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Factors affecting immunoreactivity in long-term storage of formalin-fixed paraffin-embedded tissue sections.

Author(s): Grillo F, Pigozzi S, Ceriolo P, Calamaro P, Fiocca R, Mastracci L

Publication: Histochem Cell Biol, 2015, Vol. 144, Page 93-9

PubMed ID: 25757745 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This paper investigated the stability of immunohistochemical staining of 11 antigens using 14 antibodies after slide-mounted FFPE tissue sections were stored for 1 to 36 months unaltered at room temperature or 4°C, or after being wrapped in parafilm or coated in paraffin and stored at room temperature. Tissue specimens included normal prostate, thyroid, ovary, lymph node, and esophagus specimens; a breast carcinoma; a gastrointestinal stromal tumor; a lung adenocarcinoma; and a colorectal adenocarcinoma. All tissue was collected on the same day and fixed in 10% neutral buffered formalin for 24 hours prior to processing in a single batch. Each FFPE block was serial sectioned to generate thirty 4 um-thick sections and all FFPE blocks were sectioned the same day (time 0). Potential effects introduced by storage of FFPE blocks were also examined using freshly cut sections. All FFPE blocks were stored protected from light at room temperature for 36 months. All slides were stored protected from light and those at room temperature were not stored in a temperature-controlled environment (experiments were conducted in Italy).  Antigen retrieval was antibody-specific and included heat or enzymatic pre-treatment. Immunohistochemical staining was evaluated by two expert pathologists. The intensity of immunohistochemical staining was scored on a 0 to 3+ scale (0 was an absence of staining  and 3+ was equivalent to staining observed at time 0).

   Summary of Findings:

   FFPE blocks stored for up to 12 months at room temperature protected from light generated equivalent immunohistochemical staining scores (3+) as those analyzed immediately for all 14 antibodies and 11 antigens; however, a significant reduction in CD3 immunostaining and modest reductions in Ki67, TTF-1, and K7 immunostaining were observed beginning after 24 months of storage. Storage of slide-mounted FFPE sections at room temperature induced significant reductions in the intensity of immunostaining after 6 months of storage, regardless of whether slides were unaltered, coated in paraffin, or wrapped in parafilm. When unaltered slides were stored at room temperature, significant and progressive declines in immunostaining were observed for CD3 (after 6 months), CD31 (after 12 months), CD117 (after 9 months), ER, PR, Ki67, p53, and TTF-1 (after 12 months) while immunostaining of cytoplasmic antigens (SMA, K7, K20, keratins AE1/AE3, and 34βE12) remained stable for up to 36 months.  Comparatively, FFPE slides stored at 4°C exhibited much more stable immunostaining as significant reductions were limited to CD3 after 24 months and vimentin after 36 months. FFPE slides that were either coated in paraffin or wrapped in parafilm prior to storage displayed unpredictable reductions in immunostaining for the majority of antigens investigated.

   Biospecimens

   • Tissue - Stomach
   • Tissue - Lung
   • Tissue - Colorectal
   • Tissue - Esophagus
   • Tissue - Thyroid Gland
   • Tissue - Ovary
   • Tissue - Lymph Node
   • Tissue - Prostate

   Preservative Types

   • Formalin

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	1 month 6 months 9 months 12 months 24 months 36 months
   Storage	Storage conditions	Unaltered Wrapped in parafilm Paraffin-coated
   Storage	Storage temperature	Room temperature 4°C

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