The effect of aging of formalin-fixed paraffin-embedded tissues on the in situ hybridization and immunohistochemistry signals in cervical lesions.
Author(s): Nuovo AJ, Garofalo M, Mikhail A, Nicol AF, Vianna-Andrade C, Nuovo GJ
Publication: Diagn Mol Pathol, 2013, Vol. 22, Page 164-73
PubMed ID: 23846444 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine if storage of FFPE cervical tissue specimens as slide-mounted sections, FFPE ribbon curls, or unsectioned FFPE blocks adversely impact the extent and intensity of immunohistochemical and in situ hybridization (ISH) staining, or alter microRNA levels.
Conclusion of Paper
Mean staining scores (based on both the extent and intensity of staining) were significantly lower for both ISH (human papilloma virus, HPV) and immunohistochemistry (p21, CD45) when specimens were stored as slide-mounted sections for 13-15 years at room temperature compared to slide mounted sections that were stored for 1 year or less. Significant reductions, while less robust than those observed in slide-mounted sections stored for 13-15 y, were observed in mean staining scores for p16 assessed by immunohistochemisty and HPV by ISH among slide-mounted sections stored for 5-7 y and FFPE ribbon curls stored for 13-15 y, respectively, compared to corresponding controls stored for less than 1 y. Similarly, FFPE blocks stored for 13-15 y also displayed significantly lower staining scores for ISH and immunohistochemistry than those stored for 1 year or less, although the magnitude of decline was not as severe as observed with slide-mounted sections. Conversely, normalized miRNA expression remained stable in specimens stored as ribbon curls for 13-15 years compared to those recently collected.
Studies
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Study Purpose
The purpose of this study was to determine if storage of FFPE cervical tissue specimens as slide-mounted sections or FFPE blocks adversely impact the extent and intensity of immunohistochemical and ISH staining. A total of 88 cervical intraepithelial neoplasia (CIN) specimens that previously tested positive for human papilloma virus (HPV) were used for analysis. FFPE specimens were stored as slide-mounted sections (4-5 µm thick on PLUS slides) and FFPE blocks. All specimen types were stored at room temperature and protected from light for 0-1 y (44 specimens) or 13-15 years (44 specimens). A subset of both specimen types were analyzed for HIV infection (17 specimens) or microRNA expression (15 specimens). Prior to immunohistochemical staining for p16 and CD45, specimens were subjected to 30 min of unspecified antigen retrieval. ISH analysis was preceded by protease digestion. HPV infection was assessed using an ISH probe cocktail capable of detecting 5 types of low risk and 13 types of high risk HPV. All staining was scored in a blinded fashion using a 0 to 100 scale based on the percentage of stained cells and the intensity of staining, such that a score of 100 would indicate that every cell displayed intense staining.
Summary of Findings:
When non-case-matched HPV positive specimens were compared, slide-mounted FFPE sections stored for 13- 15 years at room temperature displayed significantly lower HPV ISH staining scores than those stored similarly for 1 year or less (25.9 vs 84.1 mean score, p<0.001). Further, based on ISH staining, the HPV status changed from positive to negative following slide-mounted storage for 50% (22/44) of case-matched specimens. For 17 of the 22 specimens, pre-storage HPV ISH results as well as stored slide-mounted sections and FFPE blocks were available. HPV ISH scores were significantly lower in both stored slide-mounted sections (19.4 mean score, p<0.001) and fresh sections from the same archived FFPE blocks (41.5 mean score, p=0.016) in comparison to HPV ISH scores obtained during the initial diagnosis (89.4 mean score). The authors note that storage associated reductions in HPV ISH signal were heterogeneous, as signal intensities were comparable between the initial diagnosis and the stored specimen for approximately 11% of slide-mounted FFPE sections and 35% of FFPE blocks. Similar results of storage-induced effects were observed when 15 CIN specimens were analyzed for expression of the miRNA let 7c by ISH, as ISH scores were significantly lower among specimens stored as slide-mounted sections (27.3 mean score, p<0.001) or as FFPE blocks (65.6 mean score, p=0.003) for 13-15 years compared to results collected during the original diagnosis (89.7 mean score). When 18 non-case matched CIN specimens were compared following immunohistochemistry, slide-mounted FFPE sections stored for 13-15 years displayed significantly lower staining scores than those stored for 1 year or less for both p16 (10.2 vs. 84.2 mean score, p<0.001) and CD45 (10.9 vs. 92.9 mean score, p<0.001).
