Leica Biosystems HER2 FISH System - 30 Test - Manual (Page 9)

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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013
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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:
• 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.
• Background: The background should be relatively free of fluorescent
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.
• 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).
1. Assess
Slide Quality
- Probe Signal
Intensity
- Background
2. Recognise
Target Signals
- Tissue Overview
- Depth of Focus
3. Nuclei
Suitability
- Nuclei Selection
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.
• 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.
• Locate all signals present in the nucleus by focusing up and down (Z
axis).
• 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.
• 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.
4. Signal
Enumeration
- Tumor Overview
- Counting