Immunohistochemistry on old archival paraffin blocks: is there an expiry date?
Author(s): Grillo F, Bruzzone M, Pigozzi S, Prosapio S, Migliora P, Fiocca R, Mastracci L
Publication: J Clin Pathol, 2017, Vol. 70, Page 988-993
PubMed ID: 28596153 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the stability of 12 antigens in formalin-fixed, paraffin-embedded (FFPE) blocks that were stored for approximately 5- 55 years. Lengthening the duration of heat-mediated antigen retrieval (30 versus 60-90 min) and deep sectioning the FFPE block were evaluated individually and in combination for their potential to mitigate storage-associated reductions in immunostaining for the 12 antigens: smooth muscle actin (SMA), cytokeratins (CK) AE1/AE3, CK CAM 5.2, CK 5 & 6, cytokeratin 7, vimentin, desmin, CD31, CD34, leucocyte common antigen (LCA), S-100 protein and Ki67.
Conclusion of Paper
Of the 12 antigens evaluated by immunohistochemistry, four (Ki67, CD31, CD34, LCA) displayed variable immunopositive staining among the FFPE blocks examined; diminished and variable KI67 immunostaining was observed among FFPE blocks collected prior to 2010 while CD31, CD34, and LCA antigenicity was reduced in FFPE blocks collected in 1960 and 1970. The remaining antigens yielded homogenous and intense immunopositive staining regardless of FFPE block age/storage duration or whether enzymatic retrieval (CK AE1/AE3, CK CAM5.2, desmin, SMA) or heat-mediated antigen retrieval (CK 7, CK 5 & 6, S-100 protein and vimentin) was required.
Deep sectioning the FFPE block (to a depth of 500 or 1000 µm) before sections were used and a longer heat-mediated antigen retrieval treatment each individually improved the intensity of nuclear Ki67 immunopositive staining; however, the strongest and clearest immunopositive staining for Ki67 and CD31 was observed when deep sectioning (both depths yielded similar results) and longer heat-mediated treatment were used in combination, with the exception of FFPE blocks collected in 1970, which remained negative. Although an absence of records precludes conclusions, the authors postulate that different fixation and processing conditions or the storage conditions of the FFPE blocks (exposed to light, dust, and humidity) may be contributing factors to the loss of antigenicity that was observed in blocks collected in 1970. The authors note that an edge effect was observed, with stronger immunostaining observed around the periphery of sections relative to the center, although they did not share what storage durations or antigens this was observed with.
Studies
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Study Purpose
The purpose of this study was to determine the stability of 12 antigens in FFPE blocks that were stored for approximately 5- 55 years. Lengthening the duration of heat-mediated antigen retrieval (30 versus 60-90 min) and deep sectioning the FFPE block before sections were collected were evaluated individually and in combination for their potential to mitigate storage-associated reductions in immunostaining. A total of 74 FFPE blocks containing skin, myometrium, or unspecified tissue types that were collected in 1960, 1970, 1980, 1990, 2000, and 2010 were evaluated (10-15 blocks per storage timepoint) in the study by immunohistochemistry for 12 antigens (smooth muscle actin (SMA), cytokeratins (CK) AE1/AE3, CK CAM 5.2, CK 5 & 6, CK7, vimentin, desmin, CD31, CD34, leucocyte common antigen (LCA), S-100 protein and Ki67). Information on patient diagnosis was not available and thus was not provided. Specimens collected between 1960 and 1990 were re-embedded in paraffin to circumvent a mechanistic incompatibility in mounting the block to the microtome. All FFPE blocks were stored at room temperature, but the conditions of storage differed among the time points examined. FFPE blocks from 1960 and 2000-2010 were stored in plastic bags within cardboard boxes (protected from light); FFPE blocks from 1970 were stored in torn plastic bags and thus were exposed to light, dust, and humidity; blocks from 1980-1990 were stored in plastic bags (exposed to light) and transferred to cardboard boxes (dry, protected from light) after 2000. All specimens