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Other diagnoses
- Neoplastic - Benign
Platform:
Analyte Technology Platform DNA In situ hybridization Protein Immunohistochemistry RNA In situ hybridization Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0-1 y
13-15 y
In situ hybridization Specific Targeted nucleic acid HPV (DNA)
miRNA let-7c
Storage Storage conditions FFPE block
Slide-mounted FFPE section
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Study Purpose
The purpose of this study was to compare the intensity of immunohistochemical staining for the nuclear protein p16 between FFPE specimens stored for 5-7 years as slide-mounted sections and recently collected specimens. A total of 54 cervical cancer specimens were represented on a commercial tissue microarray from specimens preserved and processed 5-7 years prior. Tissue microarray sections were mounted on glass slides at least 3 years before analysis. Controls consisted of cervical biopsies from eight high-grade CINs and two cervical cancers that were recently collected (a specific time frame was not provided). Prior to p16 immunohistochemical staining specimens were subjected to 30 min of unspecified antigen retrieval. Staining was scored in a blinded fashion using a 0 to 100 scale based on the percentage of stained cells and the intensity of staining, such that a score of 100 would indicate that every cell displayed intense staining.
Summary of Findings:
When p16 immunohistochemical staining of non-case matched cervical lesion specimens were compared, slide-mounted FFPE sections stored for 5-7 years displayed significantly lower staining scores than those collected recently (48.7 vs. 82.3 mean score, p<0.001).
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Benign
- Other diagnoses
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Tissue microarray Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Immunohistochemistry Specific Targeted peptide/protein p16
Storage Storage duration Recent (< 1 y was assumed)
5-7 y
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Study Purpose
The purpose of this study was to determine if storage of FFPE cervical tissue specimens as FFPE ribbon curls or an FFPE block adversely impact the extent and intensity of ISH staining or levels of microRNA quantified by real-time qRT-PCR. An specified number of cervical intraepithelial neoplasia (CIN) specimens that previously tested positive for HPV were used for analysis. FFPE specimens were stored as FFPE ribbon curls (25 µm thick stored in an Eppendorf tube) and FFPE blocks. Alls specimen types were stored at room temperature and protected from light for 0-1 y or 13-15 years. ISH analysis was preceded by protease digestion. HPV infection was assessed using an ISH probe cocktail capable of detecting 5 types of low risk and 13 types of high risk HPV. All staining was scored in a blinded fashion using a 0 to 100 scale based on the percentage of stained cells and the intensity of staining, such that a score of 100 would indicate that every cell displayed intense staining. MicroRNA extracted from FFPE specimens by an unspecified method was analyzed by real-time qRT-PCR using the comparative CT method and U44 and U48 rRNA for normalization.
Summary of Findings:
When non-case-matched HPV positive specimens were compared, FFPE ribbon curls stored in an Eppendorf tube for 13- 15 years at room temperature (190.5 mean score) and fresh sections from FFPE blocks stored for the same duration (211.5 mean score) both displayed significantly lower HPV ISH staining scores than FFPE ribbon curls stored for 1 year or less (13,341.1 mean score, p<0.001 for both). However, differences in miRNA let-7 c, miR-99a, miR-26a, miR-34a, and miR-23 normalized expression did not differ significantly between non-case-matched specimens stored as FFPE ribbon curls for 13-15 y or less than 1 year.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Other diagnoses
- Neoplastic - Benign
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR DNA In situ hybridization Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0-1 y
13-15 y
Real-time qRT-PCR Specific Targeted nucleic acid miRNA let-7c
miR-99a
miR-26a
miR-34a
miR-23
In situ hybridization Specific Targeted nucleic acid HPV
Storage Storage conditions FFPE block
FFPE ribbon curl in an Eppendorf tube