Factors Influencing the Degradation of Archival Formalin-Fixed Paraffin-Embedded Tissue Sections

Abstract

The loss of antigenicity in archival formalin-fixed paraffin-embedded (FFPE) tissue sections negatively affects both diagnostic histopathology and advanced molecular studies. The mechanisms underlying antigenicity loss in FFPE tissues remain unclear. The authors hypothesize that water is a crucial contributor to protein degradation and decrement of immunoreactivity in FFPE tissues. To test their hypothesis, they examined fixation time, processing time, and humidity of storage environment on protein integrity and antigenicity by immunohistochemistry, Western blotting, and protein extraction. This study revealed that inadequate tissue processing, resulting in retention of endogenous water in tissue sections, results in antigen degradation. Exposure to high humidity during storage results in significant protein degradation and reduced immunoreactivity, and the effects of storage humidity are temperature dependent. Slides stored under vacuum with desiccant do not protect against the effects of residual water from inadequate tissue processing. These results support that the presence of water, both endogenously and exogenously, plays a central role in antigenicity loss. Optimal tissue processing is essential. The parameters of optimal storage of unstained slides remain to be defined, as they are directly affected by preanalytic variables. Nevertheless, minimization of exposure to water is required for antigen preservation in FFPE tissue sections. This article contains online supplemental material at http://www.jhc.org . Please visit this article online to view these materials.

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“…Grillo F et al demonstrated that antigen loss or decay was seen in IHC of CD3, CD 31, CD117, estrogen and progesterone receptors, Ki67, p53, TTF-1, vimentin, but not in IHC of smooth muscle actin, keratins 7, 20, AE1/AE3, 34βE12, and they also showed cold storage is suitable methods for long term storage of tissue sections. 19 The importance of cold storage (at minus degrees rather than at 4 o C) was reported by other researchers, 20 and these observations were consistent with our observation of figure 3.…”

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“…We also previously demonstrated that the possible oxidation of endogenous and exogenous water is a key factor in the loss of antigenicity during storage of FFPE tissue. 28 On the basis of our histomorphological observations of overbleached FFPE tissue sections (data not shown), the H 2 O 2 -based bleaching method may be linked with tissue oxidation. Clearly, the exact mechanism of the H 2 O 2 -based bleaching procedure is an area for further study.…”

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“…When using formalin-fixed, paraffin-embedded (FFPE) tissue specimens as starting material, the FFPE processing causes degradation of RNA that generates challenges in using expression patterns as a clinical biomarker; the oxygen and hydroxyl radicals in formalin crosslink RNA, and the high temperatures of the wax involved in embedding the sample cause irreversible damage to RNA, with fragmentation into 150 to 200 bases long oligonucleotides. 5 Run-to-run variations in processing parameters, as well as processing and storage variations from one institution to the next, can also affect RNA levels and degradation rates in FFPE tissue specimens. Tumor heterogeneity and the limited amount of tumor in biopsy material further affect expression analyses, introducing multiple, sometimes discordant, expression signatures.…”