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A new method for real-time evaluation of pepsin digestion of paraffin-embedded tissue sections, prior to fluorescence in situ hybridisation

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Abstract

Fluorescence in situ hybridisation (FISH) is a molecular cytogenetic technique, which is regularly applied to formalin-fixed paraffin-embedded (FFPE) tissue sections of a variety of cancers to assess chromosomal aberrations. However, high-quality FISH requires optimal enzymatic digestion, and insufficient digestion is not noted until the hybridisation signals are evaluated in the fluorescence microscope. As a consequence, FISH results may be unreliable, and the experiment might have to be repeated. To solve this problem, we developed a new method for real-time evaluation of enzymatic tissue digestion. Termination of enzyme activity at the proper time facilitates successful hybridisation, and experiments do not have to be repeated. We first performed FISH on 20 FFPE samples, which had been pepsin digested for different times, and this revealed distinct morphological changes within the nucleus and perinuclear space that were detectable by light microscopy. These observations suggested that the presence of intact and clear bare nuclei, surrounded by a translucent perinuclear space, might serve as an indicator of adequate digestion. We developed a protocol for assessment of this indicator, based on morphological features, and applied this to a collection of 400 tissue samples, partly of breast cancer and partly of different types of lymphoma, prior to FISH. The FISH success rate was 99.5% (398/400), which was significantly higher than that of the conventional method. In all successful cases, morphological signs of adequate digestion were paralleled by easily interpretable FISH signals. This new method for the real-time assessment of digestion quality improved the success rate of FISH and in addition was simple and rapid.

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Authors and Affiliations

  1. Department of Pathology, Beijing Friendship Hospital, Capital Medical University (Lymphoma Diagnostic Center, Beijing Institute of Clinical Medicine), Beijing, 100050, People’s Republic of China

    Xiaojing Teng, Shuhong Zhang, Wei Liu, Kuo Bi & Lei Zhang

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  1. Xiaojing Teng
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  2. Shuhong Zhang
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  3. Wei Liu
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  4. Kuo Bi
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  5. Lei Zhang
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Correspondence to Xiaojing Teng.

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All FFPE tissue blocks from our hospital and consultation sections from other hospitals were collected for routine pathological diagnosis and not for the purpose of this specific study. Research protocols were approved by the ethics review board of our centre.

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The authors declare that they have no conflict of interest.

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Teng, X., Zhang, S., Liu, W. et al. A new method for real-time evaluation of pepsin digestion of paraffin-embedded tissue sections, prior to fluorescence in situ hybridisation. Virchows Arch 470, 567–573 (2017). https://doi.org/10.1007/s00428-017-2097-z

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  • DOI: https://doi.org/10.1007/s00428-017-2097-z

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