NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Methods for restoration of ki67 antigenicity in aged paraffin tissue blocks.

Author(s): Grillo F, Campora M, Pigozzi S, Bonadio S, Valle L, Ferro J, Paudice M, Dose B, Mastracci L

Publication: Histochem Cell Biol, 2021, Vol. 156, Page 183-190

PubMed ID: 33837842 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to evaluate Ki-67 immunohistochemical staining in sections from archived formalin-fixed paraffin-embedded (FFPE) blocks that underwent the following additional processing steps: maintaining or reversing specimen orientation during re-embedding, re-processing archived specimens using a routine or hybrid (uses heat and microwaves during dehydration and clearing) tissue processor, sectioning at different block depths, and different durations of heat-mediated antigen retrieval. KI-67 immunostaining was also compared to on-slide control sections of recently processed FFPE tissue specimens.

Conclusion of Paper

The intensity of Ki-67 immunohistochemical staining in sections from FFPE blocks that had been stored under controlled conditions (protected from dark at 20-25°C) for 40 years significantly improved following deep sectioning of the block (1,000 µm into the block) and antigen retrieval at 98°C for 64 min rather than 34 min. Re-embedding, specimen orientation, and re-processing (regardless of whether a routine or hybrid tissue processor was used) did not significantly affect the intensity of Ki-67 immunostaining of 40-year-old specimens.

Studies

  1. Study Purpose

    The purpose of this paper was to evaluate Ki-67 immunohistochemical staining in sections from archived FFPE blocks that underwent the following additional processing steps relative to immunostaining in on-slide controls from recently processed FFPE tissue: maintaining or reversing specimen orientation during re-embedding, re-processing archived specimens using a routine or hybrid (uses heat and microwaves during dehydration and clearing) tissue processor, sectioning at different block depths, and different durations of heat-mediated antigen retrieval.  In total, 40 FFPE blocks containing a gastroenteropancreatic neuroendocrine tumor that were archived for 40 y were used in the study; all archived FFPE blocks were stored at 20-25°C in cardboard boxes and protected from light. No further details of fixation or processing were provided. Twenty archival FFPE blocks were re-embedded due to an inconsistency between the block mounts and the microtome; the orientation of the specimen within the block was flipped in half of the specimens while the original orientation was maintained in the remaining half. The remaining 20 archival FFPE blocks were re-processed using a routine automated tissue processor (10 FFPE blocks) or a hybrid automated tissue processor that uses heat and microwaves during all stages (10 FFPE blocks) and re-embedded in the original orientation. For each FFPE block, two sections were cut after the block was faced (for H&E staining and KI-67 immunohistochemistry), and two sections were cut 500 µm and 1,000 µm into the block.  Paired sections were used to evaluate antigen retrieval at 98°C for 34 min and 64 min.  Ki-67 immunostaining was determined by immunohistochemistry with an anti-Ki-67 antibody (clone 30.9) on an automated immunostainer.  The maximum Ki-67 staining intensity observed on a slide was graded using a color scale (negative, light beige, dark beige, light brown, dark brown).  A recently collected and processed FFPE specimen served as an on-slide control during immunohistochemisty; no additional details on the fixation, processing, or storage of the specimen were provided.

    Summary of Findings:

    Antigen retrieval at 98°C for 34 min yielded reduced Ki-67 staining intensity in the archival specimen relative to on-slide controls of a recently processed FFPE specimen regardless of specimen orientation within the block (original or flipped) or whether the specimen was re-processed and what type of processor was used (routine or hybrid processor); the staining intensity of surface sections from archived FFPE blocks with a 34 min antigen retrieval ranged from negative to light brown staining. Ki-67 immunostaining intensity improved among sections taken 1,000 µm into the FFPE block relative to sections that were taken at shallower depths (block surface or 500 µm into the block) when antigen retrieval was at 98°C for 34 min, with 45% of cases displaying a change in intensity from light brown to dark brown.  Extending antigen retrieval at 98°C from 34 min to 64 min in deep-sectioned specimens (1,000 µm into the FFPE block) further improved Ki-67 staining intensity, with 85% of cases displaying brown to dark brown nuclear staining without non-specific staining. No differences in Ki-67 staining intensity were observed among re-embedded versus re-processed archived specimens or those re-processed with a routine or hybrid tissue processor when specimens were matched by the depth of sectioning and the duration of antigen retrieval (98°C for either 34 or 64 min).  Notably, the authors observed staining heterogeneity between the center and periphery of large tissue sections that occurred independent of the processing measures investigated.

    Biospecimens
    Preservative Types
    • Formalin
    Diagnoses:
    • Neoplastic - Carcinoma
    Platform:
    AnalyteTechnology Platform
    Protein Immunohistochemistry
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Storage Storage duration Recently collected
    40 y
    Analyte Extraction and Purification Antigen retrieval 98°C for 34 min
    98°C for 64 min
    Biospecimen Aliquots and Components Aliquot sequential collection Section collected at the surface of the FFPE block
    Section collected 500 µm into FFPE block
    Section collected 1,000 µm into FFPE block
    Biospecimen Preservation Clearing duration/condition Routine Tissue Processor
    Hybrid Tissue Processor (Heat and Microwaves Used)
    Biospecimen Preservation Embedding duration/condition Within block orientation maintained
    Within block orientation reversed
    Biospecimen Preservation Dehydration duration/condition Routine Tissue Processor
    Hybrid Tissue Processor (Heat and Microwaves Used)

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