The effect of delay in fixation on HER2 expression in invasive carcinoma of the breast assessed with immunohistochemistry and in situ hybridisation.
Author(s): Lee AH, Key HP, Bell JA, Kumah P, Hodi Z, Ellis IO
Publication: J Clin Pathol, 2014, Vol. 67, Page 573-5
PubMed ID: 24737400 PubMed Review Paper? No
Purpose of Paper
This paper explored potential effects of a room temperature delay to formalin fixation (30 min to 24 h) on HER2 quantification by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in surgically-resected breast carcinoma specimens. Immunostaining produced with three different HER2 antibodies was also compared in a subset of specimens.
Conclusion of Paper
Of the nine breast carcinoma biospecimens analyzed, two displayed an observable reduction in the extent and intensity of immunostaining after a room temperature delay of 8 h compared to a delay of 0 or 1 h. Differences in immunostaining among the remaining seven carcinoma biospecimens were less pronounced. Comparable results were obtained with the three IHC antibodies evaluated. Determination of HER gene copy number by FISH was not affected by a room temperature delay to fixation of up to 24 h.
Studies
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Study Purpose
This study investigated effects of a room temperature delay to fixation on quantification of HER2 by FISH and IHC. Nine breast carcinoma biospecimens collected by surgical resection were dissected into small aliquots (8 x 3 x 3 mm) and subjected to a delay of 0, 1, 2, 4, 8, or 24 h at room temperature. Experimental delay to fixation timepoints did not include delays experienced in the operating theater (not specified) or the time required for specimen transport from the operating theater to the laboratory (estimated between 10-30 min). Biospecimen aliquots were fixed in either 10% phosphate buffered formalin (n=5) or 10% neutral buffered formalin (n=4) for a minimum of 10 hours prior to paraffin processing, sectioning, and analysis. FISH analysis for HER2 copy number was conducted using the PathVysion HER2 DNA FISH assay. IHC results were obtained using the Ventana Pathway Her2/neu rabbit monoclonal primary antibody and antibodies against HER2 contained in the Hercept Test kit and the Leica Oracle HER2 staining kit. Each IHC slide was scored by two of three observers for intensity and the percentage of membrane that was immunostained. Statistical analysis was not performed.
Summary of Findings:
Of the nine breast carcinoma biospecimens analyzed, two displayed an observable reduction in the extent and intensity of immunostaining after a room temperature delay of 8 h compared to those that experienced a delay of 0 or 1 h and these differences were observed with each of the three different HER2 antibodies evaluated. Differences in immunostaining among the remaining seven carcinoma biospecimens were less pronounced, but comparable results were obtained with the three IHC antibodies evaluated. Determination of HER gene copy number by FISH was not affected by a room temperature delay to fixation of up to 24 h.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Immunohistochemistry DNA FISH Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Immunohistochemistry Specific Type of antibody HercepTest
Oracle
4BS
Biospecimen Acquisition Cold ischemia time 0 h
1 h
2 h
4 h
8 h
24 h