Paraffin section storage and immunohistochemistry. Effects of time, temperature, fixation, and retrieval protocol with emphasis on p53 protein and MIB1 antigen.
Author(s): Wester K, Wahlund E, Sundström C, Ranefall P, Bengtsson E, Russell PJ, Ow KT, Malmström PU, Busch C
Publication: Appl Immunohistochem Mol Morphol, 2000, Vol. 8, Page 61-70
PubMed ID: 10937051 PubMed Review Paper? No
Purpose of Paper
This paper evaluated the stability of immunohistochemical staining when slide-mounted, formalin-fixed paraffin-embedded (FFPE) tissue sections were stored unaltered or after being coated with paraffin or xylene-based spray glue for up to 16 weeks at temperatures that ranged between -20°C and 40°C.
Conclusion of Paper
Both the extent and intensity of immunohistochemical staining were adversely affected by storage of slide-mounted tissue sections for two weeks or longer for the majority of the antigens investigated, although some antigens (pRb and PSA) were stable for up to 16 weeks. While antigens were most stable when slides were stored at -20°C, detachment of tissue sections from the slide was also more prevalent. No significant differences in the extent or intensity of immunostaining were observed when slide-mounted tissue sections were stored unaltered at 4°C or 20°C; although the extent of immunostaining was reduced when slides were coated with paraffin or a xylene-based glue prior to storage at 20°C or when stored unaltered at 40°C.
Studies
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Study Purpose
This study evaluated the stability of immunohistochemical staining when slides were stored unaltered or after being coated with paraffin or xylene-based spray glue for up to 16 weeks at temperatures that ranged between -20°C and 40°C. Biopsies from four urinary bladder carcinomas, three breast carcinomas, and two benign prostatic hyperplasias were fixed in 10% neutral buffered formalin prior to processing and paraffin embedding. Sections (4 µm thick) were mounted to electrostatically-charged slides and analyzed immediately by immunohistochemistry or coated in paraffin or xylene-based spray glue prior to storage. A total of 11 different antigens were assessed, many of which required enzyme- or heat-mediated antigen retrieval. Potential effects introduced by tissue heterogeneity were investigated by comparing p53 or MIB1 immunostaining in alternating serial sections (20 sections in total) for each of four urinary bladder carcinoma biopsy specimens.
Summary of Findings:
Based on the variability observed as a result of tumor heterogeneity, the authors identified <80% immunostaining as the threshold for reduced staining. The percentage of biopsy specimens that displayed reduced immunostaining after slide storage for 16 weeks was influenced by storage temperature and slide coating when compared to those analyzed immediately. When all 11 antigens were considered, immunostaining remained stable in approximately 80% of biopsies when slides were stored at -20°C, 60% when slides were stored at 20 or 40°C, and 25% when slides were coated in paraffin or xylene-spray prior to storage at 20°C. The authors reported that while slide storage at -20°C produced the most stable immunostaining, it adversely affected tissue-slide adhesion. Storage-induced effects were also antigen-specific, as reduced immunostaining was limited to p53, p21, cyclin A, cyclin B1, high molecular weight cytokeratin, estrogen receptor, and pan cytokeratin. Immunostaining was stable following slide storage for pRB and PSA, while results for MIB1 and Factor VIII were inconclusive. Immunostaining of p21, p53, and MIB1 in 18 biopsies after 2, 4, 8, and 16 weeks of storage at 20°C revealed that approximately 60% of biopsy specimens remained stable after 2 and 4 weeks of slide storage and 20% after storage for 8 and 16 weeks compared to slides that were analyzed immediately.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Benign
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration Fresh (unspecified)
2 weeks
4 weeks
8 weeks
16 weeks
Storage Storage temperature -20 degrees C
4 degrees C
20 degrees C
40 degrees C
Storage Storage conditions Slide was unaltered
Slide was coated in paraffin
Slide was coated in xylene-based spray glue
Immunohistochemistry Specific Targeted peptide/protein p53
MIB1
p21
cyclin A
cyclin B1
High molecular weight cytokeratin
Estrogen receptor
pan cytokeratin
pRB
PSA
Factor VII
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Study Purpose
This study evaluated the stability of immunohistochemical staining when slides were stored unaltered for 2, 4, 8, and 12 weeks at 4°or 20°C. Biopsies from 20 urinary bladder carcinomas were fixed in 10% neutral buffered formalin prior to processing and paraffin embedding. Sections (4 µm thick) were mounted to electrostatically-charged slides and analyzed immediately by immunohistochemistry for p53 and MIB1 or subjected to storage prior to analysis.
Summary of Findings:
Storage of FFPE slide-mounted sections resulted in significant declines in both the percentage of p53 immunopositive cells (p<0.001) and the intensity of immunostaining (p<0.01) beginning after 2 weeks for both 4°C and 20°C when compared to slides that were analyzed immediately and progressive reductions were observed in both the percentage of p53 immunopositive cells (p<0.05) and the intensity of immunostaining (p value not provided) following storage for 4-12 weeks at 20°C. Similar effects of slide storage were observed for MIB1, with significant declines in both the percentage of MIB1 immunopositive cells (p<0.001) and the intensity of immunostaining (p=0.0001) after 2 weeks of storage at 4°C or 20°C, and progressive declines reported after 4-12 weeks of storage when slides were stored at 4°C (percentage of immnopositive cells, p<0.05) or 20°C (percentage of immunopositive cells, p<0.001). Slides stored at 4°C did not differ significantly from those stored at 20°C in the extent or intensity of p53 or MIB1 immunostaining. Notably, storage-attributable declines in immunostaining were completely attenuated for p53 and partially attenuated for MIB1 when stored slides were subjected to antigen retrieval, specifically incubation in heated boric acid.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration Fresh (not specified)
2 weeks
4 weeks
8 weeks
12 weeks
Immunohistochemistry Specific Targeted peptide/protein p53
MIB1
Analyte Extraction and Purification Antigen retrieval Citrate buffer
Boric acid
Storage Storage temperature 4 degrees C
20 degrees C