were fixed in formalin for a minimum of 24 h, except those collected in 1970 for which no information was available. Tissues collected after 1985 were fixed in 4% buffered formalin; information on the fixation of specimens collected earlier than 1985 was not available; no additional details on fixation, processing, or paraffin embedding were provided. Each antibody was evaluated in three FFPE blocks for each storage timepoint/decade of collection. All sections were cut on the same day by the same laboratory technician, and immunohistochemistry was performed within one week of sectioning using an automated immunostainer, which included heat (30 min at 100°C) and enzymatic (treatment with protease 1 for 4-32 min) antigen retrieval depending on the antibody used, quenching endogenous peroxidase activity (5% H2O2 for 10 min), and a hematoxylin counterstaining. Immunohistochemical staining was evaluated by two histopathologists who were blinded to the age of the FFPE blocks; immunopositive staining was graded as +/+, when the intensity was equal to the positive control; +/−, when the intensity was less than the positive control; −/−, when no staining was present. Alternative approaches were evaluated for their ability to mitigate a loss of antigenicity, which included deep sectioning the FFPE block at a depth 500 μm and 1000 μm prior to section collection and extending the duration of heat-mediated retrieval from 30 min to 60 or 90 min.
Summary of Findings:
Homogenous and intense immunopositive staining was observed for SMA, cytokeratins AE1–AE3, cytokeratins CAM 5.2, cytokeratins 5 & 6, cytokeratin 7, desmin, S-100 protein and vimentin, regardless of the storage duration of the FFPE blocks examined.
Four antigens (Ki67, CD31, CD34, LCA) displayed variable immunostaining (16 weak reactions and 7 negative reactions), with weaker and negative staining intensity occurring more frequently among older blocks (collected in 1960 and 1970). FFPE blocks from 1970 displayed weak or negative immunostaining for the following antigens: Ki-67, CD31, CD34, and LCA. Ki67 was particularly sensitive to block storage as weak and/or negative immunostaining relative to the positive control was observed for all blocks collected prior to 2010. Immunohistochemistry was not adversely affected by FFPE block storage for the remaining cytoplasmic antigens, regardless of whether they required enzymatic retrieval (CK AE1/AE3, CK CAM5.2, desmin, SMA), or heat-mediated antigen retrieval (CK 7, CK 5 & 6, S-100 protein and vimentin).
Deep sectioning the FFPE block to a depth of 500 or 1000 µm before sections were used and a longer heat-mediated antigen retrieval treatment each individually improved the intensity of nuclear Ki67 immunopositive staining; however, the strongest and clearest immunopositive staining for Ki67 and CD31 was observed when deep sectioning (both depths yielded similar results) and longer heat-mediated treatment were used in combination, with the exception of FFPE blocks collected in 1970, which remained negative. Although an absence of records precludes conclusions, the authors postulate that different fixation and processing conditions or the storage conditions of the FFPE blocks (exposed to light, dust, and humidity) may be contributing factors to the loss of antigenicity that was observed in blocks collected in 1970. The authors note that an edge effect was observed, with stronger immunostaining observed around the periphery of sections relative to the center, although they did not share what storage durations or antigens this was observed with.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Not specified
Platform:
Analyte Technology Platform Glycoprotein Immunohistochemistry Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 5 y
15 y
25 y
35 y
45 y
55 y
Immunohistochemistry Specific Targeted peptide/protein SMA
CK AE1/AE3
CK CAM 5.2
CK 5 & 6
CK 7
CD31
vimentin
desmin
CD34
LCA
S-100 protein
Ki67
Analyte Extraction and Purification Antigen retrieval 30 min at 100°C
60 min at 100°C
90 min at 100°C
Biospecimen Aliquots and Components Aliquot sequential collection Section collected 500 µm into FFPE block
Section collected 1,000 µm into FFPE block