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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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K. Between Observer Precision Study
The between observer precision study was performed in a randomized and blinded fashion.
Between observer reproducibility testing of the Leica HER2 FISH System - 30 Test was
evaluated between three investigational sites. A single experienced observer at each
investigational site was used. Eighteen whole section breast cancer cases were used for
between observer precision, reecting samples types used in the clinical setting.
On enumeration of the slides stained in the Between Observer Precision Study, 53/54 cases
evaluated demonstrated a concordant result giving an overall concordance of 98.15% with
a lower 95% CI of 90.11%.
L. Lot-to-Lot Precision Study
The lot-to-lot precision study was performed in a randomized and blinded fashion. Lot-to-Lot
precision was determined on three independently manufactured lots of Leica HER2 FISH
System - 30 Test, manufactured under Good Manufacturing Practice (GMP). Each lot was
tested at a single investigational site on 540 previously HER2 characterized TMA samples
containing formalin-xed parafn-embedded breast cancer cases. The use of TMAs for the
determination of lot-to-lot reproducibility enables a larger volume of cases covering a wider
range of HER2 expression to be tested between lots.
On enumeration of the slides stained in the Lot-to-Lot Precision Study, 540/540 cases
evaluated demonstrated a concordant result giving an overall concordance of 100% with a
lower 95% CI of 99.45%.
Assay Robustness
Robustness studies were performed on the Leica BOND-MAX and BOND-III System to determine
the assay tolerance range for heat retrieval time and temperature; enzyme retrieval
time, temperature and concentration; denaturation time and temperature; hybridization time and
temperature; and stringency wash time and temperature. Robustness studies using the default
Leica BOND-MAX and BOND-III System protocol were also performed outside the
recommended limits as dened in the FDA/ORA guidance document ORA LAB5.3 Rev1.7 for
temperature and humidity.
• No difference in amplication status was observed when the default temperature for each
heat dependent step was raised by 4 °C or decreased by 4 °C, when compared to the default
Leica HER2 FISH System - 30 Test protocol. Highest quality ratings were observed at the default
temperatures and these temperatures are recommended.
• No difference in amplication status was observed when the heat induced epitope retrieval
(HIER) time was performed for 20 minutes and 30 minutes at 97 °C with Leica BOND ER1
solution, when compared to the default Leica HER2 FISH System - 30 Test protocol. Highest
quality ratings were observed at the default time of 25 minutes and this incubation time is
recommended.
• No difference in amplication status was observed when the enzyme induced epitope retrieval
(EIER) time was performed for 15 minutes and 35 minutes at 37 °C, when compared to the
default Leica HER2 FISH System - 30 Test protocol. Highest quality ratings were observed at
the default time of 25 minutes and this incubation time is recommended.
• No difference in amplication status was observed when the enzyme induced epitope retrieval
(EIER) enzyme concentration was performed with enzyme concentrate/enzyme diluent
ratios of 1:200 and 1:500 using the default Leica HER2 FISH System - 30 Test protocol.
Highest quality ratings were observed at the default concentration of 1:300 and this dilution
is recommended.
• No difference in amplication status was observed when the denaturation time was performed
for 5 minutes and 15 minutes, when compared to the default Leica HER2 FISH System - 30
Test protocol. Highest quality ratings were observed at the default time of 10 minutes and this