully automated, advanced staining
system(s)
• BOND Universal CovertilesTM (product code S21.2001 or S21.4583)
• BOND Mixing Stations (product code S21.1971)
• Drying oven, capable of maintaining 60 °C
• Light microscope (4–40x objective magnication)
• Slides (Leica BOND Plus Slides – product code S21.2113)
• Coverslips
• BOND Slide Label & Print Ribbon (product code S21.4564)
• BOND Aspirating Probe Cleaning System (product code CS9100)
C. Methodology
• Prior to undertaking this methodology, users must be trained in BOND fully automated
immunohistochemical techniques.
• Each test section to be stained with the HER2 Primary Antibody will require an identical
section for staining with the HER2 Negative Control. The negative control section allows
differentiation between specic and nonspecic staining at the antigen site. Each BOND
staining run should include a HER2 Control Slide. At the end of the staining protocol, if
the cell lines do not demonstrate the correct staining patterns (refer to Bond Oracle HER2
IHC Systems Interpretation Guide), the run should be regarded as invalid.
D. Slide Layout
A new BOND Universal Covertile (product code S21.2001 or S21.4583) should be used with
each slide. The use of BOND Universal Covertiles which have been previously utilized for
either immunohistochemical or in situ hybridization staining has not been validated with this
test.
The slide tray layout (Table 2) enables optimal performance of the Bond Oracle HER2 IHC
System and the full 60 tests to be obtained.
English
Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN-CE-Rev_E 18/06/2013
Page 6 of 23
Slide
Slide Description Reagent Tissue
Position
English
1 Case 1 *HER2 Negative Control Test
2 Case 2 *HER2 Negative Control Test
3 Case 3 *HER2 Negative Control Test
4 Case 4 *HER2 Negative Control Test
5 Case 1 *HER2 Primary Antibody Test
6 Case 2 *HER2 Primary Antibody Test
7 Case 3 *HER2 Primary Antibody Test
8 Case 4 *HER2 Primary Antibody Test
9 HER2 Control Slide *HER2 Primary Antibody Positive
10 In-house Tissue Control *HER2 Primary Antibody Positive
Table 2. Slide tray layout, showing tissue type and reagent
Slide Icon
Type
E. Procedure Steps
Follow the steps below to set up a slide tray with the layout described in Table 2. These
instructions should be read in conjunction with the BOND System User Manual.
1. On the BOND instrument, 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, BOND Dewax Solution
(supplied as ready-to-use), BOND Epitope Retrieval Solution 1 (supplied as ready-to-use)
and BOND Wash Solution (supplied as x10 concentrate) in the bulk reagent containers to
perform the required staining runs.
3. Ensure that a clean BOND Mixing Station is installed.
4. Turn on the BOND fully automated, advanced staining system.
5. Turn on the BOND Controller attached to the BOND fully automated, advanced staining
system.
6. Open the BOND software.
7. For a new Bond Oracle HER2 IHC System, scan the reagent tray barcodes with the
handheld scanner to enter the system into the BOND reagent inventory.
8. Go to the Slide setup screen and click Add case.
Page 7 of 23
Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN-CE-Rev_E 18/06/2013
Loading...