Leica TA9217 사용자 설명서

  

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Leica Biosystems  Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013

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).

-  Probe Signal 

Intensity

- Background

- Tissue Overview
- Depth of Focus

- 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.

•  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.

- Tumor Overview
- Counting