Effect of ischemic time, fixation time, and fixative type on HER2/neu immunohistochemical and fluorescence in situ hybridization results in breast cancer.
Author(s): Moatamed NA, Nanjangud G, Pucci R, Lowe A, Shintaku IP, Shapourifar-Tehrani S, Rao N, Lu DY, Apple SK
Publication: Am J Clin Pathol, 2011, Vol. 136, Page 754-61
PubMed ID: 22031314 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of fixation parameters on the evaluation of HER2 status in breast cancer specimens by IHC. A portion of a single grade 3 invasive ductal carcinoma was fixed in 10% NBF for 12 h, and the remainder was stored for 4 days at 4 degrees C prior to fixation for 0-168 h in each of the tested fixatives. IHC staining was performed using the HercepTest and was scored using the American society of clinical oncologists (ASCO)/ College of American pathologists (CAP) guidelines.
Summary of Findings:
In the specimen that was immediately fixed in 10% NBF for 12 h the IHC score was 3+ and. The authors report that the 4 days of cold ischemia time at 4 degrees C prior to fixation did not impact IHC results or quality of morphology. The most consistent results were obtained when the specimen was fixed with Pen-FIX resulting in scores of 3+ for all fixation times (1-168 h). When the specimens were fixed in 10% NBF, fixation for 1 h yielded a HER2 score of 1+, and fixation for 2-48 h led to variable scores of 2+ or 3+, but fixation for 72 h or longer resulted in consistent HER2 scores of 3+. Fixation in 15% NBF, SMF, and zinc formalin generally led to a HER2 score of 3+, but specimens fixed in 15% NBF for 10-24 h, SMF for 1 or 12 h, or zinc formalin for 1, 24 or 168 h scored 2+. Fixation in Bouin's fixative for 1 h led to a HER2 score of 1+, and longer fixation times led to scores of 2+ or 3+ with equal frequency and no trends were observed.
Biospecimens
Preservative Types
- Formalin
- Other Preservative
- None (Fresh)
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Immunohistochemistry Morphology H-and-E microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Bouin's fixative
Formalin (buffered)
None (fresh)
Pen-fix
Sakura molecular fixative
Zinc formalin
Biospecimen Preservation Time in fixative 0 h
1 h
2 h
3 h
4 h
5 h
6 h
7 h
8 h
9 h
10 h
11 h
12 h
24 h
48 h
72 h
168 h
Immunohistochemistry Specific Targeted peptide/protein HER2
Biospecimen Acquisition Cold ischemia time < 1 h
4 days
Biospecimen Preservation Concentration of fixative 10% NBF
15% NBF
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Study Purpose
The purpose of this study was to determine the effects of fixation parameters on the assessment of HER2 status of breast cancer specimens by FISH. A portion of a single grade 3 invasive ductal carcinoma was fixed in 10% NBF for 12 h and the remainder was stored for 4 days at 4 degrees C prior to fixation for 0-168 h in each of the tested fixatives. A HER2/chromosome enumeration probe 17 (CEP17) ratio of greater than 2.2 was defined as positive.
Summary of Findings:
In all specimens showing successful hybridization, the HER2/CEP17 ratio was greater than 7.8 which was observed in the specimen fixed immediately in 10% NBF for 12 h. Successful FISH hybridization was observed in all specimens fixed in 10% NBF, regardless of fixation duration, and in specimens fixed in 15% NBF for 1-9 h or 12-168 h. FISH hybridization was also successful in all specimens fixed for 7 h or less in Pen-Fix, SMF, or zinc formalin, but specimens fixed for longer than 7 h in these three fixatives often showed no hybridization. The single specimen fixed for 1 h in Bouin's fixative yielded successful hybridization, but specimens fixed in Bouin's for longer than 1 h failed to hybridize. No hybridization was observed in the unfixed specimen.
Biospecimens
Preservative Types
- Formalin
- None (Fresh)
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA FISH Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Cold ischemia time < 1 h
4 days
Biospecimen Preservation Type of fixation/preservation Bouin's fixative
Formalin (buffered)
None (fresh)
Pen-fix
Sakura molecular fixative
Zinc formalin
Biospecimen Preservation Time in fixative 0 h
1 h
2 h
3 h
4 h
5 h
6 h
7 h
8 h
9 h
10 h
11 h
12 h
24 h
48 h
72 h
168
FISH Specific Targeted nucleic acid HER2/neu
Biospecimen Preservation Concentration of fixative 10% NBF
15% NBF