Effects of long-term storage on the detection of proteins, DNA, and mRNA in tissue microarray slides.
Author(s): Karlsson C, Karlsson MG
Publication: J Histochem Cytochem, 2011, Vol. 59, Page 1113-21
PubMed ID: 22147608 PubMed Review Paper? No
Purpose of Paper
This paper examined whether storage of slide-mounted formalin-fixed paraffin-embedded (FFPE) tissue sections leads to reductions in clinically relevant biomarkers when analyzed by immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), or chromogenic in situ hybridization (CISH). FFPE slides from a tissue microarray that were left unaltered or coated in paraffin prior to storage at room temperature and 4°C for different durations (1 week or 6 months) were compared.
Conclusion of Paper
Storage of FFPE slides (unaltered or paraffin-coated) for up to one year at either room temperature or 4°C produced FISH and CISH results that were comparable to FFPE slides analyzed immediately, but antigen-specific effects of FFPE slide storage durations, temperatures, and condition (unaltered vs. paraffin-coated slides) were observed with IHC analysis. The duration of FFPE slide storage significantly affected p53, Ki-67, CK7, and HER2 IHC scores; storage temperature significantly affected CK7 IHC scores; and paraffin coating of slides significantly affected p53, CK7, and HER2 IHC scores. The authors conclude that paraffin coating FFPE slides offered no protective benefit and that while detection of gene copy number and chromosomal translocations were possible using FFPE slides stored for up to one year, FFPE slide storage duration and temperature may modestly, albeit significantly, affect immunohistochemical analysis of some antigens.
Studies
-
Study Purpose
This paper examined whether storage of slide-mounted formalin-fixed paraffin-embedded (FFPE) tissue sections leads to reductions in clinically relevant biomarkers when analyzed by immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), or chromogenic in situ hybridization (CISH) using a tissue microarray. The tissue microarray used for analysis contained one case of prostate adenocarcinoma, five cases of non-small cell lung cancer (NSCLC) adenocarcinoma, five cases of NSCLC squamous cell carcinoma, 10 cases of breast adenocarcinoma, five cases of thyroid papillary adenocarcionma, seven cases of colon adenocarcinoma, seven cases of lymph node B chronic lymphatic leukemia, two cases of lymph node mantel cell lymphoma, two cases each of normal lung and thyroid, and one case each of normal tonsil, prostate, pancreas, lung, and colon. FFPE sections (4 µm thick) were mounted on Capillary Gap Microscope slides for immunohistochemistry and SuperFrost Plus slides for FISH and CISH. FFPE slides were left unaltered or deparaffinized in xylene, air dried, and coated in paraffin prior to storage for 1 week or 1, 3, 6, or 12 months at room temperature or 4°C or analyzed immediately (0 d). FFPE slides were subjected to 30 min of antigen retrieval by microwave heating in a TRIS-EDTA, pH 9.0 buffer prior to IHC. IHC staining was scored based on intensity (range 0-255, 0=black, 255=white) and determined using automated software. IHC staining was considered positive for scores between 0-100. Twenty tumor cells were assessed by FISH.
Summary of Findings:
Multifactor analysis revealed antigen-specific effects of FFPE slide storage durations, temperatures, and condition (unaltered vs. paraffin-coated slides) when analyzed by IHC. While storage temperature did not affect IHC scores, p53 immunoscores declined significantly when FFPE slides were dipped in paraffin prior to storage and these declines were compounded further by storage duration (7 U, p<0.01),. Although Ki-67 IHC scores declined over time for all storage temperatures and conditions (unaltered, paraffin-coated) when compared to slides that were immediately analyzed (p<0.001), significantly lower IHC scores were observed for FFPE slides stored at room temperature compared to 4°C (1.5 U, p<0.001) and those coated in paraffin compared to unaltered slides (1.5 U, p<0.001). CK7 scores were significantly reduced among FFPE slides stored at room temperature compared to 4°C (1.1 U, p=0.027) and when dipped in paraffin (1.5 U, p<0.01) and time-dependent declines were also observed (p<0.001). Conversely, HER2 IHC scores increased with storage duration (p<0.0001), as FFPE slides stored at 4°C for 12 months had a score 10 U higher than immediately analyzed slides. While significantly lower HER2 IHC scores were observed among paraffin-coated FFPE slides compared to those that were unaltered (3.3 U, p=0.0003), FISH detection of HER2 amplification or translocation in mantel cell lymphoma specimens was unaltered by storage duration, temperature, or condition. Likewise, CISH analysis for kappa and gamma light chain mRNA were comparable among FFPE slides analyzed immediately and those stored for up to one year, regardless of storage temperature or condition.
Biospecimens
- Tissue - Prostate
- Tissue - Lung
- Tissue - Breast
- Tissue - Tonsil
- Tissue - Colorectal
- Tissue - Lymph Node
- Tissue - Thyroid Gland
Preservative Types
- Formalin
Diagnoses:
- Normal
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Immunohistochemistry DNA FISH RNA Tissue microarray Protein Tissue microarray DNA Tissue microarray RNA In situ hybridization Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0 days
1 week
1 month
3 months
6 months
12 months
Storage Storage temperature Room temperature
4°C
In situ hybridization Specific Targeted nucleic acid Kappa light chain mRNA
Gamma light chain mRNA
Immunohistochemistry Specific Targeted peptide/protein p53
Ki-67
CK7
Her-2
FISH Specific Targeted nucleic acid HER2/CEP17
Storage Storage conditions Unaltered FFPE slide
Paraffin-coated FFPE slide