Leica Biosystems HER2 FISH System - 30 Test - Manual (Page 13)

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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013
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.
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.
Cell Line Bond Oracle
HER2 IHC
System Profile HER2
Receptor Load
per cell*
Leica HER2 FISH System - 30 Test
HER2:CEP17 Acceptance Criteria
SKBr-3
3+ 4.3 x 10 5 HER2 amplification is observed
MDA-MB-453
2+ 1.4 x 10 5
HER2/CEP17 gene ratio should be between
1.5 – 2.5
MDA-MB-175
1+ 6.3 x 10 4 HER2 amplification is not observed
MDA-MB-231
0 9.3 x 10 3 HER2 amplification is not observed
*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).