Leica Biosystems Bond Oracle HER2 IHC System - Manual (Page 12)

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Leica Biosystems Bond Oracle HER2 IHC System Instructions for Use TA9145 EN-CE-Rev_E 18/06/2013
Slide Screening Order Rationale
Slides should be screened in the following order:
1. HER2 Control Slide – HER2 Primary Antibody
A valid assay with the Oracle HER2 Control Slide shows the following:
• Presence of strong brown, complete cell membrane staining in the 3+ Control Cell Line
SK-BR-3.
• Presence of weak to moderate brown, complete cell membrane staining in the 2+ Control
Cell Line, MDA-MB-453.
• Presence of faint/barely perceptible brown, incomplete cell membrane staining in the
1+ Control Cell Line, MDA-MB-175.
• No staining in the 0 Control Cell Line MDA-MB-231.
Important note: A feature of the MDA-MB-175 1+ control cell line is a distinct growth pattern in
which the cells form clusters. These clusters give rise to a continuous luminal brush border
region across the cell cluster. This brush border staining will be stronger than that of the rest
of the cell membrane. It is the faint/barely perceptible incomplete cell membrane staining that
is the correct HER2 oncoprotein 1+ staining pattern. Dot-like immunostaining of the Golgi
region in the cytoplasm may also be observed in this cell line.
2. In-house Positive Control Tissue – HER2 Primary Antibody
The PRESENCE of brown membrane staining should be observed corresponding to the
known HER2 oncoprotein status of the chosen positive control.
3. In-house Negative Control Tissue Component – HER2 Positive Control
The ABSENCE of membrane staining should be observed. A negative control tissue
component confirms the lack of detection system cross-reactivity to specifically targeted cells/
cellular components. If membrane staining occurs in a negative control tissue component,
results with the patient specimen should be considered invalid.
4. Patient Tissue – stained using the HER2 Negative Control
The ABSENCE of membrane staining verifies the specific labeling of the target antigen by the
primary antibody. Other brown staining occurring in the cytoplasm of the specimen treated
with the HER2 Negative Control, such as in connective tissue, leukocytes, erythrocytes,
or necrotic tissue, should be considered nonspecific background staining and should be
noted.
5. Patient Tissue – stained using the HER2 Primary Antibody
HER2 oncoprotein expression levels are determined by the criteria defined in both Table 4
and in the Bond Oracle HER2 IHC System Interpretation Guide.
Limitations
A. General Limitations
Immunohistochemistry is a laboratory based, multi-step technique, used to aid in the
interpretation and determination of histopathological characteristics. It is a technique which
requires specialized training in all aspects of procedure (including the selection of appropriate
reagents, tissue, fixation, processing and IHC slide preparation) and interpretation.
Immunohistochemical staining of tissue 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 artifact,
antibody trapping, or false negative results. Inconsistent results may be due to variations in
fixation, embedding methods, or to inherent irregularities within the tissue (15). Excessive or
incomplete counterstaining may also compromise correct interpretation of the results.