Optimizing Ventana chromogenic dual in-situ hybridization for mucinous epithelial ovarian cancer.
Author(s): Li X, Chew SH, Chay WY, Lim-Tan SK, Goh LK
Publication: BMC Res Notes, 2013, Vol. 6, Page 562
PubMed ID: 24373486 PubMed Review Paper? No
Purpose of Paper
This paper examined whether the length of formalin-fixed paraffin-embedded (FFPE) block storage affects successful detection of HER-2 genomic amplification by dual in situ hybridization (DISH).
Conclusion of Paper
Overall, evaluation of HER-2 genomic amplification status by DISH was successful for 79 of 92 (86%) FFPE ovarian tumor specimens when an optimized protocol was used. DISH optimization was multi-facetted and tailored to the specific issue observed. Analysis of specimens stored for longer than 1 y necessitated optimization and resulted in successful DISH analysis for HER-2 genomic amplification for 100% of specimens stored for 10 y or less. However, the percentage of FFPE specimens for which DISH analysis was unsuccessful increased progressively with FFPE block storage beginning after 11 y (1%) and peaking after storage for 22 y (11%), the oldest timepoint examined.
Studies
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Study Purpose
This study examined whether the duration of FFPE block storage affects successful detection of HER-2 genomic amplification by DISH. Ovarian specimens were surgically obtained from 92 patients diagnosed with epithelial ovarian cancer and fixed in neutral buffered formalin for unspecified durations. FFPE specimens stored for 10 y or less were stored at the same site as collection, while those stored for longer than 10 y were stored offsite. FFPE sections (3 µm thick) were used for DISH staining with a commercial probe cocktail and an automated stainer. When necessary, the efficiency of different optimization techniques was evaluated using archival FFPE specimens. These optimization steps focused on durations of cell conditioning, chromagen incubation, counterstain incubation, as well as the use and duration of protease 2 or 3 incubations, and the temperature of the stringency wash.
Summary of Findings:
Overall, evaluation of HER-2 genomic amplification status by DISH was successful for 79 of 92 (86%) FFPE ovarian tumor specimens when an optimized protocol was used. DISH analysis of FFPE specimens stored for longer than 1 year required optimization due to non-specific background staining, sub-optimal counterstaining, an absence of ISH signal with well-preserved nuclear morphology, or a "fuzzy" ISH signal. Nuclear bubbling was occasionally observed and addressed by increasing the duration of deparaffinization. Unsuccessful DISH analysis was first observed among FFPE blocks stored for 11 y (1% of specimens were unsuccessfully analyzed by DISH) even when optimization was employed and increased progressively with FFPE block storage to a maximum of 14% of specimens stored for 22 years. It is unclear if storage conditions were a contributing factor as FFPE blocks stored for 11 y or older were stored off-site while those stored for 10 y or less were stored onsite. Optimization techniques were tailored to the specific issue encountered: "fuzzy" ISH signals necessitated an increase in the temperature of the stringency wash from 72 to 76°C, the duration of counterstain was altered to mitigate suboptimal staining intensity, and duration of hybridization and chromagen incubations were shortened when non-specific staining was observed. Older FFPE blocks required a greater number of deviations from the original DISH protocol with FFPE blocks stored for 5 y requiring deviations in four parameters, those stored for 10 y requiring deviations in eight parameters, and those stored for 20 years requiring deviations in 11 parameters.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA In situ hybridization Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration <1 y
2 y
3 y
4 y
5 y
6 y
7 y
8 y
9 y
10 y
11 y
12 y
13 y
14 y
15 y
16 y
17 y
18 y
19 y
20 y
21 y
22 y