Assessment of 2-Year Storage Conditions on Protein, RNA, and DNA in Unstained Human Tissue Sections, Including a Novel Multiplex Digital Gene Expression Profiling Method with Implications for Biobanking.
Author(s): Ramsower C, Wisner L, Zellner K, Glinsmann-Gibson B, Larsen B, McGrath M, Maguire A, Rimsza L
Publication: Biopreserv Biobank, 2021, Vol. , Page
PubMed ID: 34591685 PubMed Review Paper? No
Purpose of Paper
This paper assessed the potential effects of the duration, humidity, and the use of inert gas during storage of unstained formalin-fixed, paraffin-embedded (FFPE) slide-mounted sections on the stability of immunohistochemical staining, in situ hybridization (ISH) signal, DNA and RNA quantity and quality, and mRNA expression profiles in multiple tumor types.
Conclusion of Paper
Overall, storage of unstained FFPE slides for up to 24 months did not affect immunohistochemical or ISH staining, the concentration of DNA and RNA extracted, or gene expression profiles for the antigens and genes evaluated regardless of the storage temperature (room temperature or 4°C), the use of desiccant, or replacement of air with nitrogen gas. Notably, loss of CD20 immunohistochemical staining and loss of U6 RNA signal each occurred in slides from one specimen that were stored at room temperature, which the authors attributed to tissue-specific differences. When nucleic acid quantification methods were compared, DNA (p=0.031) and RNA (p=0.003) concentrations were both significantly lower when quantified by Qubit fluorometry than Nanodrop spectrophotometry.
Studies
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Study Purpose
This study assessed the potential effects of the duration, humidity, and the use of inert gas during storage of unstained formalin-fixed, paraffin-embedded (FFPE) slide-mounted sections on the stability of immunohistochemical staining, in situ hybridization (ISH) signal, DNA and RNA quantity and quality, and mRNA expression profiles in multiple tumor types. In total, nine FFPE tissue blocks containing lymph node (three specimens); skin (three specimens); and spleen, tonsil, and breast tumors (one specimen of each) were used. Patient diagnoses included diffuse large B cell lymphoma, Hodgkin lymphoma, follicular lymphoma, adenocarcinoma, Kaposi’s sarcoma, or benign hyperplasia. Six of the nine FFPE specimens were fixed in 10% neutral buffered formalin for 4-24 h and stored as FFPE blocks for 15-23 y prior to sectioning, while processing details of the remaining three specimens were unknown. Approximately 20-30 µm was removed from the face of each tissue block prior to the generation of 4 µm-thick sections that were slide-mounted. Slides were analyzed immediately (time 0 control) or underwent experimental storage at (1) room temperature on a bench top without desiccant, (2) room temperature on a bench top with desiccant, (3) 4°C with desiccant, and (4) 4°C with desiccant and under nitrogen gas for 3, 6, 12, and 24 months. Immunohistochemistry (cytokeratin, CD20, Ki67, U6) and ISH (U6 ISH assay) staining was quantified by a pathologist using the Allred scoring system (staining intensity score (0-3) x the percentage of positively stained cells). DNA and RNA were co-extracted using the Qiagen All Prep DNA/RNA FFPE Extraction Kit, and then each was quantified with a Nanodrop spectrophotometer and a Qubit fluorometer. Gene expression profiles were generated with PlexSet assay and the nCounter platform; levels of the 23 genes assessed were normalized to β-actin.
Summary of Findings:
Immunohistochemical staining of cytoplasmic (cytokeratin), membrane (CD20), and nuclear (Ki67) antigens were not significantly or adversely affected by the duration, temperature, or conditions of unstained FFPE slide storage, as no difference in the extent, intensity or localization of immunopositive staining was observed among the storage conditions for the antigens evaluated. However, CD20 staining (an oncogene that was expressed in two of the three lymphoid specimens) declined in slides from one specimen after 6 months of storage at room temperature, and declined in all slides from that specimens after 1 y regardless of storage conditions. CD20 staining in the remaining two lymphoid specimens were unaffected by storage duration and conditions. No clear trends in U6 mRNA ISH staining were observed across the durations or conditions of FFPE slide storage evaluated, with moderate to strong staining observed in all but one instance (a loss of U6 signal in slides from one individual that were stored at room temperature). Storage of unstained FFPE slides did not significantly affect the concentration of extracted DNA or RNA at any of the storage temperatures, conditions, or durations evaluated when quantified by a spectrophotometer or a fluorometer. The authors did note that both DNA (p=0.031) and RNA (p=0.003) concentrations were significantly lower when quantified by a Qubit fluorometer compared to quantification with a Nanodrop spectrophotometer. The FFPE slide storage durations and conditions evaluated also did not alter PlexSet gene expression when levels were normalized to β-actin, as no significant differences in β-actin normalized expression of the 23 genes evaluated were observed between baseline controls (0 d) and slides stored for 24 months under each condition.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Benign
- Neoplastic - Sarcoma
- Neoplastic - Carcinoma
- Neoplastic - Lymphoma
Platform:
Analyte Technology Platform Protein Immunohistochemistry DNA Fluorometry DNA Spectrophotometry RNA Fluorometry RNA Spectrophotometry RNA In situ hybridization RNA DNA microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature Room temperature
4°C
Storage Storage conditions Desiccant
No desiccant
Ambient atmosphere
Under nitrogen gas
Storage Storage duration Analyzed immediately
3 months
6 months
12 months
24 months